CNS Synapses Research Articles

Graded control of Purkinje cell outputs by cAMP through opposing actions on axonal action potential and transmitter release.

All-or-none signalling by action potentials (APs) in neuronal axons is pivotal for the precisely timed and identical size of outputs to multiple distant targets. However, technical limitations with respect to measuring the signalling in small intact axons have hindered the evaluation of high-fidelity signal propagation. Here, using direct recordings from axonal trunks and/or terminals of cerebellar Purkinje cells in slice and culture, we demonstrate that the timing and amplitude of axonal outputs are gradually modulated by cAMP depending on the length of axon. During the propagation in long axon, APs were attenuated and slowed in conduction by cAMP via specifically decreasing axonal Na+ currents. Consequently, the Ca2+ influx and transmitter release at distal boutons are reduced by cAMP, counteracting its direct facilitating effect on release machinery as observed at various CNS synapses. Together, our tour de force functional dissection has unveiled the axonal distance-dependent graded control of output timing and strength by intracellular signalling. KEY POINTS: The information processing in the nervous system has been classically thought to rely on the axonal faithful and high-speed conduction of action potentials (APs). We demonstrate that the strength and timing of axonal outputs are weakened and delayed, respectively, by cytoplasmic cAMP depending on the axonal length in cerebellar Purkinje cells (PCs). Direct axonal patch clamp recordings uncovered axon-specific attenuation of APs by cAMP through reduction of axonal Na+ currents. cAMP directly augments transmitter release at PC terminals without changing presynaptic Ca2+ influx or readily releasable pool of vesicles, although the extent is weaker compared to other CNS synapses. Two opposite actions of cAMP on PC axons, AP attenuation and release augmentation, together give rise to graded control of synaptic outputs in a manner dependent on the axonal length.

Dynamics of Changes in Acetylcholinesterase Activity in the Brain and Bood of Rats During Pharmacological Correction of Seizures Caused by Carbamate

To simulate a convulsive syndrome, phenylcarbamate was administered intraperitoneally at a dose of 1 mg/kg. Valproic acid aminoester (43 mg/kg), caramiphene (50 mg/kg), and diferidine (2 mg/kg) were investigated as potential anticonvulsant drugs. Blood and brain tissue for the determination of acetylcholinesterase (AСhE) were taken at 10, 30, 60 minutes, 6 and 24 hours after administration of the xenobiotic. In case of poisoning with reversible inhibitors of AChE and accumulation of acetylcholine in the CNS synapses (due to inhibition of AChE activity), only the initial manifestations of convulsive activity are caused, and other mechanisms not associated with AChE inhibition are responsible for the processes of further maintenance and recurrence of seizures.

Polymorphism in the LASP1 gene promoter region alters cognitive functions of patients with schizophrenia

Schizophrenia’s pathogenesis remains elusive. Cognitive dysfunction is the endophenotype and outcome predictor of schizophrenia. The LIM and SH3 domain protein (LASP1) protein, a component of CNS synapses and dendritic spines, has been related to the N-methyl-D-aspartate receptor (NMDAR) dysfunction hypothesis and schizophrenia. A single-nucleotide polymorphism (rs979607) in the LASP1 gene promoter region has been also implicated in schizophrenia susceptibility. The aim of this study was to investigate the role of the LASP1 rs979607 polymorphism in the cognitive functions of patients with schizophrenia. Two hundred and ninety-one Han Taiwanese patients with schizophrenia were recruited. Ten cognitive tests and two clinical rating scales were assessed. The scores of cognitive tests were standardized to T-scores. The genotyping of the LASP1 rs979607 polymorphism was performed using TaqMan assay. Among the 291 patients, 85 were C/C homozygotes of rs979607, 141 C/T heterozygotes, and 65 T/T homozygotes, which fitted the Hardy-Weinberg equilibrium. After adjusting age, gender, and education with general linear model, the C/C homozygotes performed better than C/T heterozygotes in overall composite score (p = 0.023), Category Fluency test (representing processing speed and semantic memory) (p = 0.045), and Wechsler Memory Scale (WMS)-III backward Spatial Span test (p = 0.025), albeit without correction for multiple comparisons for the latter two individual tests. To the best of our knowledge, this is the first study suggesting that the genetic variation of LASP1 may be associated with global cognitive function, category verbal fluency, and spatial working memory of patients with schizophrenia. The finding also lends support to the NMDAR dysfunction hypothesis of schizophrenia. More studies with longitudinal designs are warranted.

Distinct Nanoscale Calcium Channel and Synaptic Vesicle Topographies Contribute to the Diversity of Synaptic Function

The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses.

The role of DJ-1 in CNS synapses

The role of DJ-1 in CNS synapses

Microglia Sculpt Sex Differences in Social Behavior

Microglia are increasingly recognized as developmental sculptors of neural circuits. In this issue of Neuron, VanRyzin etal. (2019) demonstrate a novel mechanism by which endocannabinoids drive microglia to phagocytose newborn astrocytes in the medial amygdala of male rats, promoting sex differences in social play behavior.

The in vivo specificity of synaptic G\u03b2 and G\u03b3 subunits to the \u03b12a adrenergic receptor at CNS synapses

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, β, and γ subunits, with 16 Gα, 5 Gβ and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gβγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gβγ specificity of presynaptic α2a-adrenergic receptors (α2aARs) in both adrenergic (auto-α2aARs) and non-adrenergic neurons (hetero-α2aARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits, and co-immunoprecipitation of tagged α2aARs from mouse models including transgenic FLAG-α2aARs and knock-in HA-α2aARs, we investigated the in vivo specificity of Gβ and Gγ subunits to auto-α2aARs and hetero-α2aARs activated with epinephrine to understand the role of Gβγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gβ2, Gγ2, Gγ3, and Gγ4 with activated auto α2aARs, whereas we found Gβ4 and Gγ12 preferentially interacted with activated hetero-α2aARs. Further understanding of in vivo Gβγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gβ and Gγ, and the mechanisms underlying GPCR signaling through Gβγ subunits.

Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

Control of synapse number and function in the developing central nervous system is critical to the formation of neural circuits. Astrocytes play a key role in this process by releasing factors that promote the formation of excitatory synapses. Astrocyte‐secreted thrombospondins (TSPs) induce the formation of structural synapses, which however remain post‐synaptically silent, suggesting that completion of early synaptogenesis may require a two‐step mechanism. Here, we show that the humoral innate immune molecule Pentraxin 3 (PTX3) is expressed in the developing rodent brain. PTX3 plays a key role in promoting functionally‐active CNS synapses, by increasing the surface levels and synaptic clustering of AMPA glutamate receptors. This process involves tumor necrosis factor‐induced protein 6 (TSG6), remodeling of the perineuronal network, and a β1‐integrin/ERK pathway. Furthermore, PTX3 activity is regulated by TSP1, which directly interacts with the N‐terminal region of PTX3. These data unveil a fundamental role of PTX3 in promoting the first wave of synaptogenesis, and show that interplay of TSP1 and PTX3 sets the proper balance between synaptic growth and synapse function in the developing brain.

Near infrared light decreases synaptic vulnerability to amyloid beta oligomers

Synaptic dysfunction due to the disrupting binding of amyloid beta (Aβ) and tau oligomers is one of the earliest impairments in Alzheimer’s Disease (AD), driving initial cognitive deficits and clinical manifestation. Consequently, there is ample consensus that preventing early synaptic dysfunction would be an effective therapeutic strategy for AD. With this goal in mind, we investigated the effect of a treatment of mice with near infrared (NIR) light on synaptic vulnerability to Aβ oligomers. We found that Aβ oligomer binding to CNS synaptosomes isolated from wild type (wt) mice treated with NIR light was significantly reduced and the resulting suppression of long term potentiation (LTP) by Aβ oligomers was prevented. Similarly, APP transgenic mice treated with NIR showed a significant reduction of endogenous Aβ at CNS synapses. We further found that these phenomena were accompanied by increased synaptic mitochondrial membrane potential in both wt and Tg2576 mice. This study provides evidence that NIR light can effectively reduce synaptic vulnerability to damaging Aβ oligomers, thus furthering NIR light therapy as a viable treatment for AD.

CNS synapses are stabilized trans-synaptically by laminins and laminin-interacting proteins.

The retina expresses several laminins in the outer plexiform layer (OPL), where they may provide an extracellular scaffold for synapse stabilization. Mice with a targeted deletion of the laminin β2 gene (Lamb2) exhibit retinal disruptions: photoreceptor synapses in the OPL are disorganized and the retinal physiological response is attenuated. We hypothesize that laminins are required for proper trans-synaptic alignment. To test this, we compared the distribution, expression, association and modification of several pre- and post-synaptic elements in wild-type and Lamb2-null retinae. A potential laminin receptor, integrin α3, is at the presynaptic side of the wild-type OPL. Another potential laminin receptor, dystroglycan, is at the post-synaptic side of the wild-type OPL. Integrin α3 and dystroglycan can be co-immunoprecipitated with the laminin β2 chain, demonstrating that they may bind laminins. In the absence of the laminin β2 chain, the expression of many pre-synaptic components (bassoon, kinesin, among others) is relatively undisturbed although their spatial organization and anchoring to the membrane is disrupted. In contrast, in the Lamb2-null, β-dystroglycan (β-DG) expression is altered, co-localization of β-DG with dystrophin and the glutamate receptor mGluR6 is disrupted, and the post-synaptic bipolar cell components mGluR6 and GPR179 become dissociated, suggesting that laminins mediate scaffolding of post-synaptic components. In addition, although pikachurin remains associated with β-DG, pikachurin is no longer closely associated with mGluR6 or α-DG in the Lamb2-null. These data suggest that laminins act as links among pre- and post-synaptic laminin receptors and α-DG and pikachurin in the synaptic space to maintain proper trans-synaptic alignment.

Not just amyloid: physiological functions of the amyloid precursor protein family.

Amyloid precursor protein (APP) gives rise to the amyloid-β peptide and thus has a key role in the pathogenesis of Alzheimer disease. By contrast, the physiological functions of APP and the closely related APP-like proteins (APLPs) remain less well understood. Studying these physiological functions has been challenging and has required a careful long-term strategy, including the analysis of different App-knockout and Aplp-knockout mice. In this Review, we summarize these findings, focusing on the in vivo roles of APP family members and their processing products for CNS development, synapse formation and function, brain injury and neuroprotection, as well as ageing. In addition, we discuss the implications of APP physiology for therapeutic approaches.

Membrane-associated guanylate kinase dynamics reveal regional and developmental specificity of synapse stability.

The membrane-associated guanylate kinase (MAGUK) family of synaptic scaffolding proteins anchor glutamate receptors at CNS synapses. MAGUK removal via RNAi-mediated knockdown in the CA1 hippocampal region in immature animals causes rapid and lasting reductions in glutamatergic transmission. In mature animals, the same manipulation has little acute effect. The hippocampal dentate gyrus, a region with ongoing adult neurogenesis, is sensitive to MAGUK loss in mature animals, behaving like an immature CA1. Over long time courses, removal of MAGUKs in CA1 causes reductions in glutamatergic transmission, indicating that synapses in mature animals require MAGUKs for anchoring glutamate receptors, but are much more stable. These results demonstrate regional and developmental control of synapse stability and suggest the existence of a sensitive period of heightened hippocampal plasticity in CA1 of pre-adolescent rodents, and in dentate gyrus throughout maturity. Fast excitatory transmission in the brain requires localization of glutamate receptors to synapses. The membrane-associated guanylate kinase (MAGUK) family of synaptic scaffolding proteins is critical for localization of glutamate receptors to synapses. Although the MAGUKs are well-studied in reduced preparations and young animals, few data exist on their role in adult animals. Here, we present a detailed developmental study of the role of the MAGUKs during rat development. We first confirmed by knockdown experiments that MAGUKs are essential for glutamatergic transmission in young animals and cultured slices, and an increase in postsynaptic density protein 95 (PSD-95) by overexpression caused correlated increases in glutamatergic transmission. We found that CA1 synapses in adults, in contrast, were largely unaffected by overexpression of MAGUKs, and although adult CA1 synapses required MAGUKs to the same degree as synapses in young animals, this was only apparent over long time scales of knockdown. We additionally showed that overexpression of MAGUKs is likely to function to accelerate the developmental strengthening of excitatory transmission. Finally, we showed that adult dentate gyrus appears similar to immature CA1, demonstrating regional and developmental control of MAGUK dynamics. Together, these results demonstrate a period of juvenile instability at CA1 synapses, followed by a period of adult stability in which synapses are acutely unaffected by changes in MAGUK abundance.

Survey of numerical electrostimulation models

This paper evaluates results of a survey of electrostimulation models of myelinated nerve. Participants were asked to determine thresholds of excitation for 18 cases involving different characteristics of the neuron, the stimulation waveform, and the electrode arrangement. Responses were received from 7 investigators using 10 models. Excitation thresholds differed significantly among these models. For example, with a 2 ms monophasic stimulus pulse and an electrode/fiber distance of 1 cm, thresholds from the least to greatest value differed by a factor of 8.3; with a 5 μs pulse, thresholds differed by the factor 3.8. Significant differences in reported simulations point to the need for experimental validation. Additional efforts are needed to develop computational models for unmyelinated C-fibers, A-delta fibers, CNS neurons, and CNS Synapses.

A Synaptotagmin Isoform Switch during the Development of an Identified CNS Synapse

Various Synaptotagmin (Syt) isoform genes are found in mammals, but it is unknown whether Syts can function redundantly in a given nerve terminal, or whether isoforms can be switched during the development of a nerve terminal. Here, we investigated the possibility of a developmental Syt isoform switch using the calyx of Held as a model synapse. At mature calyx synapses, fast Ca(2+)-driven transmitter release depended entirely on Syt2, but the release phenotype of Syt2 knockout (KO) mice was weaker at immature calyces, and absent at pre-calyceal synapses early postnatally. Instead, conditional genetic inactivation shows that Syt1 mediates fast release at pre-calyceal synapses, as well as a fast release component resistant to Syt2 deletion in immature calyces. This demonstrates a developmental Syt1-Syt2 isoform switch at an identified synapse, a mechanism that could fine-tune the speed, reliability, and plasticity of transmitter release at fast releasing CNS synapses.

An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse.

The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses.

Mechanisms of facilitation and depression in CNS synapses: Presynaptic and postsynaptic components

Here we review the mechanisms of evoked synchronous, asynchronous, and spontaneous release of mediators. Special attention is paid to the relationship between the presynaptic level of calcium and the release probability of neuromediators as a key link whose modulation results in short-term synaptic alterations that lead to the development of facilitation and depression in different CNS synapses. We also review postsynaptic mechanisms that may be responsible for short-term facilitation or depression in synapses.

Dynamic control of 26S proteasome function at CNS synapses (478.3)

The selective strengthening and weakening of synapses in response to past experience and activity is known as synaptic plasticity and is fundamental to all brain processes such as the ability to learn, form memories and modify behavior. In contrast, impairments in synaptic plasticity are considered to contribute to neuropsychiatric disorders and neurodegenerative diseases. Synaptic plasticity is largely mediated by both morphological and functional modifications to synapses. Local protein synthesis and protein degradation are known to contribute to these modifications. It is now clear that regulated proteolysis via the ubiquitin proteasome system (UPS) plays a major role in the growth and development, maintenance and remodeling of synaptic connections in the brain. We and others have previously found a novel form of regulation for the 26S proteasome in neurons involving the plasticity kinase Ca2+/calmodulin‐dependent protein kinases II alpha (CaMKIIα). CaMKIIα phosphorylates the ATPase 19S regulatory particle (RP) subunit, Rpt6, at serine 120 (S120) in an activity‐dependent fashion to modulate the activity and control the distribution of proteasomes in neurons (Djakovic et al., 2009; 2012; Bingol et al., 2010). We find that Rpt6 phosphorylation and proteasome function regulates synaptic strength and activity‐dependent generation of dendritic spines (Djakovic et al., 2012; Hamilton et al., 2012). I will discuss our more recent work and the functional relevance of proteasome phosphorylation in neurons using newly generated Rpt6 Ser120Ala and Ser120Asp (phospho‐dead and phospho‐mimetic, respectively) genetically modified mice.

Synaptic selection by dopamine neurotransmission (468.3)

Electrophysiological and electrochemical recordings provide time resolution sufficient to measure quantal neurotransmission at CNS synapses, but do not resolve activity of individual synapses within populations. Modulation at the level of individual synapses, however, may be required in habit learning and decision making.We will discuss means to resolve the modulation of subgroups of individual synapses using fluorescent false neurotransmitters (FFNs) that act as synaptic vesicle transporter substrates as well as endocytic probes. These optical approaches indicate not only presynaptic release probability and reuptake kinetics but also new forms of presynaptic plasticity, including the rapid movement of presynaptic release sites, and transfer between silent and active states. We will show how such selection of corticostriatal synapses by dopamine and cannabinoid might enable normal motor function.

Cav2.1 Channels Control Multivesicular Release by Relying on Their Distance from Exocytotic Ca2+Sensors at Rat Cerebellar Granule Cells

The concomitant release of multiple numbers of synaptic vesicles [multivesicular release (MVR)] in response to a single presynaptic action potential enhances the flexibility of synaptic transmission. However, the molecular mechanisms underlying MVR at a single CNS synapse remain unclear. Here, we show that the Cav2.1 subtype (P/Q-type) of the voltage-gated calcium channel is specifically responsible for the induction of MVR. In the rat cerebellar cortex, paired-pulse activation of granule cell (GC) ascending fibers leads not only to a facilitation of the peak amplitude (PPFamp) but also to a prolongation of the decay time (PPPdecay) of the EPSCs recorded from molecular layer interneurons. PPFamp is elicited by a transient increase in the number of released vesicles. PPPdecay is highly dependent on MVR and is caused by dual mechanisms: (1) a delayed release and (2) an extrasynaptic spillover of the GC transmitter glutamate and subsequent pooling of the glutamate among active synapses. PPPdecay was specifically suppressed by the Cav2.1 channel blocker ω-agatoxin IVA, while PPFamp responded to Cav2.2/Cav2.3 (N-type/R-type) channel blockers. The membrane-permeable slow Ca(2+) chelator EGTA-AM profoundly reduced the decay time constant (τdecay) of the second EPSC; however, it only had a negligible impact on that of the first, thereby eliminating PPPdecay. These results suggest that the distance between presynaptic Cav2.1 channels and exocytotic Ca(2+) sensors is a key determinant of MVR. By transducing presynaptic action potential firings into unique Ca(2+) signals and vesicle release profiles, Cav2.1 channels contribute to the encoding and processing of neural information.

Bassoon-disruption slows vesicle replenishment and induces homeostatic plasticity at a CNS synapse

Endbulb of Held terminals of auditory nerve fibers (ANF) transmit auditory information at hundreds per second to bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). Here, we studied the structure and function of endbulb synapses in mice that lack the presynaptic scaffold bassoon and exhibit reduced ANF input into the AVCN. Endbulb terminals and active zones were normal in number and vesicle complement. Postsynaptic densities, quantal size and vesicular release probability were increased while vesicle replenishment and the standing pool of readily releasable vesicles were reduced. These opposing effects canceled each other out for the first evoked EPSC, which showed unaltered amplitude. We propose that ANF activity deprivation drives homeostatic plasticity in the AVCN involving synaptic upscaling and increased intrinsic BC excitability. In vivo recordings from individual mutant BCs demonstrated a slightly improved response at sound onset compared to ANF, likely reflecting the combined effects of ANF convergence and homeostatic plasticity. Further, we conclude that bassoon promotes vesicular replenishment and, consequently, a large standing pool of readily releasable synaptic vesicles at the endbulb synapse.