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Social defeat stress (SDS) plays a major role in the pathogenesis of psychiatric disorders like anxiety and depression. Sleep is generally considered to involve recovery of the brain from prior experience during wakefulness and is altered after acute SDS. However, the effect of acute SDS on sleep/wake behavior in mice varies between studies. In addition, whether sleep changes in response to stress contribute to anxiety is not well established. Here, we first investigated the effects of acute SDS on sleep/wake states in the active period in mice. Our results showed that total sleep time (time in rapid eye-movement [REM] and non-REM [NREM] sleep) increased in the active period after acute SDS. NREM sleep increased mainly during the first 3 h after SDS, while REM sleep increased at a later time. Then, we demonstrated that the increased NREM sleep had an anxiolytic benefit in acute SDS. Mice deprived of sleep for 1 h or 3 h after acute SDS remained in a highly anxious state, while in mice with ad libitum sleep the anxiety rapidly faded away. Altogether, our findings suggest an anxiolytic effect of NREM sleep, and indicate a potential therapeutic strategy for anxiety.

Microglia constantly survey the brain microenvironment and rapidly adopt different phenotypes in response to environmental stimuli. Such dynamic functions require a unique metabolism and bioenergetics. However, little is known about the basic metabolism of microglia and how metabolic changes regulate microglia function. Here, we uncover that microglia activation is accompanied by extensive transcriptional changes in glucose and lipid metabolism-related genes. Using metabolic flux assays, we found that LPS, a prototype of the pathogen-associated molecular patterns (PAMPs), significantly enhanced glycolysis but suppressed oxidative phosphorylation (OXPHOS) in primary cultured microglia. By contrast, ATP, a known damage-associated molecular pattern (DAMPs) that triggers sterile activation of microglia, boosted both glycolysis and OXPHOS. Importantly, both LPS and ATP activated the mechanistic target of rapamycin (mTOR) pathway and enhanced the intracellular reactive oxygen species (ROS). Inhibition of mTOR activity suppressed glycolysis and ROS production in both conditions but exerted different effects on OXPHOS: it attenuated the ATP-induced elevation of OXPHOS, yet had no impact on the LPS-induced suppression of OXPHOS. Further, inhibition of mTOR or glycolysis decreased production of LPS-induced proinflammatory cytokines and ATP-induced tumor necrosis factor-α (TNF-α) and brain derived neurotrophic factor (BDNF) in microglia. Our study reveals a critical role for mTOR in the regulation of metabolic programming of microglia to shape their distinct functions under different states and shed light on the potential application of targeting metabolism to interfere with microglia-mediated neuroinflammation in multiple disorders.

Summary: Overeating is a serious issue in modern society, causing many health problems, including obesity. Although the hypothalamus has been previously identified as the key brain structure that regulates body weight homeostasis, the downstream pathways and non-canonical neural circuitry involved in feeding behavior remain largely uncharacterized. Here, we discover that suppressing the activity of GABAergic cells in the anterior ventrolateral periaqueductal gray (vlPAG), whether directly or through long-projection GABAergic inputs from either the bed nucleus of the stria terminalis (BNST) or the lateral hypothalamus (LH), is sufficient to promptly induce feeding behavior in well-fed mice. In contrast, optogenetic activation of these cells interrupts food intake in starved mice. Long-term chemogenetic manipulation of vlPAG GABAergic cell activity elicits a corresponding change in mouse body weight. Our studies reveal distinct midbrain GABAergic pathways and highlight an important role of GABAergic cells in the anterior vlPAG in feeding behavior. : Hao et al. uncovered an important role of the GABAergic cells in the anterior ventrolateral periaqueductal gray (vlPAG) and two distinct midbrain GABAergic pathways (the lateral hypothalamus-vlPAG and BNST-vlPAG) in regulation of feeding behavior. Keywords: feeding, vlPAG, GABAergic pathway, lateral hypothalamus, mice

Previous genetic fate-mapping studies have indicated that embryonic glial fibrillary acidic protein-positive (GFAP(+)) cells are multifunctional progenitor/neural stem cells that can produce astrocytes as well as neurons and oligodendrocytes throughout the adult mouse central nervous system (CNS). However, emerging evidence from recent studies indicates that GFAP(+) cells adopt different cell fates and generate different cell types in different regions. Moreover, the fate of GFAP(+) cells in the young adult mouse CNS is not well understood. In the present study, hGFAP-Cre/R26R transgenic mice were used to investigate the lineage of embryonic GFAP(+) cells in the young adult mouse CNS. At postnatal day 21, we found that GFAP(+) cells mainly generated NeuN(+) neurons in the cerebral cortex (both ventral and dorsal), hippocampus, and cerebellum. Strangely, these cells were negative for the Purkinje cell marker calbindin in the cerebellum and the neuronal marker NeuN in the thalamus. Thus, contrary to previous studies, our genetic fate-mapping revealed that the cell fate of embryonic GFAP(+) cells at the young adult stage is significantly different from that at the adult stage.

The main lysosomal protease cathepsin D (cathD) is essential for maintaining tissue homeostasis via its degradative function, and its loss leads to ceroid accumulation in the mammalian nervous system, which results in progressive neurodegeneration. Increasing evidence implies non-proteolytic roles of cathD in regulating various biological processes such as apoptosis, cell proliferation, and migration. Along these lines, we here showed that cathD is required for modulating dendritic architecture in the nervous system independent of its traditional degradative function. Upon cathD depletion, class I and class III arborization (da) neurons in Drosophila larvae exhibited aberrant dendritic morphology, including over-branching, aberrant turning, and elongation defects. Re-introduction of wild-type cathD or its proteolytically-inactive mutant dramatically abolished these morphological defects. Moreover, cathD knockdown also led to dendritic defects in the adult mushroom bodies, suggesting that cathD-mediated processes are required in both the peripheral and central nervous systems. Taken together, our results demonstrate a critical role of cathD in shaping dendritic architecture independent of its proteolytic function. Electronic supplementary material The online version of this article (10.1007/s12264-020-00479-6) contains supplementary material, which is available to authorized users.

The laterodorsal tegmentum (LDT) is a brain structure involved in distinct behaviors including arousal, reward, and innate fear. How environmental stimuli and top-down control from high-order sensory and limbic cortical areas converge and coordinate in this region to modulate diverse behavioral outputs remains unclear. Using a modified rabies virus, we applied monosynaptic retrograde tracing to the whole brain to examine the LDT cell type specific upstream nuclei. The LDT received very strong midbrain and hindbrain afferents and moderate cortical and hypothalamic innervation but weak connections to the thalamus. The main projection neurons from cortical areas were restricted to the limbic lobe, including the ventral orbital cortex (VO), prelimbic, and cingulate cortices. Although different cell populations received qualitatively similar inputs, primarily via afferents from the periaqueductal gray area, superior colliculus, and the LDT itself, parvalbumin-positive (PV(+)) GABAergic cells received preferential projections from local LDT neurons. With regard to the different subtypes of GABAergic cells, a considerable number of nuclei, including those of the ventral tegmental area, central amygdaloid nucleus, and VO, made significantly greater inputs to somatostatin-positive cells than to PV(+) cells. Diverse inputs to the LDT on a system-wide level were revealed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-019-00397-2) contains supplementary material, which is available to authorized users.

The globus pallidus (GP) is emerging as a critical locus of basal ganglia control of motor activity, but the exact role of GABAergic GP neurons remain to be defined. By targeted expression of channelrhodopsin 2 (ChR2) in GABAergic neurons using the VGAT-ChR2-EYFP transgenic mice, we showed that optogenetic stimulation of GABAergic neurons in the right GP produced hyperkinesia. Optogenetic stimulation of GABAergic GP neurons increased c-Fos-positive cells in GP, M1 cortex, and caudate-putamen (CPu), and decreased c-Fos-positive cells in entopeduncular nucleus (EPN), compared to the contralateral hemisphere. In agreement with the canonical basal ganglia model. Furthermore, we delivered AAV-CaMKIIα-ChR2-mCherry virus to the excitatory neurons of the subthalamic nucleus (STN) and selectively stimulated glutamatergic afferent fibers from the STN onto the GP. This optogenetic stimulation produced abnormal movements, similar to the behaviors that observed in the VGAT-ChR2-EYFP transgenic mice. Meanwhile, we found that the c-Fos expression pattern in the GP, M1, STN, EPN, and CPu produced by optogenetic activation of glutamatergic afferent fibers from the STN in GP was similar to the c-Fos expression pattern in the VGAT-ChR2-EYFP transgenic mice. Taken together, our results suggest that excess GP GABAergic neurons activity could be the neural substrate of abnormal involuntary movements in hyperkinetic movement disorders. The neural circuitry underlying the abnormal involuntary movements is associated with excessive GP, M1, CPu activity, and reduced EPN activity. Inhibition of GP GABAergic neurons represents new treatment targets for hyperkinetic movement disorder.

Here we described an experimental protocol for in vivo imaging of macropinocytosis and subsequent intracellular events. By microinjection, we delivered fluorescence dextrans together with or without ATPγS into transparent Drosophila melanogaster embryos. Using a confocal microscope for live imaging, we monitored the generation of dextran-positive macropinosomes and subsequent intracellular events. Our protocol provides a continent and reliable way for investigating macropinocytosis and its underlying mechanisms, especially when combined with genetic strategies.

Endocytosis is a basic cellular process that describes a form of active transport across the plasma membrane into the cell. The endocytic pathway consists of distinct membrane compartments; internalized molecules are delivered to early endosomes, and some of them are recycled back to the surface, whereas other molecules are sent to late endosomes and lysosomes for degradation. However, little is known about how mitochondria are involved in the endocytic pathway. Here, we report that FM dyes, membrane-impermeant fluorescent lipid probes, can traffic to mitochondria directly from the plasma membrane by clathrin-mediated endocytosis. FM dye entry into mitochondria uses microtubule-dependent active transport, but the mechanism is different from the classical endocytic pathway. Hence, this study reveals a previously unrealized lipid trafficking pathway from the plasma membrane to mitochondria.

Innate fear has a critical role in survival of animals. Unlike conditioned fear, the neuronal circuitry underlying innate fear is largely unknown. We found that the laterodorsal tegmentum (LDT) and lateral habenula (LHb) are specifically activated by the mouse predator odorant trimethylthiazoline (TMT). Using optogenetics to selectively stimulate GABAergic neurons in the LDT immediately produced fear-like responses (freezing, accelerated heart rate and increased serum corticosterone), whereas prolonged stimulation caused anxiety-like behaviors. Notably, although selective stimulation of parvalbumin (PV)-positive interneurons similarly induced fear-like responses, stimulation of somatostatin-positive interneurons or inhibition of PV neurons in the LDT suppressed TMT-induced fear-like responses without affecting conditioned fear. Finally, activation of LHb glutamatergic inputs to LDT interneurons was sufficient to generate fear-like responses. Thus, the LHb-LDT pathway is important for regulating olfactory cue-induced innate fear. Our results provide a potential target for therapeutic intervention for anxiety disorder.

Summary Presynaptic endosomes reportedly participate in synaptic vesicle (SV) recycling. However, it remains unclear whether they differentially regulate SV biogenesis and synaptic transmission in different types of synapses and how they are implicated in diseases. Using cryo-electron tomography and endocytic tracing, we uncover different endocytic modes and dynamics associated with distinct SV morphology between glutamatergic and GABAergic synapses. We further find that cathepsin D (CatD), a lysosomal storage disease (LSD) protein, is selectively located in GABAergic presynaptic endosomes. Inactivation of CatD results in enlarged presynaptic endosomes, reduces the readily releasable pool, and impairs synaptic transmission in GABAergic, but not glutamatergic, synapses. Moreover, CatD-deficient mice exhibit hyperactivity and increased sensitivity to seizure, mimicking epileptic behavior in CatD-related LSD patients. These data reveal an important role for presynaptic endosomal CatD in regulating GABAergic SV biogenesis and provide mechanistic insights for understanding the synaptic pathology and behavioral defects in CatD-associated LSD.

In addition to cortical areas, the thalamus also displays plasticity during a critical period in early life. Since most sensory information is transmitted to the cortex via the thalamus, it will be of significant interest to understand the precise time window and underlying mechanisms of this critical period in the thalamus. By using in vitro whole-cell patch recording in acute brain slices, we found that VPm relay synapses were only sensitive to whisker deprivation from postnatal day 11 (P11) to P14. Whisker deprivation initiated within the P11 to P14 window significantly reduced the amplitude of AMPAR-EPSCs, but not NMDAR-EPSCs when recorded 24 hours after whisker removal. From P10 to P11, the timing for entry into the critical period and the kinetics underlying NMDAR-EPSCs function were significantly altered. At P11, NMDAR-EPSCs were less sensitive to ifenprodil, a selective blocker of NR2B-containing NMDAR, and the protein level of NR2A was significantly increased compared to those at P10. At the end of the critical period there were no obvious changes in synaptic properties when compared between P14 and P15. Using calcium imaging, we found that fewer P15 VPm neurons could be excited by the GABAa receptor agonist, muscimol, when compared to P14 VPm neurons; this correlated to an increase in KCC2 expression. Our studies revealed a precise critical period of sensory experience-dependent plasticity in the thalamus featuring distinct molecular mechanisms which occur at the start and end of this critical window.

Multipolar-to-bipolar transition (MBT) is crucial for the neuronal migration and positioning in the neocortex. Reelin-Disabled-1 (Dab1) signaling plays a pivotal role in neuronal migration, yet how Dab1 coordinatively regulates downstream molecules to affect MBT remains unclear. We have previously found that alternative splicing produces multiple Dab1 isoforms with different tyrosine motifs and differential ability to recruit downstream effectors. Here, we report that splicing of Dab1 exons 7 and 8 and 9bc dynamically regulates the inclusion and activities of Dab1 tyrosine motifs in the neocortex. By in utero electroporation, we show that expression of Dab1 isoforms missing exons 7 and 8 or retaining exons 9bc in WT neurons resulted in neuronal migration defects with attenuated Dab1 tyrosine phosphorylation, disrupted leading process extension, and disorientated multipolar neurons in the multipolar accumulation zone. Introducing the canonical Dab1 form, but not those missing exons 7 and 8 or retaining exons 9bc, into Dab1-deficient neurons promoted MBT and rescued neuronal migration defects, suggesting that alternative splicing of Dab1 modulates the tyrosine motif switch and mediates MBT of cortical neurons. Our study reveals a critical mechanism by which Dab1 alternative splicing coordinately controls MBT and neuronal migration in a spatiotemporal manner.

Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum–Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body–exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.

Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear. Using inositol 1,4,5-trisphosphate receptor type 2 knockout (Itpr2−/−) mice to specifically disturb somatic Ca2+ signaling in astrocytes, we showed that developmental elimination of the ventral posteromedial nucleus relay synapse was impaired. Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2−/− mice. Further studies showed that developmental synapse elimination was also impaired in P2ry1−/− mice and was not rescued by ATP, indicating a possible role of purinergic signaling. This hypothesis was confirmed by MRS-2365, a selective P2Y1 agonist, could also rescue the deficient of synapse elimination in Itpr2−/− mice. Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination. DOI: http://dx.doi.org/10.7554/eLife.15043.001, eLife digest Neighbouring neurons connect to each other and share information through structures known as synapses. As the brain develops, many synapses turn out to be redundant. Just like trees in a garden that need to be trimmed, these redundant synapses must be pruned in order to form the right pattern of connections between different neurons. Brain cells called astrocytes play a key role in synaptic pruning, but it is unclear exactly how astrocytes coordinate this process. One important way in which astrocytes communicate with neurons is through a process called calcium signaling, in which the movement of calcium ions into or out of the cell sets off a cascade of activity inside the astrocytes. Yang et al. have now studied developing mice that lacked a gene that is essential for calcium signaling in astrocytes. Two weeks after they were born, these mice still had redundant synapses that are normally lost after birth. However, injecting the developing brain with a substance called ATP prevented this defect and allowed synapses to be correctly pruned. This is likely to be because astrocytes also use ATP to communicate with neurons, and ATP compensated for the missing calcium signaling. The experiments also uncovered the specific structure – called the P2Y1 receptor – on the outer surface of a neuron that ATP latches on to in order to help remove synapses. Further work is now needed to reveal how activating the P2Y1 receptor coordinates synaptic removal. DOI: http://dx.doi.org/10.7554/eLife.15043.002

Corrigendum: Laterodorsal tegmentum interneuron subtypes oppositely regulate olfactory cue-induced innate fear