Structure, biophysics, and circuit function of a "giant" cortical presynaptic terminal.

The hippocampal mossy fiber synapse, formed between axons of dentate gyrus granule cells and dendrites of CA3 pyramidal neurons, is a key synapse in the trisynaptic circuitry of the hippocampus. Because of its comparatively large size, this synapse is accessible to direct presynaptic recording, allowing a rigorous investigation of the biophysical mechanisms of synaptic transmission and plasticity. Furthermore, because of its placement in the very center of the hippocampal memory circuit, this synapse seems to be critically involved in several higher network functions, such as learning, memory, pattern separation, and pattern completion. Recent work based on new technologies in both nanoanatomy and nanophysiology, including presynaptic patch-clamp recording, paired recording, super-resolution light microscopy, and freeze-fracture and "flash-and-freeze" electron microscopy, has provided new insights into the structure, biophysics, and network function of this intriguing synapse. This brings us one step closer to answering a fundamental question in neuroscience: how basic synaptic properties shape higher network computations.

Layer 5 of cortex innervates the thalamic reticular nucleus in mice.

Neurons in the thalamic reticular nucleus (TRN) are a primary source of inhibition to the dorsal thalamus and, as they are innervated in part by the cortex, are a means of corticothalamic regulation. Previously, cortical inputs to the TRN were thought to originate solely from layer 6 (L6), but we recently reported the presence of putative synaptic terminals from layer 5 (L5) neurons in multiple cortical areas in the TRN [J. A. Prasad, B. J. Carroll, S. M. Sherman, J. Neurosci. 40, 5785-5796 (2020)]. Here, we demonstrate with electron microscopy that L5 terminals from multiple cortical regions make bona fide synapses in the TRN. We further use light microscopy to localize these synapses relative to recently described TRN subdivisions and show that L5 terminals target the edges of the somatosensory TRN, where neurons reciprocally connect to higher-order thalamus, and that L5 terminals are scarce in the core of the TRN, where neurons reciprocally connect to first-order thalamus. In contrast, L6 terminals densely innervate both edge and core subregions and are smaller than those from L5. These data suggest that a sparse but potent input from L5 neurons of multiple cortical regions to the TRN may yield transreticular inhibition targeted to higher-order thalamus.

Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse.

Rab3A-interacting molecule (RIM) is crucial for fast Ca2+-triggered synaptic vesicle (SV) release in presynaptic active zones (AZs). We investigated hippocampal giant mossy fiber bouton (MFB) AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1α-/- and wild-type mice. In RIM1α-/-, AZs are larger with increased synaptic cleft widths and a 3-fold reduced number of tightly docked SVs (0-2 nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron-dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1α-/- is more heterogeneous. Thus, RIM1α, besides its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs.

Sublamina-Specific Dynamics and Ultrastructural Heterogeneity of Layer 6 Excitatory Synaptic Boutons in the Adult Human Temporal Lobe Neocortex.

Synapses "govern" the computational properties of any given network in the brain. However, their detailed quantitative morphology is still rather unknown, particularly in humans. Quantitative 3D-models of synaptic boutons (SBs) in layer (L)6a and L6b of the temporal lobe neocortex (TLN) were generated from biopsy samples after epilepsy surgery using fine-scale transmission electron microscopy, 3D-volume reconstructions and electron microscopic tomography. Beside the overall geometry of SBs, the size of active zones (AZs) and that of the three pools of synaptic vesicles (SVs) were quantified. SBs in L6 of the TLN were middle-sized (~5 µm2), the majority contained only a single but comparatively large AZ (~0.20 µm2). SBs had a total pool of ~1100 SVs with comparatively large readily releasable (RRP, ~10 SVs L6a), (RRP, ~15 SVs L6b), recycling (RP, ~150 SVs), and resting (~900 SVs) pools. All pools showed a remarkably large variability suggesting a strong modulation of short-term synaptic plasticity. In conclusion, L6 SBs are highly reliable in synaptic transmission within the L6 network in the TLN and may act as "amplifiers," "integrators" but also as "discriminators" for columnar specific, long-range extracortical and cortico-thalamic signals from the sensory periphery.

Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.

Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing.

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. Here, we have investigated Drosophila motor neuron synaptic terminals during ageing. We observed cumulative fragmentation of presynaptic structures accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, we find that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. Our results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections.

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals.

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer's disease, and suggest that misregulated EV traffic may be an underlying defect.

Presynaptic coupling by electrical synapses coordinates a rhythmic behavior by synchronizing the activities of a neuron pair.

Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in Caenorhabditis elegans, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that inx-1/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of inx-1 leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.

Presynaptic α2δ subunits are key organizers of glutamatergic synapses.

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

Ground state depletion microscopy as a tool for studying microglia-synapse interactions.

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co-localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction-limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super-resolution microscopy to the analysis of routine formalin-fixed paraffin sections of archival human brain. The investigation of microglia-synapse interactions in dementia will be of special interest in this context.

Lowering Synaptogyrin-3 expression rescues Tau-induced memory defects and synaptic loss in the presence of microglial activation.

Tau is a major driver of neurodegeneration and is implicated in over 20 diseases. Tauopathies are characterized by synaptic loss and neuroinflammation, but it is unclear if these pathological events are causally linked. Tau binds to Synaptogyrin-3 on synaptic vesicles. Here, we interfered with this function to determine the role of pathogenic Tau at pre-synaptic terminals. We show that heterozygous knockout of synaptogyrin-3 is benign in mice but strongly rescues mutant Tau-induced defects in long-term synaptic plasticity and working memory. It also significantly rescues the pre- and post-synaptic loss caused by mutant Tau. However, Tau-induced neuroinflammation remains clearly upregulated when we remove the expression of one allele of synaptogyrin-3. Hence neuroinflammation is not sufficient to cause synaptic loss, and these processes are separately induced in response to mutant Tau. In addition, the pre-synaptic defects caused by mutant Tau are enough to drive defects in cognitive tasks.

Symmetrical arrangement of proteins under release-ready vesicles in presynaptic terminals.

Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca2+ influx. This Ca2+-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially "tethered" to the PM by a variable number of protein densities (∼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the "primed" vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca2+ influx.

Development of γ-aminobutyric acid-, glycine-, and glutamate-immunopositive boutons on the rat genioglossal motoneurons.

Detailed information about the development of excitatory and inhibitory synapses on the genioglossal (GG) motoneuron may help to understand the mechanism of fine control of GG motoneuron firing and the coordinated tongue movement during postnatal development. For this, we investigated the development of γ-aminobutyric acid (GABA)-immunopositive (GABA +), glycine + (Gly +), and glutamate + (Glut +) axon terminals (boutons) on the somata of rat GG motoneurons at a postnatal day 2 (P2), P6 and P18 by retrograde labeling of GG motoneurons with horseradish peroxidase, electron microscopic postembedding immunogold staining with GABA, Gly, and Glut antisera, and quantitative analysis. The number of boutons per GG motoneuron somata and the mean length of bouton apposition, measures of bouton size and synaptic covering percentage, were significantly increased from P2/P6 to P18. The number and fraction of GABA + only boutons of all boutons decreased significantly, whereas those of Gly + only boutons increased significantly from P2/P6 to P18, suggesting developmental switch from GABAergic to glycinergic synaptic transmission. The fraction of mixed GABA +/Gly + boutons of all boutons was the highest among inhibitory bouton types throughout the postnatal development. The fractions of excitatory and inhibitory boutons of all boutons remained unchanged during postnatal development. These findings reveal a distinct developmental pattern of inhibitory synapses on the GG motoneurons different from that on spinal or trigeminal motoneurons, which may have an important role in the regulation of the precise and coordinated movements of the tongue during the maturation of the oral motor system.

Maturation of Complex Synaptic Connections of Layer 5 Cortical Axons in the Posterior Thalamic Nucleus Requires SNAP25.

Synapses are able to form in the absence of neuronal activity, but how is their subsequent maturation affected in the absence of regulated vesicular release? We explored this question using 3D electron microscopy and immunoelectron microscopy analyses in the large, complex synapses formed between cortical sensory efferent axons and dendrites in the posterior thalamic nucleus. Using a Synaptosome-associated protein 25 conditional knockout (Snap25 cKO), we found that during the first 2 postnatal weeks the axonal boutons emerge and increase in the size similar to the control animals. However, by P18, when an adult-like architecture should normally be established, axons were significantly smaller with 3D reconstructions, showing that each Snap25 cKO bouton only forms a single synapse with the connecting dendritic shaft. No excrescences from the dendrites were formed, and none of the normally large glomerular axon endings were seen. These results show that activity mediated through regulated vesicular release from the presynaptic terminal is not necessary for the formation of synapses, but it is required for the maturation of the specialized synaptic structures between layer 5 corticothalamic projections in the posterior thalamic nucleus.

A Presynaptic Perspective on Transport and Assembly Mechanisms for Synapse Formation.

Neurons are highly polarized cells with a single axon and multiple dendrites derived from the cell body to form tightly associated pre- and postsynaptic compartments. As the biosynthetic machinery is largely restricted to the somatodendritic domain, the vast majority of presynaptic components are synthesized in the neuronal soma, packaged into synaptic precursor vesicles, and actively transported along the axon to sites of presynaptic biogenesis. In contrast with the significant progress that has been made in understanding synaptic transmission and processing of information at the post-synapse, comparably little is known about the formation and dynamic remodeling of the presynaptic compartment. We review here our current understanding of the mechanisms that govern the biogenesis, transport, and assembly of the key components for presynaptic neurotransmission, discuss how alterations in presynaptic assembly may impact nervous system function or lead to disease, and outline key open questions for future research.

Presynaptic vesicular accumulation is required for antipsychotic efficacy in psychotic-like rats.

BACKGROUND: The therapeutic effects of antipsychotic drugs (APDs) are mainly attributed to their postsynaptic inhibitory functions on the dopamine D2 receptor, which, however, cannot explain the delayed onset of full therapeutic efficacy. It was previously shown that APDs accumulate in presynaptic vesicles during chronic treatment and are released like neurotransmitters in an activity-dependent manner triggering an auto-inhibitory feedback mechanism. Although closely mirroring therapeutic action onset, the functional consequence of the APD accumulation process remained unclear. AIMS: Here we tested whether the accumulation of the APD haloperidol (HAL) is required for full therapeutic action in psychotic-like rats. METHODS: We designed a HAL analog compound (HAL-F), which lacks the accumulation property of HAL, but retains its postsynaptic inhibitory action on dopamine D2 receptors. RESULTS/OUTCOMES: By perfusing LysoTracker fluorophore-stained cultured hippocampal neurons, we confirmed the accumulation of HAL and the non-accumulation of HAL-F. In an amphetamine hypersensitization psychosis-like model in rats, we found that subchronic intracerebroventricularly delivered HAL (0.1 mg/kg/day), but not HAL-F (0.3-1.5 mg/kg/day), attenuates psychotic-like behavior in rats. CONCLUSIONS/INTERPRETATION: These findings suggest the presynaptic accumulation of HAL may serve as an essential prerequisite for its full antipsychotic action and may explain the time course of APD action. Targeting accumulation properties of APDs may, thus, become a new strategy to improve APD action.

Monitoring Activity-Dependent Bulk Endocytosis in Primary Neuronal Culture Using Large Fluorescent Dextrans.

The efficient recycling of synaptic vesicles (SVs) during neuronal activity is central for sustaining brain function. During intense neuronal activity, the dominant mechanism of SV retrieval is activity-dependent bulk endocytosis (ADBE). Here, we describe a method to monitor ADBE in isolation from other SV endocytosis modes, via the uptake of large fluorescent fluid-phase markers in primary neuronal culture. Furthermore, we outline how to monitor ADBE using this approach across a field of neurons or in individual neurons.

Combining Single Molecule Super-Resolution Imaging Techniques to Unravel the Nanoscale Organization of the Presynapse.

The fusion of synaptic vesicles with the plasma membrane underpins neurotransmission. A number of presynaptic proteins play a critical role in overcoming the energy barrier inherent to the fusion of the negatively charged vesicular and plasma membranes. Emerging concepts suggest that this process is hierarchical and dependent on rapid and transient reorganization of proteins in and out of small nanoclusters located in the active zones of nerve terminals. Examining the nanoscale organization of presynaptic molecules requires super-resolution microscopy to overcome the limits of conventional light microscopy. In this chapter, we describe three super-resolution techniques that allow for the examination of the nanoscale organization of proteins within live hippocampal nerve terminals. We used (1) single-particle tracking photoactivated localization microscopy (sptPALM) to resolve the mobility and clustering of syntaxin1A (STX1A), (2) universal Point Accumulation Imaging in Nanoscale Topography (uPAINT) to study the mobility of a pool of vesicular-associated membrane protein 2 (VAMP2) transiting on the plasma membrane, and (3) subdiffractional Tracking of Internalized Molecules (sdTIM) to track VAMP2-positive recycling synaptic vesicles in conjunction with Cholera Toxin subunit B (CTB), which has recently been shown to be trafficked retrogradely from the presynapse to the cell body via signaling endosomes.

A novel structure associated with aging is augmented in the DPP6-KO mouse brain.

In addition to its role as an auxiliary subunit of A-type voltage-gated K+ channels, we have previously reported that the single transmembrane protein Dipeptidyl Peptidase Like 6 (DPP6) impacts neuronal and synaptic development. DPP6-KO mice are impaired in hippocampal-dependent learning and memory and exhibit smaller brain size. Using immunofluorescence and electron microscopy, we report here a novel structure in hippocampal area CA1 that was significantly more prevalent in aging DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appeared as clusters of large puncta that colocalized NeuN, synaptophysin, and chromogranin A. They also partially labeled for MAP2, and with synapsin-1 and VGluT1 labeling on their periphery. Electron microscopy revealed that these structures are abnormal, enlarged presynaptic swellings filled with mainly fibrous material with occasional peripheral, presynaptic active zones forming synapses. Immunofluorescence imaging then showed that a number of markers for aging and especially Alzheimer's disease were found as higher levels in these novel structures in aging DPP6-KO mice compared to WT. Together these results indicate that aging DPP6-KO mice have increased numbers of novel, abnormal presynaptic structures associated with several markers of Alzheimer's disease.

The Input-Output Relation of Primary Nociceptive Neurons is Determined by the Morphology of the Peripheral Nociceptive Terminals.

The output from the peripheral terminals of primary nociceptive neurons, which detect and encode the information regarding noxious stimuli, is crucial in determining pain sensation. The nociceptive terminal endings are morphologically complex structures assembled from multiple branches of different geometry, which converge in a variety of forms to create the terminal tree. The output of a single terminal is defined by the properties of the transducer channels producing the generation potentials and voltage-gated channels, translating the generation potentials into action potential (AP) firing. However, in the majority of cases, noxious stimuli activate multiple terminals; thus, the output of the nociceptive neuron is defined by the integration and computation of the inputs of the individual terminals. Here, we used a computational model of nociceptive terminal tree to study how the architecture of the terminal tree affects the input-output relation of the primary nociceptive neurons. We show that the input-output properties of the nociceptive neurons depend on the length, the axial resistance (Ra), and location of individual terminals. Moreover, we show that activation of multiple terminals by a capsaicin-like current allows summation of the responses from individual terminals, thus leading to increased nociceptive output. Stimulation of the terminals in simulated models of inflammatory or neuropathic hyperexcitability led to a change in the temporal pattern of AP firing, emphasizing the role of temporal code in conveying key information about changes in nociceptive output in pathologic conditions, leading to pain hypersensitivity.SIGNIFICANCE STATEMENT Noxious stimuli are detected by terminal endings of primary nociceptive neurons, which are organized into morphologically complex terminal trees. The information from multiple terminals is integrated along the terminal tree, computing the neuronal output, which propagates toward the CNS, thus shaping the pain sensation. Here, we revealed that the structure of the nociceptive terminal tree determines the output of nociceptive neurons. We show that the integration of noxious information depends on the morphology of the terminal trees and how this integration and, consequently, the neuronal output change under pathologic conditions. Our findings help to predict how nociceptive neurons encode noxious stimuli and how this encoding changes in pathologic conditions, leading to pain.