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The Hong Kong Zhuhai Macao Bridge (HZMB) link is currently one of the largest fixed links ever realised. The link comprises various bridges, causeways, artificial islands and tunnels and has a total length of more than 55 km with an investment of about RMB 120billion (approx. €15 billion). The Link accommodates a dual carriageway with three traffic lanes in each direction. The construction of the Link started at the end of 2010 and was opened to traffic in October 2018. The HZMB Link is internationally regarded as one of the most challenging sea crossing projects in China and a demonstration of China's comprehensive breakthrough in technology, scientific research and other related fields. The offshore part of the HZMB Link, crossing the Pearl River Estuary involves one of the world's longest and deepest immersed tunnels. The tunnel was designed and constructed to meet state-of-the-art requirements and challenging structural, geotechnical and marine/offshore conditions. The HZMB tunnel won the ITA Tunnelling Award Major Project of the Year 2018. This paper presents the project from the Client perspective. The paper will give a general introduction of the project, will provide information regarding the challenging environment in which the tunnel had to be realized and the high demanding design criteria that were applicable. Furthermore, the evaluation of the various technical options will be discussed, ultimately leading to the selection of the immersed tunnel option. The paper will also look into the background of selecting the Design & Build contract approach for this project and the experiences that were built up using this type of contract, new to China on this scale. Finally, the paper introduces how operation and maintenance of the tunnel is planned and executed so as to guarantee the 120-year design lifetime. In Ref. (The Islands and Tunnel Project of HZM Link et al., 2022) more details of the project are discussed from the D&B contractors' perspective. [ABSTRACT FROM AUTHOR]

A major stumbling block to understanding neural circuits is the extreme anatomical and functional diversity of interneurons. Subsets of interneurons can be targeted for manipulation using Cre mouse lines, but Cre expression is rarely confined to a single interneuron type. It is essential to have a strategy that further restricts labeling in Cre driver lines. We now describe an approach that combines Cre driver mice, recombinant adeno-associated virus, and rabies virus to produce sparse but binary labeling of select interneurons--frequently only a single cell in a large region. We used this approach to characterize the retinal amacrine and ganglion cell types in five GABAergic Cre mouse (Mus musculus) lines, and identified two new amacrine cell types: an asymmetric medium-field type and a wide-field type. We also labeled several wide-field amacrine cell types that have been previously identified based on morphology but whose connectivity and function had not been systematically studied due to lack of genetic markers. All Cre-expressing amacrine cells labeled with an antibody to GABA. Cre-expressing RGCs lacked GABA labeling and included classically defined as well as recently identified types. In addition to the retina, our technique leads to sparse labeling of neurons in the cortex, lateral geniculate nucleus, and superior colliculus, and can be used to express optogenetic tools such as channelrhodopsin and protein sensors such as GCaMP. The Cre drivers identified in this study provide genetic access to otherwise hard to access cell types for systematic analysis including anatomical characterization, physiological recording, optogenetic and/or chemical manipulation, and circuit mapping. [ABSTRACT FROM AUTHOR]

The mechanisms that contribute to the extinction of previously acquired memories are not well understood. These processes, often referred to as inhibitory learning, are thought to be parallel learning mechanisms that require a reacquisition of new information and suppression of previously acquired experiences in order to adapt to novel situations. Using newly generated metabotropic glutamate receptor 5 (mGluR5) knock-out mice,weinvestigated the role ofmGluR5in the acquisition and reversal of an associative conditioned task and a spatial reference task.Wefound that acquisition of fear conditioning is partially impaired in mice lacking mGluR5. More markedly, we found that extinction of both contextual and auditory fear was completely abolished in mGluR5 knock-out mice. In the Morris Water Maze test (MWM), mGluR5 knock-out mice exhibited mild deficits in the rate of acquisition of the regular water maze task, but again had significant deficits in the reversal task, despite overall spatial memory being intact. Together, these results demonstrate that mGluR5 is critical to the function of neural circuits that are required for inhibitory learning mechanisms, and suggest that targeting metabotropic receptors may be useful in treating psychiatric disorders in which aversive memories are inappropriately retained. [ABSTRACT FROM AUTHOR]

Because synaptic vesicle exocytosis is a nano-mechanical process. it should be influenced by the mechanical properties of the cell membrane to which the vesicle fuses. By dissolving surfactants at various concentrations in the neuronal membrane, we have perturbed mechanical properties of the membrane and have found that dissolved surfactants lower the probability that a synaptic vesicle will open its fusion pore when the fusion machinery of the vesicle is activated by binding calcium. By using standard theories from the physics and chemistry of surfaces, we can account for this decrease in fusion probability and can infer that a vesicle, when activated, opens its fusion pore ∼3 times out of 4 and that the area of the fusion pore is ∼4 nm². [ABSTRACT FROM AUTHOR]

Genetically encoded sensors of glutamate concentration are based on FRET between cyan and yellow fluorescent proteins bracketing a bacterial glutamate-binding protein. Such sensors have yet to find quantitative applications in neurons, because of poor response amplitude in physiological buffers or when expressed on the neuronal cell surface. We have improved our glutamate-sensing fluorescent reporter (GIuSnFR) by systematic optimization of linker sequences and glutamate affinities. Using SuperGluSnFR. which exhibits a 6.2-fold increase in response magnitude over the original GIuSnFR. we demonstrate quantitative optical measurements of the time course of synaptic glutamate release, spillover, and reuptake in cultured hippocampal neurons with centisecond temporal and spine-sized spatial resolution. During burst firing, functionally significant spillover persists for hundreds of milliseconds. These glutamate levels appear sufficient to prime NMDA receptors, potentially affecting dendritic spike initiation and computation. Stimulation frequency-dependent modulation of spillover suggests a mechanism for nonsynaptic neuronal communication. [ABSTRACT FROM AUTHOR]

Investigates the potassium (K)-binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels through the use of single-channel kinetics of extracellular magnesium (Mg) block. Effects of intracellular K on the apparent rates of Mg dissociation and permeation; Equilibrium dissociation constant and rate constants for Mg dissociation and permeation; Qualitative assessment of the affinity and selectivity of the internal site.

Investigates the effect of extracellular and intracellular sodium (Na) on the single-channel kinetics of magnesium (Mg) block in recombinant NR1-NR2B NMDA receptor channels. Association rate constant of Mg; Locations and apparent affinities of the Na-binding sites; Dissociation rate constant of Mg.

Neuronal nicotinic acetylcholine receptors (nAChRs) belong to a family of ligand-gated ion channels that play important roles in central and peripheral nervous systems. The subcellular distribution of neuronal nAChRs has important implications for function and is not well understood. Here, we analyzed the targeting of two major types of neuronal nAChRs by expressing epitope-tagged subunits in cultured hippocampal neurons. Surprisingly, the α7 nAChR (α7) and α4/β2 nAChR (α4β2) displayed distinct patterns of expression, with α7 targeted preferentially to the somatodendritic compartments, whereas α4β2 was localized to both axonal and dendritic domains. When fused to CD4 or IL2RA (interleukin 2 receptor α subunit) proteins, which are normally distributed ubiquitously, the M3-M4 intracellular loop from the α7 subunit promoted dendritic expression, whereas the homologous M3-M4 loop from the α4 subunit led to surface axonal expression. Systemic screening and alanine substitution further identified a 25-residue leucine motif ([DE]XXXL[LI]) containing an axonal targeting sequence within the α4 loop and a 48-residue dileucine and tyrosine motif (YXXØ) containing a dendritic targeting sequence from the α7 loop. These results provide valuable information in understanding diverse roles of neuronal nAChRs in mediating and modulating synaptic transmission, synaptic plasticity, and nicotine addiction. [ABSTRACT FROM AUTHOR]

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-bindlng residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/ KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafticking of intracellular kainate receptors following multimeric assembly. [ABSTRACT FROM AUTHOR]

We developed two-photon scanning patterned illumination microscopy (2P-SPIM) for super-resolution two-photon imaging. Our approach used a traditional two-photon microscopy setup with temporally modulated excitation to create patterned illumination fields. Combing nine different illuminations and structured illumination reconstruction, super-resolution imaging was achieved in two-photon microscopy. Using 2P-SPIM we achieved a lateral resolution of 141 nm, which represents an improvement by a factor of 1.9 over the corresponding diffraction limit. We further demonstrated super-resolution cellular imaging by 2P-SPIM to image actin cytoskeleton in mammalian cells and three-dimensional imaging in highly scattering retinal tissue. [ABSTRACT FROM AUTHOR]

Metaplasticity regulates the threshold for modification of synaptic strength and is an important regulator of learning rules; however, it is not known whether these cellular mechanisms for homeostatic regulation of synapses contribute to particular forms of learning. Conditional ablation of mGluR5 in CAI pyramidal neurons resulted in the inability of low-frequency trains of afferent activation to prime synapses for subsequent theta burst potentiation. Priming-induced metaplasticity requires mGluR5-mediated mobilization of endocannabinoids during the priming train to induce long-term depression of inhibition (I-LTD). Mice lacking priming-induced plasticity had no deficit in spatial reference memory tasks, but were impaired in an associative task with a temporal component. Conversely, enhancing endocannabinoid signaling facilitated temporal associative memory acquisition and, after training animals in these tasks, ex vivo I-LTD was partially occluded and theta burst LTP was enhanced. Together, these results suggest a link between metaplasticity mechanisms in the hippocampus and the formation of temporal associative memories. [ABSTRACT FROM AUTHOR]

Intracellular trafficking of ionotropic glutamate receptors is regulated predominantly by determinants in the cytoplasmic C-terminal domain of the subunit proteins. Although AMPA receptors are found at the vast majority of excitatory synapses, synaptic kainate receptors exhibit a much more restricted distribution, suggesting that specific mechanisms exist for selective trafficking of these receptor proteins. In this report, we define a critical forward trafficking motif that is necessary for surface expression of the glutamate receptor 6 (GluR6) kainate receptor as well as chimeric proteins containing only the GluR6 C-terminal domain. The trafficking determinant was identified by tracking surface expression of green fluorescent protein-tagged GluR6 receptors with confocal immunofluorescence in COS-7 cells and cultured neurons and patch-clamp electrophysiology in human embryonic kidney 293 cells. Serial truncation and alanine site mutagenesis of the GluR6 subunit C terminus localized the critical motif to a seven amino acid stretch of predominantly basic residues. Alanine mutation of the trafficking motif reduced kainate receptor current amplitudes by >90% and resulted in retention of the mutated receptors in the endoplasmic reticulum. This forward trafficking domain is the first such identified for kainate receptors. [ABSTRACT FROM AUTHOR]