Activated somatostatin interneurons orchestrate memory microcircuits.

Despite recent advancements in identifying engram cells, our understanding of their regulatory and functional mechanisms remains in its infancy. To provide mechanistic insight into engram cell functioning, we introduced a novel local microcircuit labeling technique that enables the labeling of intraregional synaptic connections. Utilizing this approach, we discovered a unique population of somatostatin (SOM) interneurons in the mouse basolateral amygdala (BLA). These neurons are activated during fear memory formation and exhibit a preference for forming synapses with excitatory engram neurons. Post-activation, these SOM neurons displayed varying excitability based on fear memory retrieval. Furthermore, when we modulated these SOM neurons chemogenetically, we observed changes in the expression of fear-related behaviors, both in a fear-associated context and in a novel setting. Our findings suggest that these activated SOM interneurons play a pivotal role in modulating engram cell activity. They influence the expression of fear-related behaviors through a mechanism that is dependent on memory cues.

Hippocampal engram networks for fear memory recruit new synapses and modify pre-existing synapses in vivo.

As basic units of neural networks, ensembles of synapses underlie cognitive functions such as learning and memory. These synaptic engrams show elevated synaptic density among engram cells following contextual fear memory formation. Subsequent analysis of the CA3-CA1 engram synapse revealed larger spine sizes, as the synaptic connectivity correlated with the memory strength. Here, we elucidate the synapse dynamics between CA3 and CA1 by tracking identical synapses at multiple time points by adapting two-photon microscopy and dual-eGRASP technique in vivo. After memory formation, synaptic connections between engram populations are enhanced in conjunction with synaptogenesis within the hippocampal network. However, extinction learning specifically correlated with the disappearance of CA3 engram to CA1 engram (E-E) synapses. We observed "newly formed" synapses near pre-existing synapses, which clustered CA3-CA1 engram synapses after fear memory formation. Overall, we conclude that dynamics at CA3 to CA1 E-E synapses are key sites for modification during fear memory states.

Synaptic ensembles between raphe and D1R-containing accumbens shell neurons underlie postisolation sociability in males.

Social animals expend considerable energy to maintain social bonds throughout their life. Male and female mice show sexually dimorphic behaviors, yet the underlying neural mechanisms of sociability and their dysregulation during social disconnection remain unknown. Dopaminergic neurons in dorsal raphe nucleus (DRNTH) is known to contribute to a loneliness-like state and modulate sociability. We identified that activated subpopulations in DRNTH and nucleus accumbens shell (NAcsh) during 24 hours of social isolation underlie the increase in isolation-induced sociability in male but not in female mice. This effect was reversed by chemogenetically and optogenetically inhibiting the DRNTH-NAcsh circuit. Moreover, synaptic connectivity among the activated neuronal ensembles in this circuit was increased, primarily in D1 receptor-expressing neurons in NAcsh. The increase in synaptic density functionally correlated with elevated dopamine release into NAcsh. Overall, specific synaptic ensembles in DRNTH-NAcsh mediate sex differences in isolation-induced sociability, indicating that sex-dependent circuit dynamics underlie the expression of sexually dimorphic behaviors.

Development of ß-Cyclodextrin/Konjac-Based Emulsion Gel for a Pork Backfat Substitute in Emulsion-Type Sausage.

Emulsion gel has been used to replace animal fats in meat products. Konjac is a widely used gelling agent; however, its low emulsion stability limits its use in meat products. This study aimed to examine the quality characteristics of ß-cyclodextrin (CD)-supplemented konjac-based emulsion gel (KEG) (CD-KEG) and its application as a fat substitute in emulsion-type sausages. The supplementation of CD increased hydrogen bonds and hydrophobic interactions with konjac and oil in the gels, respectively. Additionally, CD increased the structural complexity and strength of KEG. Since adding more than 6% of CD to KEG did not increase the gel strength, 6% CD-added KEG was adopted to substitute for pork backfat in manufacturing low-fat emulsion-type sausages. The following formulations of the sausages were prepared: pork backfat 20% (PF20); pork backfat 10% + KEG 10% (KEG10); KEG 20% (KEG20); pork backfat 10% + CD-KEG 10% (CD-KEG10); CD-KEG 20% (CD-KEG20); and pork backfat 5% (PF5). The CD-KEG20 formulation exhibited higher viscosity and viscoelasticity than KEG20, which suggested that CD improves the rheological properties and the thermal stability of meat batter. Additionally, CD-KEG20 showed similar emulsion stability, cooking yield and texture parameters compared with PF20. Therefore, 6% CD-added KEG is a suitable fat substitute for preparing low-fat emulsion-type sausages.

Toxicity Assessment of a Single Dose of Poly(ethylene glycol) Diglycidyl Ether (PEGDE) Administered Subcutaneously in Mice.

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used to cross-link polymers, particularly in the pharmaceutical and biomaterial sectors. However, the subcutaneous toxicity of PEGDE has not yet been assessed. PEGDE samples (500-40,000 µg/mouse) were subcutaneously injected into the paraspinal dorsum of BALB/c male mice. Cage-side observations were carried out with measurement of organ weight, body weight variation, and feed intake, as well as histopathological characterization on day 28 post-exposure. Mice that received 40,000 µg of PEGDE showed severe toxic response and had to be euthanized. Subcutaneous injection of PEGDE did not alter feed intake and organ weight; however, the body weight variation of mice injected with 20,000 µg of PEGDE was significantly lower than that of the other groups. Exposure to 10,000 and 20,000 µg of PEGDE induced epidermal ulcer formation and hair loss. The histology of skin tissue in mice administered with 20,000 µg of PEGDE showed re-epithelialized or unhealed wounds. However, the liver, spleen, and kidneys were histologically normal. Collectively, PEGDE, particularly above 10,000 µg/mouse, caused subcutaneous toxicity with ulceration, but no toxicity in the other organs. These results may indicate the optimal concentration of subcutaneously injected PEGDE.

Synaptic correlates of associative fear memory in the lateral amygdala.

Successful adaptation to the environment requires an accurate response to external threats by recalling specific memories. Memory formation and recall require engram cell activity and synaptic strengthening among activated neuronal ensembles. However, elucidation of the underlying neural substrates of associative fear memory has remained limited without a direct interrogation of extinction-induced changes of specific synapses that encode a specific auditory fear memory. Using dual-eGRASP (enhanced green fluorescent protein reconstitution across synaptic partners), we found that synapses among activated neuronal ensembles or activated synaptic ensembles showed a significantly larger spine morphology at auditory cortex (AC)-to-lateral amygdala (LA) projections after auditory fear conditioning in mice. Fear extinction reversed these enhanced synaptic ensemble spines, whereas re-conditioning with the same tone and shock restored the spine size of the synaptic ensemble. We suggest that synaptic ensembles encode and represent different fear memory states.

Super-Resolution Three-Dimensional Imaging of Actin Filaments in Cultured Cells and the Brain via Expansion Microscopy.

Actin is an essential protein in almost all life forms. It mediates diverse biological functions, ranging from controlling the shape of cells and cell movements to cargo transport and the formation of synaptic connections. Multiple diseases are closely related to the dysfunction of actin or actin-related proteins. Despite the biological importance of actin, super-resolution imaging of it in tissue is still challenging, as it forms very dense networks in almost all cells inside the tissue. In this work, we demonstrate multiplexed super-resolution volumetric imaging of actin in both cultured cells and mouse brain slices via expansion microscopy (ExM). By introducing a simple labeling process, which enables the anchoring of an actin probe, phalloidin, to a swellable hydrogel, the multiplexed ExM imaging of actin filaments was achieved. We first showed that this technique could visualize the nanoscale details of actin filament organizations in cultured cells. Then, we applied this technique to mouse brain slices and visualized diverse actin organizations, such as the parallel actin filaments along the long axis of dendrites and dense actin structures in postsynaptic spines. We examined the postsynaptic spines in the mouse brain and showed that the organizations of actin filaments are highly diverse. This technique, which enables the high-throughput 60 nm resolution imaging of actin filaments and other proteins in cultured cells and thick tissue slices, would be a useful tool to study the organization of actin filaments in diverse biological circumstances and how they change under pathological conditions.

The small GTPase ARF6 regulates GABAergic synapse development.

ADP ribosylation factors (ARFs) are a family of small GTPases composed of six members (ARF1-6) that control various cellular functions, including membrane trafficking and actin cytoskeletal rearrangement, in eukaryotic cells. Among them, ARF1 and ARF6 are the most studied in neurons, particularly at glutamatergic synapses, but their roles at GABAergic synapses have not been investigated. Here, we show that a subset of ARF6 protein is localized at GABAergic synapses in cultured hippocampal neurons. In addition, we found that knockdown (KD) of ARF6, but not ARF1, triggered a reduction in the number of GABAergic synaptic puncta in mature cultured neurons in an ARF activity-dependent manner. ARF6 KD also reduced GABAergic synaptic density in the mouse hippocampal dentate gyrus (DG) region. Furthermore, ARF6 KD in the DG increased seizure susceptibility in an induced epilepsy model. Viewed together, our results suggest that modulating ARF6 and its regulators could be a therapeutic strategy against brain pathologies involving hippocampal network dysfunction, such as epilepsy.

Dysfunction of Mitochondrial Ca2+ Regulatory Machineries in Brain Aging and Neurodegenerative Diseases.

Calcium ions (Ca2+) play critical roles in neuronal processes, such as signaling pathway activation, transcriptional regulation, and synaptic transmission initiation. Therefore, the regulation of Ca2+ homeostasis is one of the most important processes underlying the basic cellular viability and function of the neuron. Multiple components, including intracellular organelles and plasma membrane Ca2+-ATPase, are involved in neuronal Ca2+ control, and recent studies have focused on investigating the roles of mitochondria in synaptic function. Numerous mitochondrial Ca2+ regulatory proteins have been identified in the past decade, with studies demonstrating the tissue- or cell-type-specific function of each component. The mitochondrial calcium uniporter and its binding subunits are major inner mitochondrial membrane proteins contributing to mitochondrial Ca2+ uptake, whereas the mitochondrial Na+/Ca2+ exchanger (NCLX) and mitochondrial permeability transition pore (mPTP) are well-studied proteins involved in Ca2+ extrusion. The level of cytosolic Ca2+ and the resulting characteristics of synaptic vesicle release properties are controlled via mitochondrial Ca2+ uptake and release at presynaptic sites, while in dendrites, mitochondrial Ca2+ regulation affects synaptic plasticity. During brain aging and the progress of neurodegenerative disease, mitochondrial Ca2+ mishandling has been observed using various techniques, including live imaging of Ca2+ dynamics. Furthermore, Ca2+ dysregulation not only disrupts synaptic transmission but also causes neuronal cell death. Therefore, understanding the detailed pathophysiological mechanisms affecting the recently discovered mitochondrial Ca2+ regulatory machineries will help to identify novel therapeutic targets. Here, we discuss current research into mitochondrial Ca2+ regulatory machineries and how mitochondrial Ca2+ dysregulation contributes to brain aging and neurodegenerative disease.

Silver Nanowire-Based Stretchable Transparent Electrode for Flexible Organic Light-Emitting Diode.

The new stretchable transparent electrode was proposed and fabricated based on a process which utilizes silver nanowires (AgNWs) on a Polyurethane (PU) substrate. In order to overcome the rough surface nature of the silver nanowire electrode, a titanium oxide (TiO2) buffer layer was over-coated and then followed by a heat treatment of organo-metalic sol-gel solution. The fabricated stretchable electrodes exhibit electrical sheet resistance of 24 Ω/□, transmittance of 78% at 550 nm wavelength and average surface roughness of less than 5 nm. In addition, without adding additional conductive polymer layers, the fabricated AgNW-based electrode can maintain its initial electrical resistance even if nearly 130% strain is applied. In this letter, the critical role of a TiO2 buffer layer in achieving the high performance of the AgNW stretchable electrode is discussed.