Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function.

Although cell transplantation can rescue motor defects in Parkinson's disease (PD) models, whether and how grafts functionally repair damaged neural circuitry in the adult brain is not known. We transplanted hESC-derived midbrain dopamine (mDA) or cortical glutamate neurons into the substantia nigra or striatum of a mouse PD model and found extensive graft integration with host circuitry. Axonal pathfinding toward the dorsal striatum was determined by the identity of the grafted neurons, and anatomical presynaptic inputs were largely dependent on graft location, whereas inhibitory versus excitatory input was dictated by the identity of grafted neurons. hESC-derived mDA neurons display A9 characteristics and restore functionality of the reconstructed nigrostriatal circuit to mediate improvements in motor function. These results indicate similarity in cell-type-specific pre- and post-synaptic integration between transplant-reconstructed circuit and endogenous neural networks, highlighting the capacity of hPSC-derived neuron subtypes for specific circuit repair and functional restoration in the adult brain.

Plasticity of Synaptic Transmission in Human Stem Cell-Derived Neural Networks.

Long-term potentiation and depression, inferred from analysis on brain slices, are considered the cellular processes underlying learning and memory formation. They have not so far been demonstrated in human stem cell-derived neurons. By expressing channelrhodopsin in hESCs-derived glutamate neurons and co-culturing them with GABA neurons, we found that blue light stimulation increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) and decreased the ratio of paired pulse facilitation (PPF) in non-ChR2-expressing GABA neurons, indicating a facilitating action at the presynaptic terminals. When paired with postsynaptic depolarization, the repetitive stimulation significantly increased the amplitude of light-evoked EPSCs that persisted during the period, indicating long-term potentiation (LTP). In contrast, low-frequency light stimulation induced long-term depression (LTD). These effects were blocked by N-methyl-D-aspartic acid (NMDA) receptor antagonists, suggesting NMDA receptor-mediated synaptic plasticity in human neural networks. Furthermore, induced pluripotent stem cell (iPSC)-derived neurons of patient with Down syndrome showed absence of LTP or LTD. Thus, our platform offers a versatile model for assessing human neural plasticity under physiological and pathological conditions.

Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells.

Differentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4-7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

[Serotonergic neurons in the median raphe nucleus mediate anxiety- and depression-like behavior].

Serotonin (5-hydroxtryptamine, 5-HT), one of the central neurotransmitters, is the most important modulator for emotion regulation, sensory processing, cognitive control, etc. The serotonergic neurons are limited in amount and mainly distributed in the dorsal raphe nucleus (DR) and the median raphe nucleus (MR) in the midline of the brain stem. Previous studies mainly focused on the function of 5-HT neurons in the DR, but little is known about 5-HT neurons in MR. In the present study, with Pet1-Cre transgenic mice and DREADDs technology, we specifically activated or silenced 5-HT neurons in the MR, and aimed to explore their roles in anxiety- and depressive-like behaviors. The results showed that silencing 5-HT neurons in the MR decreased anxiety-like behaviors in the open field and elevated plus maze tasks. Inhibition of 5-HT neurons in the MR decreased depressive-like behaviors in the sucrose preference and forced swim test, while activation of 5-HT neurons in the MR enhanced depressive-like behaviors in the sucrose preference test. These results suggest that the 5-HT neurons in the MR play a key role in regulating anxiety- and depression-like behaviors.

Fear Conditioning Downregulates Rac1 Activity in the Basolateral Amygdala Astrocytes to Facilitate the Formation of Fear Memory.

Astrocytes are well known to scale synaptic structural and functional plasticity, while the role in learning and memory, such as conditioned fear memory, is poorly elucidated. Here, using pharmacological approach, we find that fluorocitrate (FC) significantly inhibits the acquisition of fear memory, suggesting that astrocyte activity is required for fear memory formation. We further demonstrate that fear conditioning downregulates astrocytic Rac1 activity in basolateral amygdala (BLA) in mice and promotes astrocyte structural plasticity. Ablation of astrocytic Rac1 in BLA promotes fear memory acquisition, while overexpression or constitutive activation of astrocytic Rac1 attenuates fear memory acquisition. Furthermore, temporal activation of Rac1 by photoactivatable Rac1 (Rac1-PA) induces structural alterations in astrocytes and in vivo activation of Rac1 in BLA astrocytes during fear conditioning attenuates the formation of fear memory. Taken together, our study demonstrates that fear conditioning-induced suppression of BLA astrocytic Rac1 activity, associated with astrocyte structural plasticity, is required for the formation of conditioned fear memory.

[Optogenetic activation of dorsal hippocampal astrocytic Rac1 blocks the learning of associative memory].

Rac1 belongs to the family of Rho GTPases, and plays important roles in the brain function. It affects the cell migration and axon guidance via regulating the cytoskeleton and cellular morphology. However, the effect of its dynamic activation in regulating physiological function remains unclear. Recently, a photoactivatable analogue of Rac1 (PA-Rac1) has been developed, allowing the activation of Rac1 by the specific wavelength of light in living cells. Thus, we constructed recombinant adeno-associated virus (AAV) of PA-Rac1 and its light-insensitive mutant PA-Rac1-C450A under the control of the mouse glial fibrillary acidic protein (mGFAP) promoter to manipulate Rac1 activity in astrocytes by optical stimulation. Primary culture of hippocampal astrocytes was infected with the recombinant AAV-PA-Rac1 or AAV-PA-Rac1-C450A. Real-time fluorescence imaging showed that the cell membrane of the astrocyte expressing PA-Rac1 protruded near the light spot, while the astrocyte expressing PA-Rac1-C450A did not. We injected AAV-PA-Rac1 and AAV-PA-Rac1-C450A into dorsal hippocampus to investigate the role of the activation of Rac1 in regulating the associative learning. With optical stimulation, the PA-Rac1 group, rather than the PA-Rac1-C450A group, showed slower learning curve during the fear conditioning compared with the control group, indicating that activating astrocytic Rac1 blocks the formation of contextual memory. Our data suggest that the activation of Rac1 in dorsal hippocampal astrocyte plays an important role in the associative learning.

Post-training activation of Rac1 in the basolateral amygdala is required for the formation of both short-term and long-term auditory fear memory.

Rac1, a member of the Rho family of small GTPases, is crucial for morphological changes of the mature neuronal synapse including spine formation and activity-dependent spine enlargement, while its role in the formation of associated memories, such as conditioned fear memory, is not clear. Here, we report that selective deletion of Rac1 in excitatory neurons, but not in parvalbumin inhibitory neurons, impaired short- and long-term memories (STM and LTM) of fear conditioning. Conditional knockout of Rac1 before associative fear training in the basolateral amygdala (BLA), a key area for fear memory acquisition and storage, impaired fear memory. The expression of dominant-negative mutant of Rac1, or infusion of Rac1 inhibitor NSC23766 into BLA blocked both STM and LTM of fear conditioning. Furthermore, selective inhibition of Rac1 activation in BLA immediately following fear conditioning impaired STM and LTM, demonstrating that fear conditioning-induced Rac1 activation in BLA plays a critical role in the formation of both STM and LTM of conditioned fear.

Astroglial ß-Arrestin1-mediated Nuclear Signaling Regulates the Expansion of Neural Precursor Cells in Adult Hippocampus.

Adult hippocampal neurogenesis is crucial for preserving normal brain function, but how it is regulated by niche cells is uncertain. Here we show that ß-arrestin 1 (ß-arr1) in dentate gyrus (DG) regulates neural precursor proliferation. ß-arr1 knockout (KO) mice show reduced neural precursor proliferation in subgranular zone (SGZ) which could be rescued by selective viral expression of ß-arr1 but not its nuclear-function-deficient mutants under control of hGFAP promotor in DG. Compared with wild type astrocytes, ß-arr1 KO astrocytes nurture less neurospheres, and this may be attributed to changed activity of soluble, heat-sensitive excretive factors, such as BMP2. RNA-sequencing reveals that ß-arr1 KO DG astrocytes exhibit an aberrant gene expression profile of niche factors, including elevated transcription of Bmp2. Taken together, our data suggest that ß-arr1 mediated nuclear signaling regulates the production of excretive factors derived from niche astrocytes and expansion of neural precursors in DG, thus maintaining homeostasis of adult hippocampal neurogenesis.

[Neurons in NAc core and BLA are activated during cocaine context-associated reward memory retrieval in mice].

The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli make addiction hard to cure by contributing to cocaine seeking and relapse. So it's of great importance to examine the neurobiological basis of addiction memory. Cocaine conditioned place preference (CPP) used in this study is a form of Pavlovian conditioning which can establish associations between drug and contextual factors. c-Fos and Zif268 are commonly used immediate early gene (IEG) makers to identify neurons that are activated after a stimulus or behavioral conditioning. This study was designed to reveal neuronal c-Fos, Zif268 expression pattern in 10 brain regions following cocaine context-associated reward memory retrieval in mice, combining animal behavioral study and immunofluorescence method. C57BL/6 mice were randomly divided into 3 groups: Saline retrieval, Cocaine retrieval, and No retrieval of cocaine groups. Cocaine retrieval and No retrieval of cocaine underwent CPP training (one side paired with cocaine, and the other side with saline) except that No retrieval of cocaine group didn't undergo CPP test. Saline retrieval group received saline injections (i.p) on both sides. The results showed that: Neuronal c-Fos, Zif268 protein expression levels in nucleus accumbens (NAc) core both were elevated in Cocaine retrieval group compared with those in Saline retrieval (Control) group during cocaine context-associated reward memory retrieval. Zif268 protein expression level in basolateral amygdala (BLA) was also elevated in Cocaine retrieval group compared with that in control mice. Elevation was not seen in other regions such as hippocampus, prefrontal cortex (PFC). Thus, NAc core and BLA were activated during cocaine context-associated reward memory retrieval. The results suggest that neurons that are activated in NAc core and BLA are crucial basis of cocaine context-associated reward memory.

[Voluntary wheel running enhances cell proliferation and expression levels of BDNF, IGF1 and WNT4 in dentate gyrus of adult mice].

Adult hippocampal neurogenesis plays important roles in learning, memory and mood regulation. External factors, such as physical exercise, have been found to modulate adult hippocampal neurogenesis. Voluntary running enhances cell proliferation in subgranular zone (SGZ) and increases the number of new born neurons in rodents, but underlying mechanisms are not fully understood. In this study, we used BrdU assay to identify proliferating cells in 2-month-old C57BL/6 mice after 15 days of voluntary wheel running test. mRNA and protein levels for several neural factors in dentate gyrus, Ammon's horn, and cortex were also analyzed by RT-qPCR and Western blot assay after 15 days of voluntary wheel running. Our data show that voluntary wheel running for 15 days elevated the number of proliferation cells in dentate gyrus and significantly up-regulated the mRNA levels of Bdnf, Igf1 and Wnt4. The protein levels of BDNF and IGF1 in dentate gyrus were also increased after voluntary wheel running. These results indicate that the increase of adult hippocampal neurogenesis caused by voluntary wheel running for 15 days might be through up-regulating BDNF, IGF1 and WNT4 in dentate gyrus.

Optogenetic activation of GABAergic neurons in the nucleus accumbens decreases the activity of the ventral pallidum and the expression of cocaine-context-associated memory.

GABAergic medium-sized spiny neurons (MSNs) in the nucleus accumbens (NAc) differentially express D1 and D2 dopamine receptors. Both D2- and D1-MSNs in the NAc form projections into the ventral pallidum, whereas only D1-MSNs directly project into midbrain neurons. They are critical in rewarding and aversive learning, and understanding the function of these NAc efferents and the alteration of their targeted brain regions in responding to a reward-associated context is important. In this study, we activated the GABAergic neurons in the NAc of mice expressing channelrhodopsin-2 under the control of the vesicular GABA transporter promoter by an optogenetic approach, and examined its effects on the expression of cocaine-context-associated memory. In vivo optogenetic activation of the NAc GABAergic neurons inhibited the expression of cocaine-conditioned place preference (CPP). When tested 24 h later, these mice exhibited normal cocaine-induced CPP, indicating that the inhibitory effect on the expression of CPP was transient and reversible. Activation of the NAc GABAergic neurons also attenuated the learning of cocaine-induced reinforcement, as indicated by the results of behavioural sensitization. To explore how the cocaine-context-associated information was processed and integrated, we assessed the activity of NAc MSN-targeted brain nuclei and found that the activation of NAc GABAergic neurons during CPP expression resulted in a decrease of c-Fos+ cells in the ventral palladium. Our data suggested that the NAc GABAergic efferents inhibit the ventral palladium activity and negatively regulate the expression of motivational effects induced by cocaine-context-associated cues.

ß-Arrestin-2 inhibits preference for alcohol in mice and suppresses Akt signaling in the dorsal striatum.

In this study, we investigated the role of ß-arrestin-2 in alcohol preference using the two-bottle choice and conditioned place preference procedures in wild-type (WT) and ß-arrestin-2 knockout (KO) mice. Locomotion and righting reflex tests were performed to test alcohol sensitivity. The possible molecular signals regulated by ß-arrestin-2 were analyzed by Western blot. We found that ß-arrestin-2 KO mice showed a marked increase in voluntary alcohol consumption without significant differences in preference for saccharin or aversion to quinine. These animals also exhibited higher conditioned place preference scores for alcohol than WT mice. Meanwhile, KO mice showed reduced sensitivity to alcohol and increased blood alcohol clearance. Furthermore, after the free consumption of alcohol, the activities of protein kinase B and glycogen synthase kinase 3ß (GSK3ß) increased in the dorsal striatum of WT mice, but not in KO mice, which showed high basal activity of Akt in the dorsal striatum. These results suggest that ß-arrestin-2 negatively regulates alcohol preference and reward, likely through regulating the activation of signaling pathways including Akt/GSK3ß in the dorsal striatum.

Note: Characterization of the plasma parameters of a capillary discharge-produced plasma channel waveguide to guide an intense laser pulse.

We demonstrated the production of an optical waveguide in a capillary discharge-produced plasma using a cylindrical capillary. Plasma parameters of its waveguide were characterized by use of both a Nomarski laser interferometer and a hydrogen plasma line spectrum. A space-averaged maximum temperature of 3.3 eV with electron densities of the order of 10(17) cm(-3) was observed at a discharge time of 150 ns and a maximum discharge current of 400 A. An ultrashort, intense laser pulse was guided by use of this plasma channel.

Regulation of amygdalar PKA by beta-arrestin-2/phosphodiesterase-4 complex is critical for fear conditioning.

Beta-arrestins, key regulators of receptor signaling, are highly expressed in the central nervous system, but their roles in brain physiology are largely unknown. Here we show that beta-arrestin-2 is critically involved in the formation of associative fear memory and amygdalar synaptic plasticity. In response to fear conditioning, beta-arrestin-2 translocates to amygdalar membrane where it interacts with PDE-4, a cAMP-degrading enzyme, to inhibit PKA activation. Arrb2(-/-) mice exhibit impaired conditioned fear memory and long-term potentiation at the lateral amygdalar synapses. Moreover, expression of the beta-arrestin-2 in the lateral amygdala of Arrb2(-/-) mice, but not its mutant form that is incapable of binding PDE-4, restores basal PKA activity and rescues conditioned fear memory. Taken together, our data demonstrate that the feedback regulation of amygdalar PKA activation by beta-arrestin-2 and PDE-4 complex is critical for the formation of conditioned fear memory.

Opacity effect on extreme ultraviolet radiation from laser-produced tin plasmas.

Opacity effects on extreme ultraviolet (EUV) emission from laser-produced tin (Sn) plasma have been experimentally investigated. An absorption spectrum of a uniform Sn plasma generated by thermal x rays has been measured in the EUV range (9-19 nm wavelength) for the first time. Experimental results indicate that control of the optical depth of the laser-produced Sn plasma is essential for obtaining high conversion to 13.5 nm-wavelength EUV radiation; 1.8% of the conversion efficiency was attained with the use of 2.2 ns laser pulses.