K+ efflux through postsynaptic NMDA receptors suppresses local astrocytic glutamate uptake.

Glutamatergic transmission prompts K+ efflux through postsynaptic NMDA receptors. The ensuing hotspot of extracellular K+ elevation depolarizes presynaptic terminal, boosting glutamate release, but whether this also affects glutamate uptake in local astroglia has remained an intriguing question. Here, we find that the pharmacological blockade, or conditional knockout, of postsynaptic NMDA receptors suppresses use-dependent increase in the amplitude and duration of the astrocytic glutamate transporter current (IGluT ), whereas blocking astrocytic K+ channels prevents the duration increase only. Glutamate spot-uncaging reveals that astrocyte depolarization, rather than extracellular K+ rises per se, is required to reduce the amplitude and duration of IGluT . Biophysical simulations confirm that local transient elevations of extracellular K+ can inhibit local glutamate uptake in fine astrocytic processes. Optical glutamate sensor imaging and a two-pathway test relate postsynaptic K+ efflux to enhanced extrasynaptic glutamate signaling. Thus, repetitive glutamatergic transmission triggers a feedback loop in which postsynaptic K+ efflux can transiently facilitate presynaptic release while reducing local glutamate uptake.

Morphological profile determines the frequency of spontaneous calcium events in astrocytic processes.

Astrocytes express a complex repertoire of intracellular Ca2+ transients (events) that represent a major form of signaling within individual cells and in astrocytic syncytium. These events have different spatiotemporal profiles, which are modulated by neuronal activity. Spontaneous Ca2+ events appear more frequently in distal astrocytic processes and independently from each other. However, little is known about the mechanisms underlying such subcellular distribution of the Ca2+ events. Here, we identify the initiation points of the Ca2+ events within the territory of single astrocytes expressing genetically encoded Ca2+ indicator GCaMP2 in culture or in hippocampal slices. We found that most of the Ca2+ events start in an optimal range of thin distal processes. Our mathematical model demonstrated that a high surface-to-volume of the thin processes leads to increased amplitude of baseline Ca2+ fluctuations caused by a stochastic opening of Ca2+ channels in the plasma membrane. Suprathreshold fluctuations trigger Ca2+ -induced Ca2+ release from the Ca2+ stores by activating inositol 1,4,5-trisphosphate (IP3 ) receptors. In agreement with the model prediction, the spontaneous Ca2+ events frequency depended on the extracellular Ca2+ concentration. Astrocytic depolarization by high extracellular K+ increased the frequency of the Ca2+ events through activation of voltage-gated Ca2+ channels in cultured astrocytes. Our results suggest that the morphological profile of the astrocytic processes is responsible for tuning of the Ca2+ events frequency. Therefore, structural plasticity of astrocytic processes can be directly translated into changes in astrocytic Ca2+ signaling. This may be important for both physiological and pathological astrocyte remodeling.

Differential expression and function of nicotinic acetylcholine receptors in subdivisions of medial habenula.

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that α3 and ß4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, ß2, ß3, and α4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 µm nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.

Nicotine Dependence Reveals Distinct Responses from Neurons and Their Resident Nicotinic Receptors in Medial Habenula.

Nicotinic acetylcholine receptors (nAChRs) are the molecular target of nicotine. nAChRs in the medial habenula (MHb) have recently been shown to play a role in nicotine dependence, but it is not clear which nAChR subtypes or MHb neuron types are most important. To identify MHb nAChRs and/or cell types that play a role in nicotine dependence, we studied these receptors and cells with brain slice electrophysiology using both acute and chronic nicotine application. Cells in the ventroinferior (MHbVI) and ventrolateral MHb (MHbVL) subregions expressed functional nAChRs with different pharmacology. Further, application of nicotine to cells in these subregions led to different action potential firing patterns. The latter result was correlated with a differing ability of nicotine to induce nAChR desensitization. Chronic nicotine caused functional upregulation of nAChRs selectively in MHbVI cells, but did not change nAChR function in MHbVL. Importantly, firing responses were also differentially altered in these subregions following chronic nicotine. MHbVI neurons treated chronically with nicotine exhibited enhanced basal pacemaker firing but a blunted nicotine-induced firing response. MHbVL neurons did not change their firing properties in response to chronic nicotine. Together, these results suggest that acute and chronic nicotine differentially affect nAChR function and output of cells in MHb subregions. Because the MHb extensively innervates the interpeduncular nucleus, an area critical for both affective and somatic signs of withdrawal, these results could reflect some of the neurophysiological changes thought to occur in the MHb to the interpeduncular nucleus circuit in human smokers.

Denoising of two-photon fluorescence images with block-matching 3D filtering.

Two-photon florescence imaging is widely used to perform morphological analysis of subcellular structures such as neuronal dendrites and spines, astrocytic processes etc. This method is also indispensable for functional analysis of cellular activity such as Ca2+ dynamics. Although spatial resolution of laser scanning two-photon system is greater than that of confocal or wide field microscope, it is still diffraction limited. In practice, the resolution of the system is more affected by its signal-to-noise ratio (SNR) than the diffraction limit. Thus, various approaches aiming to increase the SNR in two-photon imaging are desirable and can potentially save on building costly super-resolution imaging system. Here we analyze the statistics of noise in the two-photon florescence images of hippocampal astrocytes expressing genetically encoded Ca2+ sensor GCaMP2 and show that it can be reasonably well approximated using the same models which are used for describing noise in images acquired with digital cameras. This allows to use denoising methods available for wide field imaging on two-photon images. Particularly we demonstrate that the Block-Matching 3D (BM3D) filter can significantly improve the quality of two-photon fluorescence images so small details such as astrocytic processes can be easier identified. Moreover, denoising of the images with BM3D yields less noisy Ca2+ signals in astrocytes when denoising of the images with Gaussian filter.

α4α6ß2* nicotinic acetylcholine receptor activation on ventral tegmental area dopamine neurons is sufficient to stimulate a depolarizing conductance and enhance surface AMPA receptor function.

Tobacco addiction is a serious threat to public health in the United States and abroad, and development of new therapeutic approaches is a major priority. Nicotine activates and/or desensitizes nicotinic acetylcholine receptors (nAChRs) throughout the brain. nAChRs in ventral tegmental area (VTA) dopamine (DA) neurons are crucial for the rewarding and reinforcing properties of nicotine in rodents, suggesting that they may be key mediators of nicotine's action in humans. However, it is unknown which nAChR subtypes are sufficient to activate these neurons. To test the hypothesis that nAChRs containing α6 subunits are sufficient to activate VTA DA neurons, we studied mice expressing hypersensitive, gain-of-function α6 nAChRs (α6L9'S mice). In voltage-clamp recordings in brain slices from adult mice, 100 nM nicotine was sufficient to elicit inward currents in VTA DA neurons via α6ß2* nAChRs. In addition, we found that low concentrations of nicotine could act selectively through α6ß2* nAChRs to enhance the function of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) receptors on the surface of these cells. In contrast, α6ß2* activation did not enhance N-methyl-D-aspartic acid receptor function. Finally, AMPA receptor (AMPAR) function was not similarly enhanced in brain slices from α6L9'S mice lacking α4 nAChR subunits, suggesting that α4α6ß2* nAChRs are important for enhancing AMPAR function in VTA DA neurons. Together, these data suggest that activation of α4α6ß2* nAChRs in VTA DA neurons is sufficient to support the initiation of cellular changes that play a role in addiction to nicotine. α4α6ß2* nAChRs may be a promising target for future smoking cessation pharmacotherapy.

Anti-tau intrabodies: From anti-tau immunoglobulins to the development of functional scFv intrabodies.

Over the last decade, there has been a growing interest in intrabodies and their therapeutic potential. Intrabodies are antibody fragments that are expressed inside a cell to target intracellular antigens. In the context of intracellular protein misfolding and aggregation, such as tau pathology in Alzheimer's disease, intrabodies have become an interesting approach as there is the possibility to target early stages of aggregation. As such, we engineered three anti-tau monoclonal antibodies into single-chain variable fragments for cytoplasmic expression and activity: PT51, PT77, and hTau21. Due to the reducing environment of the cytoplasm, single-chain variable fragment (scFv) aggregation is commonly observed. Therefore, we also performed complementarity-determining region (CDR) grafting into three different stable frameworks to rescue solubility and intracellular binding. All three scFvs retained binding to tau after cytoplasmic expression in HEK293 cells, in at least one of the frameworks. Subsequently, we show their capacity to interfere with either mouse or mutant human tau aggregation in two different primary mouse neuron models and organotypic hippocampal slice cultures. Collectively, our work extends the current knowledge on intracellular tau targeting with intrabodies, providing three scFv intrabodies that can be used as immunological tools to target tau inside cells.

Development of a fully human assay combining NGN2-inducible neurons co-cultured with iPSC-derived astrocytes amenable for electrophysiological studies.

Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a "fully-humanized" set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at https://cells.ebisc.org/.

SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System.

Astrocytes, the main supportive cell type of the brain, show functional impairments upon ageing and in a broad spectrum of neurological disorders. Limited access to human astroglia for pre-clinical studies has been a major bottleneck delaying our understanding of their role in brain health and disease. We demonstrate here that functionally mature human astrocytes can be generated by SOX9 overexpression for 6 days in pluripotent stem cell (PSC)-derived neural progenitor cells. Inducible (i)SOX9-astrocytes display functional properties comparable to primary human astrocytes comprising glutamate uptake, induced calcium responses and cytokine/growth factor secretion. Importantly, electrophysiological properties of iNGN2-neurons co-cultured with iSOX9-astrocytes are indistinguishable from gold-standard murine primary cultures. The high yield, fast timing and the possibility to cryopreserve iSOX9-astrocytes without losing functional properties makes them suitable for scaled-up production for high-throughput analyses. Our findings represent a step forward to an all-human iPSC-derived neural model for drug development in neuroscience and towards the reduction of animal use in biomedical research.

Neurokinin 1 receptor activates transient receptor potential-like currents in noradrenergic A7 neurons in rats.

Noradrenergic (NAergic) A7 neurons are involved in modulating nociception by releasing noradrenaline in the dorsal spinal cord. Since NAergic A7 neurons receive dense Substance P (Sub-P) releasing terminals from ventromedial medulla, here we tested the effect of Sub-P on them. Bath application of Sub-P induced an inward current (I(Sub-P)) in NAergic neurons, which was significantly blocked by Neurokinin 1 (NK1) receptor antagonist. The I(Sub-P) was reversed at approximately -20 mV, blocked by several TRP channel blockers, enhanced by OAG and negatively regulated by PKC. Immunohistochemistry staining showed that NAergic A7 neurons express high level of TRPC6 channel proteins, which is consistent with pharmacological properties of I(Sub-P) shown above, as TRPC6 channel is shown to be augmented by OAG and inhibited by PKC. In conclusion, the above results provide mechanism underlying postsynaptic action of Sub-P on NAergic A7 neurons and a role for TRPC6 channel in NAergic pain modulation.

Activities of daily living function and neuropsychiatric symptoms of people with dementia and caregiver burden: The mediating role of caregiving hours.

OBJECTIVE: Due to the presence of neuropsychiatric behaviors and the decreased ability for activities of daily living (ADLs), family caregivers experience high burden levels in caring for people with dementia (PWD). This study sought to test the mediating role of caregiving hours in association with PWDs' ability for basic activities of daily living (BADL) function or neuropsychiatric behaviors and caregiver burden. METHODS: This study used two waves of survey data, collected between 2013 and 2016, from 186 PWD-caregiver dyads in a dementia clinic at a teaching hospital in southern Taiwan. Two sets of multiple linear regression models were used to analyze the relationships between the changes in patients' BADL function, patients' neuropsychiatric behaviors, caregiving hours in ADL (including BADL and Instrumental ADL), and caregiver burden (measured using Zarit Burden Interview). Bootstrapping methods were used to detect the mediating effects of caregiving hours in ADL if the 95% confidence interval (CI) did not cover 0. RESULTS: Caregiving hours in BADL mediated the relationship of PWDs' BADL function and caregiver burden (effect = -0.0137, 95% bootstrap CI = -0.0379, -0.0003). However, such mediating effects were not found in the relationship of PWDs' neuropsychiatric behaviors and caregiver burden. CONCLUSIONS: In order to relieve caregiver burden, respite care services for caregivers for PWD should target services that assist with PWD's BADL.

A Potential Antifungal Effect of Chitosan Against Candida albicans Is Mediated via the Inhibition of SAGA Complex Component Expression and the Subsequent Alteration of Cell Surface Integrity.

Due to the high incidence of nosocomial Candida albicans infection, the first-line drugs for C. albicans infection have been heavily used, and the emergence of drug-resistant strains has gradually increased. Thus, a new antifungal drug or therapeutic method is needed. Chitosan, a product of chitin deacetylation, is considered to be potentially therapeutic for fungal infections because of its excellent biocompatibility, biodegradability and low toxicity. The biocidal action of chitosan against C. albicans shows great commercial potential, but the exact mechanisms underlying its antimicrobial activity are unclear. To reveal these mechanisms, mutant library screening was performed. ADA2 gene, which encodes a histone acetylation coactivator in the SAGA complex, was identified. Transmission electronic microscopy images showed that the surface of chitosan-treated ada2Δ cells was substantially disrupted and displayed an irregular morphology. Interestingly, the cell wall of ada2Δ cells was significantly thinner than that of wild-type cells, with a thickness similar to that seen in the chitosan-treated wild-type strain. Although ADA2 is required for chitosan tolerance, expression of ADA2 and several Ada2-mediated cell wall-related genes (ALS2, PGA45, and ACE2) and efflux transporter genes (MDR1 and CDR1) were significantly inhibited by chitosan. Furthermore, GCN5 encoding a SAGA complex catalytic subunit was inhibited by chitosan, and gcn5Δ cells exhibited phenotypes comparable to those of ada2Δ cells in response to chitosan and other cell surface-disrupting agents. This study demonstrated that a potential antifungal mechanism of chitosan against C. albicans operates by inhibiting SAGA complex gene expression, which decreases the protection of the cell surface against chitosan.

Label-Free Vibrational Spectroscopic Imaging of Neuronal Membrane Potential.

Detecting membrane potentials is critical for understanding how neuronal networks process information. We report a vibrational spectroscopic signature of neuronal membrane potentials identified through hyperspectral stimulated Raman scattering (SRS) imaging of patched primary neurons. High-speed SRS imaging allowed direct visualization of puff-induced depolarization of multiple neurons in mouse brain slices, confirmed by simultaneous calcium imaging. The observed signature, partially dependent on sodium ion influx, is interpreted as ion interactions on the CH3 Fermi resonance peak in proteins. By implementing a dual-SRS balanced detection scheme, we detected single action potentials in electrically stimulated neurons. These results collectively demonstrate the potential of sensing neuronal activities at multiple sites with a label-free vibrational microscope.

Economic Impact of Dementia by Disease Severity: Exploring the Relationship between Stage of Dementia and Cost of Care in Taiwan.

OBJECTIVE: Given the shortage of cost-of-illness studies in dementia outside of the Western population, the current study estimated the annual cost of dementia in Taiwan and assessed whether different categories of care costs vary by severity using multiple disease-severity measures. METHODS: This study included 231 dementia patient-caregiver dyads in a dementia clinic at a national university hospital in southern Taiwan. Three disease measures including cognitive, functional, and behavioral disturbances were obtained from patients based on medical history. A societal perspective was used to estimate the total costs of dementia according to three cost sub-categories. The association between dementia severity and cost of care was examined through bivariate and multivariate analyses. RESULTS: Total costs of care for moderate dementia patient were 1.4 times the costs for mild dementia and doubled from mild to severe dementia among our community-dwelling dementia sample. Multivariate analysis indicated that functional declines had a greater impact on all cost outcomes as compared to behavioral disturbance, which showed no impact on any costs. Informal care costs accounted for the greatest share in total cost of care for both mild (42%) and severe (43%) dementia patients. CONCLUSIONS: Since the total costs of dementia increased with severity, providing care to delay disease progression, with a focus on maintaining patient physical function, may reduce the overall cost of dementia. The greater contribution of informal care to total costs as opposed to social care also suggests a need for more publicly-funded long-term care services to assist family caregivers of dementia patients in Taiwan.

Spatiotemporal calcium dynamics in single astrocytes and its modulation by neuronal activity.

Astrocytes produce a complex repertoire of Ca2+ events that coordinate their major functions. The principle of Ca2+ events integration in astrocytes, however, is unknown. Here we analyze whole Ca2+ events, which were defined as spatiotemporally interconnected transient Ca2+ increases. Using such analysis in single hippocampal astrocytes in culture and in slices we found that spreads and durations of Ca2+ events follow power law distributions, a fingerprint of scale-free systems. A mathematical model demonstrated that such Ca2+ dynamics can arise from intracellular inositol-3-phosphate diffusion. The power law exponent (α) was decreased by activation of metabotropic glutamate receptors (mGluRs) either by specific receptor agonist or by low frequency stimulation of glutamatergic fibers in hippocampal slices. Decrease in α indicated an increase in proportion of large Ca2+ events. Notably, mGluRs activation did not increase the frequency of whole Ca2+ events. This result suggests that neuronal activity does not trigger new Ca2+ events in astrocytes (detectable by our methods), but modulates the properties of existing ones. Thus, our results provide a new perspective on how astrocyte responds to neuronal activity by changing its Ca2+ dynamics, which might further affect local network by triggering release of gliotransmitters and by modulating local blood flow.

Astrocytic Ca2+ signals are required for the functional integrity of tripartite synapses.

BACKGROUND: Neuronal activity alters calcium ion (Ca2+) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. To examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "IP3 sponge", attenuates astrocytic Ca2+ signaling. RESULTS: Attenuated Ca2+ activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal CA1 region in these animals. The decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced N-methyl-D-aspartate receptor currents in CA1 pyramidal neurons in response to burst stimulation. These mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. CONCLUSIONS: Our findings suggest that IP3-mediated astrocytic Ca2+ signaling correlates with the formation of functional tripartite synapses in the hippocampus.

Low micromolar Ba(2+) potentiates glutamate transporter current in hippocampal astrocytes.

Glutamate uptake, mediated by electrogenic glutamate transporters largely localized in astrocytes, is responsible for the clearance of glutamate released during excitatory synaptic transmission. Glutamate uptake also determines the availability of glutamate for extrasynaptic glutamate receptors. The efficiency of glutamate uptake is commonly estimated from the amplitude of transporter current recorded in astrocytes. We recorded currents in voltage-clamped hippocampal CA1 stratum radiatum astrocytes in rat hippocampal slices induced by electrical stimulation of the Schaffer collaterals. A Ba(2+)-sensitive K(+) current mediated by inward rectifying potassium channels (Kir) accompanied the transporter current. Surprisingly, Ba(2+) not only suppressed the K(+) current and changed holding current (presumably, mediated by Kir) but also increased the transporter current at lower concentrations. However, Ba(2+) did not significantly increase the uptake of aspartate in cultured astrocytes, suggesting that increase in the amplitude of the transporter current does not always reflect changes in glutamate uptake.

Retrograde synaptic signaling mediated by K+ efflux through postsynaptic NMDA receptors.

Synaptic NMDA receptors (NMDARs) carry inward Ca(2+) current responsible for postsynaptic signaling and plasticity in dendritic spines. Whether the concurrent K(+) efflux through the same receptors into the synaptic cleft has a physiological role is not known. Here, we report that NMDAR-dependent K(+) efflux can provide a retrograde signal in the synapse. In hippocampal CA3-CA1 synapses, the bulk of astrocytic K(+) current triggered by synaptic activity reflected K(+) efflux through local postsynaptic NMDARs. The local extracellular K(+) rise produced by activation of postsynaptic NMDARs boosted action potential-evoked presynaptic Ca(2+) transients and neurotransmitter release from Schaffer collaterals. Our findings indicate that postsynaptic NMDAR-mediated K(+) efflux contributes to use-dependent synaptic facilitation, thus revealing a fundamental form of retrograde synaptic signaling.