Electron-translucency and partial defects of synaptic basal lamina in the electrocyte synapse of an electric ray (Narke japonica) in 3D embedment-free section electron microscopy.

The synaptic basal lamina of the electrocytes was disclosed to be electron-translucent to some extent when viewed in an en-face direction in embedment-free section transmission electron microscopy (EFS-TEM), and synaptic vesicles located close to the presynaptic membrane were seen through the synaptic basal lamina together with the presynaptic and postsynaptic membranes. This feature of translucency has the potential to analyze possible spatial interrelations in situ between bioactive molecules in the synaptic basal lamina and the synaptic vesicles in further studies. The synaptic basal lamina, appearing as an electron-dense line sandwiched by two parallel lines representing the presynaptic and postsynaptic membranes in ultrathin sections cut right to the synaptic junctional plane in conventional TEM, was not fully continuous but randomly intermittent along its trajectory. Compatible with the intermittent line appearance, the en-face 3D view in embedment-free section TEM revealed for the first time partial irregular defects of the synaptic basal lamina. Considering the known functional significance of several molecules contained in the synaptic basal lamina in the maintenance and exertion of the synapse, its partial defects may not represent its rigid structural features, but its immature structure under remodeling or its dynamic changes in consistency such as the sol/gel transition, whose validity needs further examination. RESEARCH HIGHLIGHTS: In embedment-free section TEM, a 3D en-face view of synaptic basal lamina in situ is reliably possible. The basal lamina en-face is electron-translucent, which makes it possible to analyze spatial interrelation between pre- and post-synaptic components. Partial irregular defects in the basal lamina are revealed in Torpedo electrocytes, suggesting its remodeling or dynamic changes in consistency.

PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems.

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

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.

Gap Junctions Interconnect Different Subtypes of Parvalbumin-Positive Interneurons in Barrels and Septa with Connectivity Unique to Each Subtype.

Parvalbumin (PV)-positive interneurons form dendritic gap junctions with one another, but the connectivity among gap junction-coupled dendrites remains uninvestigated in most neocortical areas. We visualized gap junctions in layer 4 of the mouse barrel cortex and examined their structural details. PV neurons were divided into 4 types based on the location of soma and dendrites within or outside barrels. Type 1 neurons that had soma and all dendrites inside a barrel, considered most specific to single vibrissa-derived signals, unexpectedly formed gap junctions only with other types but never with each other. Type 2 neurons inside a barrel elongated dendrites outward, forming gap junctions within a column that contained the home barrel. Type 3 neurons located outside barrels established connections with all types including Type 4 neurons that were confined inside the inter-barrel septa. The majority (33/38, 86.8%) of dendritic gap junctions were within 75 µm from at least 1 of 2 paired somata. All types received vesicular glutamate transporter 2-positive axon terminals preferentially on somata and proximal dendrites, indicating the involvement of all types in thalamocortical feedforward regulation in which proximal gap junctions may also participate. These structural organizations provide a new morphological basis for regulatory mechanisms in barrel cortex.

Stretchable and conformable synapse memristors for wearable and implantable electronics.

Stretchable and conformable synapse memristors that can emulate the behaviour of the biological neural system and well adhere onto the curved surfaces simultaneously are desirable for the development of imperceptible wearable and implantable neuromorphic computing systems. Previous synapse memristors have been mainly limited to rigid substrates. Herein, a stretchable and conformable memristor with fundamental synaptic functions including potentiation/depression characteristics, long/short-term plasticity (STP and LTP), "learning-forgetting-relearning" behaviour, and spike-rate-dependent and spike-amplitude-dependent plasticity is demonstrated based on highly elastic Ag nanoparticle-doped thermoplastic polyurethanes (TPU : Ag NPs) and polydimethylsiloxane (PDMS). The memristor can be well operated even at 60% strain and can be well conformed onto the curved surfaces. The formed conductive filament (CF) obtained from the movement of Ag nanoparticle clusters under the locally enhanced electric field gives rise to resistance switching of our memristor. These results indicate a feasible strategy to realize stretchable and conformable synaptic devices for the development of new-generation artificial intelligence computers.

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity.

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor-encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an "ur-" bHLH gene.

VPS35 depletion does not impair presynaptic structure and function.

The endosomal system is proposed as a mediator of synaptic vesicle recycling, but the molecular recycling mechanism remains largely unknown. Retromer is a key protein complex which mediates endosomal recycling in eukaryotic cells, including neurons. Retromer is important for brain function and mutations in retromer genes are linked to neurodegenerative diseases. In this study, we aimed to determine the role of retromer in presynaptic structure and function. We assessed the role of retromer by knocking down VPS35, the core subunit of retromer, in primary hippocampal mouse neurons. VPS35 depletion led to retromer dysfunction, measured as a decrease in GluA1 at the plasma membrane, and bypassed morphological defects previously described in chronic retromer depletion models. We found that retromer is localized at the mammalian presynaptic terminal. However, VPS35 depletion did not alter the presynaptic ultrastructure, synaptic vesicle release or retrieval. Hence, we conclude that retromer is present in the presynaptic terminal but it is not essential for the synaptic vesicle cycle. Nonetheless, the presynaptic localization of VPS35 suggests that retromer-dependent endosome sorting could take place for other presynaptic cargo.

An easy, fast and "low-tech"-equipment-requiring alternative method to optimize immunolabelling conditions for pre-embedding immunogold electron microscopy and to correlate light and electron microscopical immunogold labelling results.

Correlating light microscopic immunolabelling results with electron microscopic data is of great interest in many fields of biomedical research but typically requires very specialized, expensive equipment and complex procedures which are not available in most labs. In this technical study, we describe an easy and "low-tech"-equipment-requiring pre-embedding immunolabelling approach that allows correlation of light microscopical immunolabelling results with electron microscopic (EM) data as demonstrated by the example of immunolabelled synaptic ribbons from retinal rod photoreceptor synapses. This pre-embedding approach does not require specialized embedding devices but only commonly available equipment. The cryostat section-based procedure allows optimization of the pre-embedding immunolabelling conditions at the less laborious and time-consuming light microscopic (LM) level before the ultrastructural analyses of the immunolabelled structures can be performed on the same sample after ultrathin sectioning without further modification. The same photoreceptor synapse that has been first studied at the light microscopic level can be subsequently analyzed with this approach at the electron microscopic level at individual ultrathin sections or serial ultrathin sections from individual, identical synapses. Higher resolution EM analyses of the immunolabelled synapses can be performed with only minor modifications of the combined LM/EM procedure. The detergent-free procedure is applicable even for weakly fixed cryostat sections which is a relevant aspect for many antibodies that do not work with more strongly fixed biological samples.

Conflicting effects by antibodies against connexin36 during the action of intracellular Cyclic-AMP onto electrical synapses of retinal ganglion cells.

Alpha-type retinal ganglion cells (alpha cells) of the same class in mammalian retina are connected by gap junctions. Electrical synapses between alpha cells were examined using combined techniques of dual patch-clamp recordings, intracellular labeling and electron microscopy in the albino rat retina. In simultaneous dual whole-cell recordings from pairs of neighboring alpha cells, bidirectional electrical synapses with symmetrical junction conductance were observed in pairs with cells of the same morphological type. Regulatory domains of gap junction protein subunit connexins in electrical synapses between alpha cells by extracellular and intracellular ligands investigated by dual whole-patch clamp recordings. I examined how passage currents through electrical synapses between alpha cells are modulated by specific antibodies against connexin36 proteins, and extracellular or intracellular application of ligands. Control conditions led us to observe large passage currents between connected cells and adequate transjunctional conductance (Gj) (1.35[Formula: see text][Formula: see text][Formula: see text]0.51[Formula: see text]nS). Experimental results show that high level of intracellular cyclic AMP within examined cells suppress electrical synapses between the neighboring cells. Gj between examined cells reduced to 0.15[Formula: see text][Formula: see text][Formula: see text]0.04[Formula: see text]nS. Under application of dopamine (1.25[Formula: see text][Formula: see text][Formula: see text]0.06[Formula: see text]nS) or intracellular cyclic GMP (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS), however, Gj also remains as in the control level. Intracellular application of an antibody against the cytoplasmic loop of connexin36 reduced Gj (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS). Cocktail of the antibody against cytoplasmic connexin36 and intracellular cyclic AMP leaves Gj as in the level by single involvement of the cytoplasmic antibody. The elimination of Gj by the cytoplasmic antibody was in a dose-dependent manner. These results suggest that binding domains against cyclic AMP may be present in the cytoplasmic sites of connexin proteins to regulate channel opening of gap junctions between mammalian retinal alpha ganglion cells.

Connexin36 expression in major centers of the auditory system in the CNS of mouse and rat: Evidence for neurons forming purely electrical synapses and morphologically mixed synapses.

Electrical synapses formed by gap junctions composed of connexin36 (Cx36) are widely distributed in the mammalian central nervous system (CNS). Here, we used immunofluorescence methods to document the expression of Cx36 in the cochlear nucleus and in various structures of the auditory pathway of rat and mouse. Labeling of Cx36 visualized exclusively as Cx36-puncta was densely distributed primarily on the somata and initial dendrites of neuronal populations in the ventral cochlear nucleus, and was abundant in superficial layers of the dorsal cochlear nucleus. Other auditory centers displaying Cx36-puncta included the medial nucleus of the trapezoid body (MNTB), regions surrounding the lateral superior olivary nucleus, the dorsal nucleus of the medial lemniscus, the nucleus sagulum, all subnuclei of the inferior colliculus, and the auditory cerebral cortex. In EGFP-Cx36 transgenic mice, EGFP reporter was detected in neurons located in each of auditory centers that harbored Cx36-puncta. In the ventral cochlear nuclei and the MNTB, many neuronal somata were heavily innervated by nerve terminals containing vesicular glutamate transporter-1 (vglut1) and Cx36 was frequently localized at these terminals. Cochlear ablation caused a near total depletion of vglut1-positive terminals in the ventral cochlear nuclei, with a commensurate loss of labeling for Cx36 around most neuronal somata, but preserved Cx36-puncta at somatic neuronal appositions. The results suggest that electrical synapses formed by Cx36-containing gap junctions occur in most of the widely distributed centers of the auditory system. Further, it appears that morphologically mixed chemical/electrical synapses formed by nerve terminals are abundant in the ventral cochlear nucleus, including those at endbulbs of Held formed by cochlear primary afferent fibers, and those at calyx of Held synapses on MNTB neurons.

Type 2 wide-field amacrine cells in TH::GFP mice show a homogenous synapse distribution and contact small ganglion cells.

In vertebrate retinas, wide-field amacrine cells represent a diverse class of interneurons, important for the extraction of selective features, like motion or objects, from the visual scene. Most types of wide-field amacrine cells lack dedicated output processes, whereas some types spatially segregate outputs from inputs. In the tyrosine hydroxylase (TH)::green fluorescent protein (GFP) mouse line, two types of GFP-expressing wide-field amacrine cells have been described: dopaminergic type 1 and γ-aminobutyric acid-ergic type 2 cells (TH2). TH2 cells possess short and long radial processes stratifying in the middle of the inner plexiform layer, where they collect excitatory and inhibitory inputs from bipolar cells and other amacrine cells, respectively. Although it was shown that these inputs lead to ON-OFF light responses, their spatial distribution along TH2 cell processes is unknown. Also, the postsynaptic targets of TH2 cells have not been identified so far. Here, we analysed the synapse distribution of these cells in TH::GFP mice and show that they form a weakly coupled network. Electrical synapses (made of connexin36) and chemical (excitatory and inhibitory) synapses are uniformly distributed along TH2 dendrites, independent of dendrite length or distance from soma. Moreover, we reveal that TH2 cells contact at least two types of small ganglion cells; one of them is the W3 cell, a ganglion cell sensitive to object motion. Contacts were often associated with markers of inhibitory synapses. Thus, TH2 wide-field amacrine cells likely provide postsynaptic inhibition to W3 ganglion cells and may contribute to object-motion detection in the mouse retina.

The transient receptor potential vanilloid-1 is localized at excitatory synapses in the mouse dentate gyrus.

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.

Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission.

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

Developmental and visual input-dependent regulation of the CB1 cannabinoid receptor in the mouse visual cortex.

The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience.

Tunneling nanotubes, an emerging intercellular communication route in development.

The development of multi-cellular organisms involves a comprehensive and tightly regulated cell-to-cell communication system to coordinate the activity and behavior of individual cells. Diverse signaling pathways ranging from receptor-mediated signal transduction to contact-dependent communication via gap junctions achieve these complex interactions. In this review, we will focus on a new type of intercellular connection, the tunneling nanotube (TNT), which has been observed in many cell types in vitro and recently also in developing embryos of different species in vivo. We will summarize the latest insights into their functional roles in cell-to-cell signaling with a particular focus on the TNT-dependent electrical coupling between developing embryonic cells. Finally, potential implications of these new findings in the light of developmental processes, particularly in cell migration, will be discussed.

[Analysis of spatial arrangement of gap junctions in respect to chemical synapses in the serial ultrathin sections of rat barrel cortex].

Electron microscopic investigation of gap junctions (GJ) on serial sections of rat barrel cortex has shown that GJ were in contact with one or both processes that formed chemical synapses, however, these connections could not traced in single sections. In the serial sections, it was possible to observe two GJ in the immediate proximity to one another, in a single field of vision, thus, each GJ was traced in two or three successive sections in a series. Considering the described variants of GJ arrangement in the cortex, it is suggested that GJ could be a structural basis for local synchronization of the bioelectrical activity not only at postsynaptic, but also at presynaptic level, and the formation of GJ occurs both before, and after the development of chemical synapses.

Label-free imaging of membrane potential using membrane electromotility.

Electrical activity may cause observable changes in a cell's structure in the absence of exogenous reporter molecules. In this work, we report a low-coherence interferometric microscopy technique that can detect an optical signal correlated with the membrane potential changes in individual mammalian cells without exogenous labels. By measuring milliradian-scale phase shifts in the transmitted light, we can detect changes in the cells' membrane potential. We find that the observed optical signals are due to membrane electromotility, which causes the cells to deform in response to the membrane potential changes. We demonstrate wide-field imaging of the propagation of electrical stimuli in gap-junction-coupled cell networks. Membrane electromotility-induced cell deformation may be useful as a reporter of electrical activity.

The connectome of a decision-making neural network.

In order to understand the nervous system, it is necessary to know the synaptic connections between the neurons, yet to date, only the wiring diagram of the adult hermaphrodite of the nematode Caenorhabditis elegans has been determined. Here, we present the wiring diagram of the posterior nervous system of the C. elegans adult male, reconstructed from serial electron micrograph sections. This region of the male nervous system contains the sexually dimorphic circuits for mating. The synaptic connections, both chemical and gap junctional, form a neural network with four striking features: multiple, parallel, short synaptic pathways directly connecting sensory neurons to end organs; recurrent and reciprocal connectivity among sensory neurons; modular substructure; and interneurons acting in feedforward loops. These features help to explain how the network robustly and rapidly selects and executes the steps of a behavioral program on the basis of the inputs from multiple sensory neurons.

Gap junctions compensate for sublinear dendritic integration in an inhibitory network.

Electrically coupled inhibitory interneurons dynamically control network excitability, yet little is known about how chemical and electrical synapses regulate their activity. Using two-photon glutamate uncaging and dendritic patch-clamp recordings, we found that the dendrites of cerebellar Golgi interneurons acted as passive cables. They conferred distance-dependent sublinear synaptic integration and weakened distal excitatory inputs. Gap junctions were present at a higher density on distal dendrites and contributed substantially to membrane conductance. Depolarization of one Golgi cell increased firing in its neighbors, and inclusion of dendritic gap junctions in interneuron network models enabled distal excitatory synapses to drive network activity more effectively. Our results suggest that dendritic gap junctions counteract sublinear dendritic integration by enabling excitatory synaptic charge to spread into the dendrites of neighboring inhibitory interneurons.