The morphology of axospinous synapses and their parent spines varies widely. Additionally, many of these synapses are contacted by multiple synapse boutons (MSBs) and show substantial variability in receptor expression. The two major axospinous synaptic subtypes are perforated and nonperforated, but there are several subcategories within these two classes. The present study used serial section electron microscopy to determine whether perforated and nonperforated synaptic subtypes differed with regard to their distribution, size, receptor expression, and connectivity to MSBs in three apical dendritic regions of rat hippocampal area CA1: the proximal and distal thirds of stratum radiatum, and stratum lacunosum-moleculare. All synaptic subtypes were present throughout the apical dendritic regions, but there were several subclass-specific differences. First, segmented, completely partitioned synapses changed in number, proportion, and AMPA receptor expression with distance from the soma beyond that found within other perforated synaptic subtypes. Second, atypically large nonperforated synapses showed NMDA receptor immunoreactivity identical to perforated synapses, levels of AMPA receptor expression intermediate to nonperforated and perforated synapses, and perforated synapse-like changes in structure with distance from the soma. Finally, MSB connectivity was highest in proximal stratum radiatum, but only for those MSBs comprised of nonperforated synapses. The immunogold data suggest that most MSBs would not generate simultaneous depolarizations in multiple neurons or spines, however, because the vast majority of MSBs are comprised of two synapses with abnormally low levels of receptor expression, or involve one synapse with a high level of receptor expression and another with only a low level.
serial section analysis; immunogold electron microscopy; AMPA receptor; NMDA receptor; multiple synapse bouton; dendritic spine; postsynaptic density
Age-related memory impairment occurs in many mammalian species including humans. Moreover, women undergoing the menopausal transition often complain of problems with memory. We recently reported that rhesus monkeys display age- and menopause-related recognition memory impairment on a hippocampus-reliant test (delayed nonmatching-to-sample; DNMS). In the same monkeys, perforated synapse densities in the dentate gyrus outer molecular layer (OML) correlated with DNMS recognition accuracy, while total axospinous synapse density was similar across age and menses groups. The current study examined whether synaptic characteristics of OML axonal boutons are coupled with age- or menopause-related memory deficits. Using serial section electron microscopy, we measured the frequencies of single-synapse boutons (SSBs), multiple-synapse boutons (MSBs), and boutons with no apparent synaptic contacts (non-synaptic boutons, NSBs) in the OML. Aged females had double the percentage of NSBs as compared to young females and this measure correlated positively and inversely with DNMS acquisition (number of trials to criterion) and delay performance (average accuracy), respectively. Aged compared to young females also had a lower frequency of MSBs and a lower number of synaptic contacts per MSB, and the latter variable inversely correlated with DNMS acquisition. Although proportions of NSBs, SSBs and MSBs were similar across menses groups, compared to pre-menopausal monkeys, peri/post-menopausal monkeys had fewer MSBs contacting one or more segmented perforated synapse and the abundance of this bouton subtype positively correlated with DNMS performance. These results suggest that age- and menopause-related shifts in OML synaptic subtypes may be coupled with deficits in task acquisition and recognition memory.
delayed nonmatching-to-sample test; menopause; multiple-synapse bouton; serial sections; recognition memory
Dendrites, axons and synapses are dynamic during circuit development, however changes in microcircuit connections as branches stabilize have not been directly demonstrated. By combining in vivo time-lapse imaging of Xenopus tectal neurons with electron microscope reconstructions of imaged neurons, we report for the first time the distribution and ultrastructure of synapses on individual vertebrate neurons and relate these synaptic properties to dynamics in dendritic and axonal arbor structure over hours or days of imaging. Dynamic dendrites have a high density of immature synapses whereas stable dendrites have sparser, mature synapses. Axons initiate contacts from multisynapse boutons on stable branches. Connections are refined by decreasing convergence from multiple inputs to postsynaptic dendrites and by decreasing divergence from multisynapse boutons to postsynaptic sites. Visual deprivation or NMDAR antagonists decreased synapse maturation and elimination, suggesting that coactive input activity promotes microcircuit development by concurrently regulating synapse elimination and maturation of remaining contacts.
in vivo imaging; ultrastructure; synapse formation; synapse maturation; 3D reconstruction; Xenopus; retinotectal system; synapse elimination
Coordinated changes at excitatory and inhibitory synapses are essential for normal brain development and function. It is well established that excitatory neurons undergo structural changes, but our knowledge about inhibitory structural plasticity is rather scarce. Here we present a quantitative analysis of the dynamics of GABAergic boutons in the dendritic region of the hippocampal CA1 area using time-lapse two-photon imaging in organotypic hippocampal cultures from GAD65-GFP mice. We show that ~20% of inhibitory boutons are not stable. They are appearing, disappearing and reappearing at specific locations along the inhibitory axon and reflect immature or incomplete synapses. Furthermore, we observed that persistent boutons show large volume fluctuations over several hours, suggesting that presynaptic content of inhibitory synapses is not constant. Our data show that inhibitory boutons are highly dynamic structures and suggest that inhibitory axons are continuously probing potential locations for inhibitory synapse formation by redistributing presynaptic material along the axon. In addition, we found that neuronal activity affects the exploratory dynamics of inhibitory axons. Blocking network activity rapidly reduces the number of transient boutons, whereas enhancing activity reduces the number of persistent inhibitory boutons, possibly reflecting enhanced competition between boutons along the axon. The latter effect requires signaling through GABAA receptors. We propose that activity-dependent regulation of bouton dynamics contributes to inhibitory synaptic plasticity.
activity-dependent plasticity; bouton dynamics; hippocampal organotypic cultures; inhibitory axons; two-photon microscopy
Caloric restriction (CR) extends lifespan and ameliorates the aging-related decline in hippocampal-dependent cognitive function. In the present study, we compared subunit levels of NMDA and AMPA types of the glutamate receptor and quantified total synapses and multiple spine bouton (MSB) synapses in hippocampal CA1 from young (10 months), middle-aged (18 months), and old (29 months) Fischer 344 x Brown Norway rats that were ad libitum (AL) fed or caloric restricted (CR) from 4 months of age. Each of these parameters has been reported to be a potential contributor to hippocampal function. Western blot analysis revealed that NMDA and AMPA receptor subunits in AL animals decrease between young and middle age to levels that are present at old age. Interestingly, young CR animals have significantly lower levels of glutamate receptor subunits than young AL animals and those lower levels are maintained across life span. In contrast, stereological quantification indicated that total synapses and MSB synapses are stable across lifespan in both AL and CR rats. These results indicate significant aging-related losses of hippocampal glutamate receptor subunits in AL rats that are consistent with altered synaptic function. CR eliminates that aging-related decline by inducing stable NMDA and AMPA receptor subunit levels.
Dietary restriction; Fischer 344 X Brown Norway rats; glutamate receptor; stratum radiatum; electron microscopy; multiple spine bouton (MSB) synapses
The high resolution structure of the cytoskeleton in dendritic spines and their precursors, dendritic filopodia, was characterized by platinum replica electron microscopy. The unexpected features of the actin architecture of these protrusions dramatically change the existing paradigm of cytoskeletal organization of elongated membrane protrusions.
Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.
N-cadherin is a cell adhesion molecule which is enriched at synapses. Binding of N-cadherin molecules to each other across the synaptic cleft has been postulated to stabilize adhesion between the presynaptic bouton and the postsynaptic terminal. N-cadherin is also required for activity-induced changes at synapses, including hippocampal long term potentiation and activity-induced spine expansion and stabilization. We hypothesized that these activity-dependent changes might involve changes in N-cadherin localization within synapses. To determine whether synaptic activity changes the localization of N-cadherin, we used structured illumination microscopy, a super-resolution approach which overcomes the conventional resolution limits of light microscopy, to visualize the localization of N-cadherin within synapses of hippocampal neurons. We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft. Furthermore, the N-cadherin localization pattern within synapses changes during KCl depolarization and after transient synaptic stimulation. During KCl depolarization, N-cadherin relocalizes away from the central region of the synaptic cleft to the periphery of the synapse. In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells. This indicates that transient synaptic stimulation modulates N-cadherin localization within the synapse. These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.
The hippocampal mossy fiber (MF) terminal is among the largest and most complex synaptic structures in the brain. Our understanding of the development of this morphologically elaborate structure has been limited because of the inability of standard electron microscopy techniques to quickly and accurately reconstruct large volumes of neuropil. Here we use serial block-face electron microscopy (SBEM) to surmount these limitations and investigate the establishment of MF connectivity during mouse postnatal development. Based on volume reconstructions, we find that MF axons initially form bouton-like specializations directly onto dendritic shafts, that dendritic protrusions primarily arise independently of bouton contact sites, and that a dramatic increase in presynaptic and postsynaptic complexity follows the association of MF boutons with CA3 dendritic protrusions. We also identify a transient period of MF bouton filopodial exploration, followed by refinement of sites of synaptic connectivity. These observations enhance our understanding of the development of this highly specialized synapse and illustrate the power of SBEM to resolve details of developing microcircuits at a level not easily attainable with conventional approaches.
Impaired gating by hippocampal dentate granule cells may promote the development of limbic epilepsy by facilitating seizure spread through the hippocampal trisynaptic circuit. The second synapse in this circuit, the dentate granule cell≫CA3 pyramidal cell connection, may be of particular importance because pathological changes occurring within the dentate likely exert their principal effect on downstream CA3 pyramids. Here, we utilized GFP-expressing mice and immunolabeling for the zinc transporter ZnT-3 to reveal the pre- and postsynaptic components of granule cell≫CA3 pyramidal cell synapses following pilocarpine-epileptogenesis. Confocal analyses of these terminals revealed that while granule cell presynaptic giant boutons increased in size and complexity one month after status epilepticus, individual thorns making up the postsynaptic thorny excrescences of the CA3 pyramidal cells were reduced in number. This reduction, however, was transient, and three months after status, thorn density recovered. This recovery was accompanied by a significant change in the distribution of thorns along pyramidal cells dendrites. While thorns in control animals tended to be tightly clustered, thorns in epileptic animals were more evenly distributed. Computational modeling of thorn distributions predicted an increase in the number of boutons required to cover equivalent numbers of thorns in epileptic vs. control mice. Confirming this prediction, ZnT-3 labeling of presynaptic giant boutons apposed to GFP-expressing thorns revealed a near doubling in bouton density, while the number of individual thorns per bouton was reduced by half. Together, these data provide clear evidence of novel plastic changes occurring within the epileptic hippocampus.
Despite considerable progress in understanding the molecular components of synapses in the central nervous system, the ultrastructural rearrangements underlying synaptic development remain unclear. We used serial section transmission electron microscopy and three-dimensional reconstructions of the optic tectal neuropil of Xenopus laevis tadpoles to detect and quantify changes in synaptic ultrastructure over a 1-week period from stages 39 and 47, during which time the visual system of Xenopus tadpoles becomes functional. Synapse density, presynaptic maturation index, and number of synapses per axon bouton increase, whereas the number of DCVs per bouton decreases, between stages 39 and 47. The width of the synaptic cleft decreased and the diameter of postsynaptic profiles increased between stages 39 and 47 and then remained relatively unchanged after stage 47. We found no significant difference in synapse maturation between GABAergic and non-GABAergic synapses. To test the effect of visual experience on synaptogenesis, animals were deprived of visual experience for 3 days from stage 42 to 47. Visual deprivation decreased synapse maturation and the number of connections per bouton. Furthermore, visual deprivation increased the number of DCVs per bouton by more than twofold. The visual-deprivation-induced decrease in synaptic connections is specific to asymmetric non-GABAergic synapses; however, both symmetric GABAergic and asymmetric synapses show comparable increases in the number DCVs with visual deprivation. In both the control and the visually deprived animals, the number of DCVs per bouton is highly variable and does not correlate with either synapse maturation or the number of connected partners per bouton. These data suggest that synaptogenesis and DCV accumulation are regulated by visual experience and further suggest a complex spatial and temporal relation between DCV accumulation and synapse formation.
dense core vesicles; multiple synapse boutons; optic tectum; ultrastructure; 3D reconstruction; visual deprivation; synaptogenesis
Accumulating evidence indicate that GABA regulates activity-dependent development of inhibitory synapses in the vertebrate brain, but the underlying mechanisms remain unclear. Here we combined live imaging of cortical GABAergic axons with single cell genetic manipulation to dissect the role of presynaptic GABAB receptors (GABABRs) in inhibitory synapse formation in mouse. Developing GABAergic axons form a significant number of transient boutons but only a subset was stabilized. Synaptic vesicles in these nascent boutons are often highly mobile in the course of tens of minutes. Activation of presynaptic GABABRs stabilized mobile vesicles in nascent boutons through the local enhancement of actin polymerization. Inactivation of GABABRs in developing basket interneurons resulted in aberrant pattern of bouton size distribution, reduced bouton density and reduced axon branching, as well as reduced frequency of miniature inhibitory currents in postsynaptic pyramidal neurons. These results suggest that GABABRs along developing inhibitory axons act as a local sensor of GABA release and promote presynaptic maturation through increased recruitment of mobile vesicle pools. Such release-dependent validation and maturation of nascent terminals is well suited to sculpt the pattern of synapse formation and distribution along axon branches.
GABAB receptor; synaptic vesicle dynamics; live cell imaging; actin polymerization; FRET; inhibitory synapses; activity-dependent development
In Saccharomyces cerevisiae, polarized growth depends on interactions between the actin cytoskeleton and the secretory machinery. Here we show that the Rab GTPase-activating proteins (GAPs) Msb3 and Msb4 interact directly with Spa2, a scaffold protein of the “polarisome” that also interacts with the formin Bni1. Spa2 is required for the polarized localization of Msb3 and Msb4 at the bud tip. We also show that Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP in vitro and that Msb3 and Rho GDP dissociation inhibitor act independently but oppositely on Cdc42. Finally, we show that Msb3 and Msb4 are involved in Bni1-nucleated actin assembly in vivo. These results suggest that Msb3 and Msb4 regulate polarized growth by multiple mechanisms, directly regulating exocytosis through their GAP activity toward Sec4 and potentially coordinating the functions of Cdc42, Rho1, and Bni1 in the polarisome through their binding to these GTPases. A functional equivalent of the polarisome probably exists in other fungi and mammals.
Cerebellar Purkinje cells (PCs) receive GABAergic input that undergoes powerful retrograde modulation by presynaptic cannabinoid and glutamate receptors. Here we examine a distinct modulatory mechanism at these synapses, which does not require postsynaptic depolarization and acts through presynaptic AMPA receptors. We find that this mechanism operates mainly in the somatic vicinity of PCs where large boutons of basket cell axons form synapses on the PC soma. We use fast confocal microscopy and detailed kinetic modeling to estimate that in these boutons an action potential opens 100-200 Ca2+ channels eliciting a brief 3-5 μM transient followed by a longer-term, 15-30 nM rise of free Ca2+ (above the resting level of ~100 nM). Brief activation of local AMPA receptors suppresses Ca2+ entry (probably by silencing 20-40 P/Q-type channels) in a sub-group of terminals that tend to show a higher dynamic range of Ca2+ signaling. The results provide the first quantitative description of presynaptic Ca2+ kinetics and its modulation by AMPA receptor activation (most likely via a glutamate spillover-mediated mechanism) at identified GABAergic synapses.
Presynaptic mechanisms; Ca2+ signaling; glutamate; spillover; AMPARs
Presynaptic GABAA receptors occur at hippocampal mossy fiber synapses. Whether and how they modulate orthodromic signaling to postsynaptic targets is poorly understood. We show that an endogenous neurosteroid selective for high-affinity δ-subunit-containing GABAA receptors depolarizes rat mossy fiber boutons, enhances action potential-dependent Ca2+ transients, and facilitates glutamatergic transmission to pyramidal neurons. Conversely, blocking GABAA receptors hyperpolarizes mossy fiber boutons, increases their input resistance, decreases spike width, and attenuates action potential-dependent presynaptic Ca2+ transients, indicating that a subset of presynaptic GABA receptors are tonically active. Blocking GABAA receptors also interferes with the induction of long-term potentiation at mossy fiber-CA3 synapses. Presynaptic GABAA receptors thus facilitate information flow to the hippocampus both directly and by enhancing LTP.
In the early postnatal hippocampus, the first synapses to appear on excitatory pyramidal neurons are formed directly on dendritic shafts. Very few dendritic spines are present at this time. By adulthood, however, the overwhelming majority of synapses are located at the tips of dendritic spines. Several models have been proposed to account for the transition from mostly shaft to mostly spinous synapses but none have been demonstrated conclusively. To investigate the cellular mechanism underlying the shaft-to-spinous synapse transition, we designed live imaging experiments to directly observe the dynamics of shaft and spinous synapses on developing dendrites. Immunofluorescent synaptic labeling of GFP-filled neurons showed that the shaft-to-spinous synapse transition in dissociated culture mirrors that in vivo. Along with electron microscopy, the fluorescent labeling also showed that veritable shaft synapses are abundant in dissociated culture and that shaft synapses are frequently adjacent to spines or other dendritic protrusions, a configuration previously observed in vivo by others. We used live long term time lapse confocal microscopy of GFP-filled dendrites and VAMP2-DsRed-labeled boutons to record the fate of shaft synapses and associated dendritic protrusions and boutons with images taken hourly for up to 31 continuous hours. Inspection of the time lapse imaging series revealed that shaft synapses can persist adjacent to either existing or newly grown dendritic protrusions. Alternatively, a shaft synapse bouton can redistribute to contact an adjacent dendritic protrusion. However, we never observed shaft synapses transforming themselves into spines or any type of dendritic protrusions. We conclude that repeated iterations of dendritic protrusion or spine outgrowth adjacent to shaft synapses is very likely to be a critical component of the shaft-to-spinous synapse transition during CNS development.
synaptogenesis; spinogenesis; synaptic plasticity; correlative light and electron microscopy (CLEM); laser scanning confocal microscopy (LSCM)
Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.
Most presynaptic terminals in the brain contain G-protein coupled receptors that function to reduce action potential-evoked neurotransmitter release. These neuromodulatory receptors, including those for glutamate, GABA, endocannabinoids and adenosine, exert a substantial portion of their effect by reducing evoked presynaptic Ca2+ transients. Many axons form synapses with multiple postsynaptic neurons, but it is unclear if presynaptic attenuation in these synapses is homogeneous, as suggested by population level Ca2+ imaging. We loaded Ca2+-sensitive dyes into cerebellar parallel fiber axons and imaged action potential-evoked Ca2+ transients in individual presynaptic boutons with application of three different neuromodulators and found that adjacent boutons on the same axon showed striking heterogeneity in their strength of attenuation. Moreover, attenuation was predicted by bouton size or basal Ca2+ response: smaller boutons were more sensitive to adenosine A1 agonist but less sensitive to CB1 agonist while boutons with high basal action potential-evoked Ca2+ transient amplitude were more sensitive to mGluR4 agonist. These results suggest that boutons within brief segment of a single parallel fiber axon can have different sensitivities towards neuromodulators and may have different capacities for both short-term and long-term plasticities.
neuromodulator; parallel fiber; cerebellum; presynaptic bouton; calcium imaging; Imaging
Although quantum dots (QDs) have provided invaluable information regarding the diffusive behaviors of postsynaptic receptors, their application in presynaptic terminals has been rather limited. In addition, the diffraction-limited nature of the presynaptic bouton has hampered detailed analyses of the behaviors of synaptic vesicles (SVs) at synapses. Here, we created a quantum-dot based presynaptic probe and characterized the dynamic behaviors of individual SVs. As previously reported, the SVs exhibited multiple exchanges between neighboring boutons. Actin disruption induced a dramatic decrease in the diffusive behaviors of SVs at synapses while microtubule disruption only reduced extrasynaptic mobility. Glycine-induced synaptic potentiation produced significant increases in synaptic and inter-boutonal trafficking of SVs, which were NMDA receptor- and actin-dependent while NMDA-induced synaptic depression decreased the mobility of the SVs at synapses. Together, our results show that sPH-AP-QD revealed previously unobserved trafficking properties of SVs around synapses, and the dynamic modulation of SV mobility could regulate presynaptic efficacy during synaptic activity.
Synaptic activity regulates the postsynaptic accumulation of AMPA receptors over timescales ranging from minutes to days. Indeed, the regulated trafficking and mobility of GluR1 AMPA receptors underlies many forms of synaptic potentiation at glutamatergic synapses throughout the brain. However, the basis for synapse-specific accumulation of GluR1 is unknown. Here we report that synaptic activity locally immobilizes GluR1 AMPA receptors at individual synapses. Using single-molecule tracking together with the silencing of individual presynaptic boutons, we demonstrate that local synaptic activity reduces diffusional exchange of GluR1 between synaptic and extraynaptic domains, resulting in postsynaptic accumulation of GluR1. At neighboring inactive synapses, GluR1 is highly mobile with individual receptors frequently escaping the synapse. Within the synapse, spontaneous activity confines the diffusional movement of GluR1 to restricted subregions of the postsynaptic membrane. Thus, local activity restricts GluR1 mobility on a submicron scale, defining a novel input-specific mechanism for regulating AMPA receptor composition and abundance.
Most existing connectomic data and ongoing efforts focus either on individual synapses (e.g., with electron microscopy) or on regional connectivity (tract tracing). An individual pyramidal cell (PC) extends thousands of synapses over macroscopic distances (∼cm). The contrasting requirements of high-resolution and large field of view make it too challenging to acquire the entire synaptic connectivity for even a single typical cortical neuron. Light microscopy can image whole neuronal arbors and resolve dendritic branches. Analyzing connectivity in terms of close spatial appositions between axons and dendrites could thus bridge the opposite scales, from synaptic level to whole systems. In the mammalian cortex, structural plasticity of spines and boutons makes these “potential synapses” functionally relevant to learning capability and memory capacity. To date, however, potential synapses have only been mapped in the surrounding of a neuron and relative to its local orientation rather than in a system-level anatomical reference. Here we overcome this limitation by estimating the potential connectivity of different neurons embedded into a detailed 3D reconstruction of the rat hippocampus. Axonal and dendritic trees were oriented with respect to hippocampal cytoarchitecture according to longitudinal and transversal curvatures. We report the potential connectivity onto PC dendrites from the axons of a dentate granule cell, three CA3 PCs, one CA2 PC, and 13 CA3b interneurons. The numbers, densities, and distributions of potential synapses were analyzed in each sub-region (e.g., CA3 vs. CA1), layer (e.g., oriens vs. radiatum), and septo-temporal location (e.g., dorsal vs. ventral). The overall ratio between the numbers of actual and potential synapses was ∼0.20 for the granule and CA3 PCs. All potential connectivity patterns are strikingly dependent on the anatomical location of both pre-synaptic and post-synaptic neurons.
rat hippocampus; potential synapses; connectivity; 3D model; CA pyramidal axons; dentate mossy fiber; CA3b interneuron axons; computational
As synapses grow at the Drosophila neuromuscular junction, they shed membrane material in an activity-dependent manner. Glia and postsynaptic muscle cells are required to engulf this debris to ensure new synaptic growth.
Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived “debris” during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.
The synapse is the fundamental unit of communication between neurons and their target cells. As the nervous system matures, synapses often need to be added, removed, or otherwise remodeled to accommodate the changing needs of the circuit. Such changes are often regulated by the activity of the circuit and are thought to entail the extension or retraction of cellular processes to form or break synaptic connections. We have explored the precise nature of new synapse formation during development of the Drosophila larval neuromuscular junction (NMJ). We find that growing synapses are actually quite wasteful and shed significant amounts of presynaptic membranes and a subset of immature (nonfunctional) synapses. The shedding of this presynaptic material is enhanced by stimulating the activity of the neuron, suggesting that its formation is dependent upon NMJ activity. Surprisingly, we find presynaptic membranes are efficiently removed from the NMJ by two surrounding cell types: glia cells (a neuronal ‘support cell’), which invade the NMJ, and the postsynaptic muscle cell itself. Blocking the ability of these cells to ingest shed presynaptic membranes dramatically reduces new synapse growth, suggesting that the shed presynaptic material is inhibitory to new synapse addition. Therefore, our data demonstrate that actively growing synapses constantly shed membrane material, that glia and muscles work to rapidly clear this from the NMJ, and that the combined efforts of glia and muscles are critical for the proper addition of new synapses to neural circuits.
The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 μm in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.
The neurotoxic effect of amyloid-β peptide (Aβ) over the central synapses has been described and is reflected in the decrease of some postsynaptic excitatory proteins, the alteration in the number and morphology of the dendritic spines, and a decrease in long-term potentiation. Many studies has been carried out to identify the putative Aβ receptors in neurons, and is still no clear why the Aβ oligomers only affect the excitatory synapses. Aβ oligomers bind to neurite and preferentially to the postsynaptic region, where the postsynaptic protein-95 (PSD-95) is present in the glutamatergic synapse, and interacts directly with the N-methyl-D-aspartate receptor (NMDAR) and neuroligin (NL). NL is a postsynaptic protein which binds to the presynaptic protein, neurexin to form a heterophilic adhesion complex, the disruption of this interaction affects the integrity of the synaptic contact. Structurally, NL has an extracellular domain homolog to acetylcholinesterase, the first synaptic protein that was found to interact with Aβ. In the present review we will document the interaction between Aβ and the extracellular domain of NL-1 at the excitatory synapse, as well as the interaction with other postsynaptic components, including the glutamatergic receptors (NMDA and mGluR5), the prion protein, the neurotrophin receptor, and the α7-nicotinic acetylcholine receptor. We conclude that several Aβ oligomers receptors exist at the excitatory synapse, which could be the responsible for the neurotoxic effect described for the Aβ oligomers. The characterization of the interaction between Aβ receptors and Aβ oligomers could help to understand the source of the neurologic damage observed in the brain of the Alzheimer’s disease patients.
Aβ oligomers; postsynaptic receptors; neuroligin-1; synaptotoxicity; Alzheimer’s disease
Complete reconstructions of vertebrate neuronal circuits on the synaptic level require new approaches. Here, serial section transmission electron microscopy was automated to densely reconstruct four volumes, totaling 670μm3, from the rat hippocampus as proving grounds to determine when axo-dendritic proximities predict synapses. First, in contrast with Peters’ rule, the density of axons within reach of dendritic spines did not predict synaptic density along dendrites because the fraction of axons making synapses was variable. Second, an axo-dendritic touch did not predict a synapse; nevertheless, the density of synapses along a hippocampal dendrite appeared to be a universal fraction, 0.2, of the density of touches. Finally, the largest touch between an axonal bouton and spine indicated the site of actual synapses with about 80% precision, but would miss about half of all synapses. Thus, it will be difficult to predict synaptic connectivity using data sets missing ultrastructural details that distinguish between axo-dendritic touches and bona fide synapses.
The ultrastructural details of presynapses formed between
substrates of submicrometer silica beads and hippocampal neurons
are visualized via cryo-electron microscopy (cryo-EM). The silica
beads are derivatized by poly-d-lysine or lipid bilayers.
Molecular features known to exist at presynapses are clearly present
at these artificial synapses, as visualized by cryo-EM. Key synaptic
features such as the membrane contact area at synaptic junctions,
the presynaptic bouton containing presynaptic vesicles, as well as
microtubular structures can be identified. This is the first report
of the direct, label-free observation of ultrastructural details of
Artificial synapses; cryo-EM; electron microscopy; synaptic vesicles; hippocampal neurons