A functional division of Drosophila sweet taste neurons that is value-based and task-specific

Sucrose is an attractive feeding substance and a positive reinforcer for Drosophila. But Drosophila females have been shown to robustly reject a sucrose-containing option for egg-laying when given a choice between a plain and a sucrose-containing option in specific contexts. How the sweet taste system of Drosophila promotes context-dependent devaluation of an egg-laying option that contains sucrose, an otherwise highly appetitive tastant, is unknown. Here, we report that devaluation of sweetness/sucrose for egg-laying is executed by a sensory pathway recruited specifically by the sweet neurons on the legs of Drosophila. First, silencing just the leg sweet neurons caused acceptance of the sucrose option in a sucrose versus plain decision, whereas expressing the channelrhodopsin CsChrimson in them caused rejection of a plain option that was “baited” with light over another that was not. Analogous bidirectional manipulations of other sweet neurons did not produce these effects. Second, circuit tracing revealed that the leg sweet neurons receive different presynaptic neuromodulations compared to some other sweet neurons and were the only ones with postsynaptic partners that projected prominently to the superior lateral protocerebrum (SLP) in the brain. Third, silencing one specific SLP-projecting postsynaptic partner of the leg sweet neurons reduced sucrose rejection, whereas expressing CsChrimson in it promoted rejection of a light-baited option during egg-laying. These results uncover that the Drosophila sweet taste system exhibits a functional division that is value-based and task-specific, challenging the conventional view that the system adheres to a simple labeled-line coding scheme.

The taste systems of many animal species are known to possess a dedicated “channel” for detecting sugars, a class of chemicals that is highly nutritious. For example, mice have been shown to encode gustatory receptors that specifically sense sugars, and the taste neurons that express these sugar receptors on their tongues generally do not express receptors that sense chemicals of another taste modality (e.g., bitterness) (1–3). Furthermore, activation of these sugar-sensing taste neurons by artificial means has been shown to be able to drive appetitive sugar-induced innate responses (e.g., licking) and act as a positive reinforcer for learning (3–5). In some recent studies, these properties of the sweet taste neurons have been found to be present in some of their central nervous system (CNS) targets (e.g., taste-sensitive neurons in the insular cortex), too (6, 7). Thus, one school of thought is that taste coding for sweetness in mice may follow the simple “labeled-line” rule: sweet taste neurons, and potentially some of their central targets, are hardwired to detect sugars specifically and drive sugar-induced reinforcing neural signals and appetitive behaviors (1–7).Drosophila melanogaster also possess sugar-detecting taste neurons. Pioneering early studies have shown that sugar-sensing taste neurons in flies are molecularly, anatomically, and functionally distinct from taste neurons that sense bitterness; sweet-sensing and bitter-sensing taste neurons express different gustatory receptors, project axons to different areas in the brain, and are required to promote different (appetitive versus aversive) behaviors (8–12). Moreover, the activation of sweet neurons by artificial means can drive appetitive behaviors and act as a positive reinforcer for learning (10, 13, 14), while artificial activation of bitter-sensing neurons can induce rejection behaviors and be used as a punishment for learning (10, 13, 15). Interestingly, while these results suggest that Drosophila sweet neurons and their mammalian counterparts have some shared properties, subsequent studies suggest that significant differences exist between them, too. First, the Drosophila genome appears to encode many more sweet receptors than mouse genome does (12, 16–23). Second, Drosophila sweet neurons appear to be able to detect some chemicals that belong to another taste modality [e.g., acetic acid (AA)] (24–27). Third, Drosophila sweet neurons can be found on several body parts (e.g., proboscis and legs) (8, 12, 18, 20, 23, 28–30). Interestingly, sweet neurons on different body parts of Drosophila do not promote identical behavioral outputs (8, 20, 23, 24, 28, 29). For example, labellar sweet neurons and esophageal sweet neurons on the proboscis have been shown to promote proboscis extension reflex (PER) and ingestion, respectively, whereas leg sweet neurons have been shown to promote PER and slowing down of locomotion (8, 12, 28, 29). Collectively, these results suggest that in contrast to the apparent homogeneity of sweet neurons in some mammals, a functional division exists among Drosophila sweet neurons, although the different behavioral responses promoted by different Drosophila sweet neurons generally appear appetitive in nature.In this work, we report yet another striking feature of Drosophila sweet neurons that sets them apart from their mammalian counterparts, namely a functional division that is value-based and task-specific. We discovered this by taking advantage of a context-dependent but highly robust sugar rejection behavior exhibited by egg-laying females (31–34). Previous studies have shown that when selecting for egg-laying site in a small enclosure (dimension ∼16 × 10 × 18 mm), Drosophila readily accept a sucrose-containing agarose for egg-laying when it is the sole option but strongly reject it when a plain option is also available (31, 32). Importantly, silencing their sweet neurons causes the females to no longer reject the sucrose option when choosing between the sucrose versus plain options (31, 32). Thus, in addition to promoting appetitive behaviors and acting as a positive reinforcer, activation of sweet neurons on an egg-laying option can also decrease the value of such an option (thereby causing its rejection over an option that does not activate sweet neurons). These observations not only suggest the existence of an apparent “antiappetitive” role of Drosophila sweet neurons when the task of animals is to select for egg-laying sites but also raise a key question as to whether such counterintuitive, value-decreasing property of sweetness detection during egg-laying may be 1) solely an emergent property of specific neurons in the brain that respond similarly to all peripheral sweet neurons but are sensitive to animals’ behavioral goal and context or 2) carried out by specific sweet neurons at the periphery and then transmitted into the brain via a unique neural pathway activated by these neurons. To disambiguate between these possibilities, we genetically targeted different subsets of sweet neurons to assess their circuit properties as well as their behavioral roles as the animals decided in either a regular or a virtual sweet versus plain decision during egg-laying, taking advantage of a high-throughput closed-loop optogenetic stimulation platform we developed recently. Our collective results support the second scenario and suggest that the value-decreasing property of sweetness/sucrose is conveyed specifically by the sweet neurons on the legs of Drosophila—and not by other sweet neurons—and the unique postsynaptic target(s) of the leg sweet neurons that send long-range projections to the superior lateral protocerebrum (SLP) in the brain. These results reveal a previously unappreciated functional and anatomical division of the Drosophila sweet taste neurons that is both task-specific and value-based, pointing to a level of complexity and sophistication that seems unmatched by their mammalian counterparts so far.     

Decidual NK cells kill Zika virus–infected trophoblasts

Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal–fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1^−/− pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection.

In the first trimester of human pregnancy, when Zika virus (ZIKV) infection is most damaging to the developing fetus (1–3), ∼70% of maternal immune cells at the maternal–fetal interface are decidual natural killer cells (dNK), which are in close contact with extravillous trophoblasts (EVT), placental cells of fetal origin which invade the decidua to orchestrate placentation. T lymphocytes and natural killer (NK) cells generally do not kill EVT, which do not express the main classical HLA molecules responsible for T cell activation (HLA-A and B) (4–7) but do express HLA-C and nonclassical HLA-E and -G, which help inhibit NK cell activation. HLA-G, whose expression is mostly restricted to the placenta, helps maintain dNK tolerance of fetal cells. There is important cross-talk between dNK and EVT—EVT regulate the maturation of dNK precursor cells into tolerant dNK (8) and dNK promote EVT migration and invasion of spiral arteries, processes essential for placentation (9–13). Invading fetal EVT are susceptible to infection by a variety of pathogens, including human cytomegalovirus (HCMV), Toxoplasma gondii, and ZIKV (1, 14–17). dNK in the placenta face a difficult problem—they must tolerate the semiforeign fetus but still protect against infection. Although dNK contain the machinery needed to recognize and kill, they have reduced cytolytic activity against classical NK target cells recognized by peripheral blood NK cells (pNK) and are not known to kill infected trophoblasts (18–23). However, dNK degranulate and secrete cytokines, including interferon (IFN)-γ, in response to HCMV-infected maternal decidual stromal cells in equal levels to pNK (19, 20). We recently demonstrated that dNK can eliminate intracellular Listeria infection in trophoblasts without degranulating or killing the host cells (24). Although ZIKV infects the placenta and fetus and can cause severe birth defects including microcephaly, brain atrophy, and subcortical and chorioretinal and optic nerve atrophy, little is known about the response of decidual immune cells to ZIKV and its effect on maternal–fetal transmission (25).     

Functional feedback from mushroom bodies to antennal lobes in the Drosophila olfactory pathway

Feedback plays important roles in sensory processing. Mushroom bodies are believed to be involved in olfactory learning/memory and multisensory integration in insects. Previous cobalt-labeling studies have suggested the existence of feedback from the mushroom bodies to the antennal lobes in the honey bee. In this study, the existence of functional feedback from Drosophila mushroom bodies to the antennal lobes was investigated through ectopic expression of the ATP receptor P2X₂ in the Kenyon cells of mushroom bodies. Activation of Kenyon cells induced depolarization in projection neurons and local interneurons in the antennal lobes in a nicotinic receptor-dependent manner. Activation of Kenyon cell axons in the βγ-lobes in the mushroom body induced more potent responses in the antennal lobe neurons than activation of Kenyon cell somata. Our results indicate that functional feedback from Kenyon cells to projection neurons and local interneurons is present in Drosophila and is likely mediated by the βγ-lobes. The presence of this functional feedback from the mushroom bodies to the antennal lobes suggests top-down modulation of olfactory information processing in Drosophila.In the fruit fly, the olfactory system is important for identifying food sources, avoiding predators, and recognizing mating partners (1). The primary Drosophila olfactory neurons are the olfactory receptor neurons (ORNs) present in the two olfactory organs of the fruit fly, the antennae and the maxillary palps (2, 3). Chemical stimuli detected by the olfactory receptors in the ORNs are converted into electrical signals, which are transmitted to the secondary neurons, the projection neurons (PNs), in the antennal lobes. The antennal lobes are important olfactory coding centers that consist of PNs as well as inhibitory and excitatory local interneurons (LNs) (4 –6). The PNs receive signals from the primary ORNs and also receive lateral inhibitory/excitatory inputs from the LNs (7 –10). After processing in the antennal lobes, olfactory information is relayed by PNs to the mushroom bodies and the protocerebrum region of the fly brain (11 –13).Feedback is important in sensory processing. For example, in the mammalian thalamocortical system, a large number of thalamus neurons are modulated by cortical feedback mechanisms (14). Such top-down cortical feedback regulation is critical for visual perception (15). Anatomical and functional studies of the mammalian olfactory system indicate that there is also functional feedback from the cortex to the olfactory bulb (16, 17). Several lines of evidence also suggest the existence of feedback in the insect olfactory pathway. Olsen and Wilson (8) showed that spontaneous excitatory postsynaptic activity in PNs is suppressed presynaptically via lateral inhibition by LNs in antennal lobes, suggesting modulation of the primary ORNs by the secondary LNs. In addition, Tanaka et al. (18) demonstrated that odor stimulation can induce spikes and subthreshold membrane potential oscillations in PNs that are phase-locked to odor-elicited local field-potential oscillations in mushroom bodies. These results indicate the existence of feedback within the antennal lobe of Drosophila. Finally, by injecting cobalt into the α-lobe of the mushroom bodies of the honey bee, Rybak and Menzel identified a projection from the α-lobe to the antennal lobe (19). A neuron (the antennal lobe feedback neurons, ALF-1) with similar projection pattern was reported by Kirschner et al. (20). The results of these studies suggest feedback from the mushroom body to the antennal lobe in the honey bee. Based on these observations, we investigated whether functional feedback from mushroom bodies to antennal lobes is present in Drosophila. Drosophila mushroom bodies are predominately composed of Kenyon cells (KCs) (21). Because of the small size of KCs, it is difficult to stimulate these cells using conventional electrophysiological methods. Taking advantage of fly genetics, and the absence of the ionotropic ATP receptor P2X₂ gene in the Drosophila genome (22), Zemelman et al. established a UAS-P2X₂ system for precise activation of specific neurons in the fly brain using exogenous ATP (23). In this study, we have used the UAS-P2X₂ system for activation of KCs in combination with the patch-clamp method for recording of PN and LN activity. Using these techniques, we showed in this study the existence of functional feedback from KCs in the mushroom bodies to PNs and LNs in the antennal lobes.     

From the Cover: A role for cortical nNOS/NK1 neurons in coupling homeostatic sleep drive to EEG slow wave activity

Although the neural circuitry underlying homeostatic sleep regulation is little understood, cortical neurons immunoreactive for neuronal nitric oxide synthase (nNOS) and the neurokinin-1 receptor (NK1) have been proposed to be involved in this physiological process. By systematically manipulating the durations of sleep deprivation and subsequent recovery sleep, we show that activation of cortical nNOS/NK1 neurons is directly related to non-rapid eye movement (NREM) sleep time, NREM bout duration, and EEG δ power during NREM sleep, an index of preexisting homeostatic sleep drive. Conversely, nNOS knockout mice show reduced NREM sleep time, shorter NREM bouts, and decreased power in the low δ range during NREM sleep, despite constitutively elevated sleep drive. Cortical NK1 neurons are still activated in response to sleep deprivation in these mice but, in the absence of nNOS, they are unable to up-regulate NREM δ power appropriately. These findings support the hypothesis that cortical nNOS/NK1 neurons translate homeostatic sleep drive into up-regulation of NREM δ power through an NO-dependent mechanism.The electrical activity of the cerebral cortex has been used to distinguish sleep vs. wakefulness since the earliest EEG studies of sleep (1). Several neural circuits have been implicated in the synchronization and desynchronization of cortical activity that distinguish non-rapid eye movement sleep (NREM) from wakefulness and rapid eye movement sleep (REM). Input from the basal forebrain (BF), likely from both cholinergic and noncholinergic neurons, is critical for the desynchronized EEG characteristic of wakefulness and REM (2, 3). Synchronization of the EEG during NREM depends on thalamic as well as intrinsic cortical oscillators (4).The firing rate of cortical neurons has generally been reported to be reduced during NREM relative to wakefulness and REM (5, 6). A few studies have reported cortical neurons with the opposite pattern. For example, 4 of 177 neurons in the monkey orbitofrontal cortex increased their firing rates during NREM (7). In the cat parietal cortex, 25% of neurons discharged in phase with NREM slow waves during up states but ceased firing during quiet wakefulness (8).Using Fos immunohistochemistry as a marker of cellular activity, we described a population of cortical GABAergic interneurons that are activated during sleep in three species (9, 10). These neurons express neuronal nitric oxide synthase (nNOS) and thus likely release nitric oxide (NO) as well as GABA (11). The percentage of activated cortical nNOS neurons was proportional to NREM δ energy (NRDE), the product of NREM time and NREM EEG δ power. Because NREM time and δ power increase in response to prolonged wakefulness through a regulated process referred to as sleep homeostasis, NRDE is an electrophysiological marker of homeostatic sleep “drive.” Consequently, activation of cortical nNOS neurons during sleep seems to be related to the sleep need that accrues during wakefulness.Cortical nNOS neurons receive cholinergic (12), monoaminergic (13), and peptidergic (14, 15) inputs. Sleep-active nNOS cells correspond to type I nNOS neurons, which are larger and less numerous than type II cells, express the neurokinin-1 receptor (NK1; 14, 15), and are likely projection neurons (16, 17). On the basis of these observations, we have proposed that cortical nNOS/NK1 neurons are fundamentally related to the homeostatic regulation of sleep and play a critical role in coordinating slow waves within the cortex, possibly through release of NO (18).To further test this hypothesis, we subjected rats to varying durations of sleep deprivation (SD) and recovery sleep opportunities (RS) and evaluated which sleep parameters were most closely correlated with cortical nNOS/NK1 neuron activation. We then asked whether those sleep parameters were altered in nNOS KO mice. Consistent with a role for cortical nNOS/NK1 neurons in sleep homeostasis, we find that these neurons are most active during consolidated NREM with increased δ power, that the absence of nNOS results in attenuation of NREM consolidation and NREM δ power, and that nNOS KO mice are unable to respond appropriately to challenges to the sleep homeostatic system.     

Electrical synapses connect a network of gonadotropin releasing hormone neurons in a cichlid fish

Initiating and regulating vertebrate reproduction requires pulsatile release of gonadotropin-releasing hormone (GnRH1) from the hypothalamus. Coordinated GnRH1 release, not simply elevated absolute levels, effects the release of pituitary gonadotropins that drive steroid production in the gonads. However, the mechanisms underlying synchronization of GnRH1 neurons are unknown. Control of synchronicity by gap junctions between GnRH1 neurons has been proposed but not previously found. We recorded simultaneously from pairs of transgenically labeled GnRH1 neurons in adult male Astatotilapia burtoni cichlid fish. We report that GnRH1 neurons are strongly and uniformly interconnected by electrical synapses that can drive spiking in connected cells and can be reversibly blocked by meclofenamic acid. Our results suggest that electrical synapses could promote coordinated spike firing in a cellular assemblage of GnRH1 neurons to produce the pulsatile output necessary for activation of the pituitary and reproduction.Development and function of the reproductive system in vertebrates depends on the timing and levels of signaling by gonadal sex steroids (1, 2). Production of these steroids is controlled by neurons expressing gonadotropin-releasing hormone (GnRH1), which comprise the final output of the brain to the hypothalamic-pituitary-gonadal axis. During vertebrate development, GnRH1 neurons originate outside the central nervous system in the olfactory placode and migrate into the basal forebrain (3–6). These neurons signal to the pituitary via the decapeptide GnRH1 to effect the release of the gonadotropins, follicle stimulating hormone and luteinizing hormone, which in turn stimulate steroid production by the gonads. It has long been known that this release depends on coordinated, pulsatile GnRH1 release, not simply elevated levels (7, 8), requiring some level of synchronization in the output of these neurons. Episodic activation of the pituitary gonadotropes has been observed in multiple vertebrate taxa, including mammals and fish (9–12), however, mechanisms that underlie this required coordinated activity of GnRH1 neurons are unknown. Synchrony could in principle derive from coincident input from a “pacemaker” neural population, from direct coupling of GnRH1 neurons, or from a combination of mechanisms. Gap junction-mediated coupling has been suspected to play a role, as synchronous firing can be observed in neurons mechanically isolated from brain slices and in cultures of embryonic mouse and primate neurons, and immortalized mouse GnRH1 neurons express the connexin proteins that constitute gap junctions (13–15). However, no evidence for gap junctions among adult GnRH1 cells in vivo has been found (16, 17).To search for the origin of synchrony among these neurons, we used a unique model system for analysis of GnRH1 neurons, Astatotilapia burtoni, a cichlid fish. GnRH1 neurons in males of this species exhibit dynamic morphological plasticity caused by changes in their social status (18–21). Here we use transgenic dominant male A. burtoni to perform paired recordings from GnRH1 neurons, and report that they are reciprocally connected by electrical synapses. These findings suggest that gap junctions contribute to the coordinated firing of these neurons necessary for reproductive function.     

Nanchung and Inactive define pore properties of the native auditory transduction channel in Drosophila

Auditory transduction is mediated by chordotonal (Cho) neurons in Drosophila larvae, but the molecular identity of the mechanotransduction (MET) channel is elusive. Here, we established a whole-cell recording system of Cho neurons and showed that two transient receptor potential vanilloid (TRPV) channels, Nanchung (NAN) and Inactive (IAV), are essential for MET currents in Cho neurons. NAN and IAV form active ion channels when expressed simultaneously in S2 cells. Point mutations in the pore region of NAN-IAV change the reversal potential of the MET currents. Particularly, residues 857 through 990 in the IAV carboxyl terminus regulate the kinetics of MET currents in Cho neurons. In addition, TRPN channel NompC contributes to the adaptation of auditory transduction currents independent of its ion-conduction function. These results indicate that NAN-IAV, rather than NompC, functions as essential pore-forming subunits of the native auditory transduction channel in Drosophila and provide insights into the gating mechanism of MET currents in Cho neurons.

Drosophila is endowed with specialized auditory receptor cells in which sound waves are transduced into electrical signals. However, the key molecules/ion channels mediating this mechanotransduction (MET) process remain obscure. In adult flies, Johnston’s organ neurons (JONs) detect sound and gravity (1–5). At the tip of the dendrite, each JON bears a cilium, in which the auditory transduction takes place. Two transient receptor potential (TRP) channels, Nanchung (NAN) and Inactive (IAV), are both localized to the proximal cilia (6), while NompC has a restricted distribution in the distal cilia (7). All three molecules are the potential candidates mediating the MET currents in JONs.NAN and IAV are required for sound sensation in JONs (6, 8, 9). Furthermore, NAN and IAV are expressed in auditory receptor neurons and likely function as heteromers (6, 8). In nan or iav null mutants, the compound action potential induced by sound is abolished (6, 8). In addition, NompC is a mechanosensitive channel responsible for touch sensation in Drosophila (7). Importantly, the antennal sound receivers in nompC null allele mutant show no nonlinear amplification to pure tone stimuli. Moreover, self-sustained oscillations of antennal sound receivers are abolished in nompC null mutant (10), but it is still unclear whether NAN, IAV, and NompC amplify subthreshold MET current or mediate the subthreshold MET current themselves in auditory receptor neurons (1, 10–13). Therefore, it is necessary to study the following: first, whether NAN, IAV, and NompC are essential for the primary MET current evoked by sound; furthermore, which of them are the pore-forming subunits of the native auditory transduction channel; and finally, the gating mechanism of MET currents.Patch clamp recordings of JONs are not feasible, because these neurons are very small and their cell bodies are embedded in a delicate antenna whose integrity is crucial for their function (13). Thus, we established patch clamp recordings of chordotonal (Cho) neurons of Drosophila larvae and showed that NAN and IAV are essential for the MET current in Cho neurons. We further found that NAN and IAV form active ion channels when coexpressed in heterologous cells. Mutations in the pore lining residues of NAN and IAV altered the permeation properties of native MET current in Cho neurons. We also identified residues 857 through 990 in the IAV carboxyl terminus that regulate the adaptation time constant of MET currents. Additionally, nompC null mutants showed a decreased adaptation time constant of the MET current, with the current amplitude comparable to that in wild-type. Moreover, a nonconducting point mutation of NompC did not affect MET currents. These findings provide convincing evidence that NAN-IAV channels form the pore of the native auditory transduction channel in Drosophila and reveal functional domains in NAN and IAV for channel gating. Intriguingly, NompC regulates auditory transduction currents independent of its ion-conducting function, which is consistent with the idea that NompC couples forces to the transduction channels by acting as the amplifier.     

miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

It is well known that the nonmalignant stromal components in tumors play pivotal roles in tumor progression and therapeutic responses (1, 2). Macrophages are a major component of tumor microenvironment and display considerable phenotypic plasticity in their effects toward tumor progression (3–5). Classically activated (M1) macrophages often exert direct tumor cytotoxic effects or induce antitumor immune responses by helping present tumor-related antigens (6, 7). In contrast, tumoral cues can polarize macrophages toward alternative activation with immunosuppressive M2 properties (6–8). Numerous studies have firmly established the protumor effects of M2-like tumor-associated macrophages (TAMs) and the association of TAMs with poor prognosis of human cancer (9–11). However, how tumors induce the coordinated molecular and phenotypic changes in TAMs for M2 polarization remains incompletely understood, impeding the designing of TAM-targeting strategies for cancer intervention. In addition, drug delivery also represents a hurdle for therapeutic macrophage reprogramming.Noncoding RNAs, including microRNAs, have been shown to play vital roles in various pathological processes of cancer (12). The microRNA miR-182 has been implicated in various developmental processes and disease conditions (13–15). Particularly, it receives extensive attention in cancer studies. Prevalent chromosomal amplification of miR-182 locus and up-regulation of its expression in tumors have been observed in numerous cancer types including breast cancer, gastric cancer, lung adenocarcinoma, colorectal adenocarcinoma, ovarian carcinoma, and melanoma (16–21). miR-182 expression is also linked to higher risk of metastasis and shorter survival of patients (20, 22–24). Functional studies showed that miR-182 expression in cancer cells plays vital roles in various aspects of cancer malignancy, including tumor proliferation (25–29), migration (30, 31), invasion (16, 32, 33), epithelial-mesenchymal transition (34–36), metastasis (21, 37, 38), stemness (30, 39, 40), and therapy resistance (41, 42). A number of target genes, including FOXO1/3 (18, 21, 43–45), CYLD (46), CADM1 (47), BRCA1 (27, 48), MTSS1 (34), PDK4 (49), and SMAD7 (35), were reported to be suppressed by miR-182 in cancer cells. Our previous work also proved that tumoral miR-182 regulates lipogenesis in lung adenocarcinoma and promotes metastasis of breast cancer (34, 35, 49). Although miR-182 was established as an important regulator of cancer cell malignancy, previous studies were limited, with analyses of miR-182 in cultured cancer cells and transplanted tumors. Thus, the consequences of miR-182 regulation in physiologically relevant tumor models, such as genetically modified mice, have not been shown. More importantly, whether miR-182 also plays a role in tumor microenvironmental cell components is unknown.In this study, we show that miR-182 expression in macrophages can be induced by breast cancer cells and regulates TAM polarization in various tumor models of mice. In addition, miR-182 inhibition with TAM-targeting exosomes demonstrates promising efficacy for cancer treatment.     

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56^bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α₄β₁ integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4⁺ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4⁺ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4⁺ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4⁺ T cells and the cytolytic activity of NK cells.Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system (CNS) (1) and one of the major causes of neurological disability in young adults (2). MS is considered to be a primarily antigen-driven T cell-mediated disease with a complex genetic background influenced by environmental factors (1, 3) that is caused by an imbalanced immune-regulatory network (4). Among other well-known players of this network such as regulatory T cells and tolerogenic dendritic cells (DCs) (1), natural killer (NK) cells have been recently identified as additional factors in controlling homeostasis of antigen-activated T cells (5, 6).Originally discovered as antigen receptor-negative innate lymphocytes that play an important role in controlling virus-infected and tumor cells (7), NK cells have also been shown to suppress activated T cells through secretion of anti-inflammatory cytokines and/or cytolytic function (5, 6, 8–12). NK cells lyse target cells in a complex process depending on cell surface expression of certain inhibitory and activating receptors on NK cells and the corresponding ligands on target cells (13). Several activating NK-cell receptors–in particular, NKG2D (CD314) (5, 8, 9, 11, 14), the receptor for MIC-A/B and ULBP1-6, and DNAM-1 (DNAX accessory molecule, CD226) (6, 12, 15), the receptor for Nectin-2 (CD112) and poliovirus receptor (PVR/CD155)−have been proposed to be involved in NK cell-mediated lysis of activated T cells. Of note, polymorphisms in the gene encoding for DNAM-1 have been consistently found in MS-association studies (16–18). Both major NK-cell subsets, namely the CD56^brightCD16^dim/− and the CD56^dimCD16⁺ subsets (here referred to as CD56^bright and CD56^dim, respectively), seem to be capable of killing activated T cells (19). CD56^dim NK cells are the major NK-cell subset in the peripheral blood (PB) (90% of NK cells) and kill target cells without prior sensitization but only secrete low levels of cytokines (7, 20, 21), whereas CD56^bright NK cells are more abundant in secondary lymphoid tissues and inflammatory lesions (75–95% of NK cells), where they produce high amounts of immune-modulating cytokines but acquire cytolytic functions only after prolonged activation (7, 20, 21).Immune-modulating therapies targeting NK-cell frequencies and cytolytic functions among others such as IFN-β (22–24), glatiramer acetate (25), natalizumab (26, 27), fingolimod (28, 29), and daclizumab (10, 30, 31) point to an immune-protective role of both NK-cell subsets in MS. Daclizumab, a humanized antibody directed against the IL-2 receptor (IL-2R) α-chain (CD25) (reviewed in ref. 4) is a promising MS therapy, which recently showed superior efficacy compared with IFN-β in a phase III study (32). Expansion of peripheral (10, 33) as well as intrathecal (34) CD56^bright NK cells under daclizumab treatment correlated positively with therapeutic response (10, 30, 35). Nevertheless, it still remains to be elucidated whether NK-cell immune-regulatory functions are impaired as part of the disease process and whether modulation of the IL-2R with daclizumab restores these deficits or simply boosts NK-cell activity (4). Furthermore, the distribution and function of NK cells in active MS lesions is still poorly understood. Resolving the molecular basis of NK cell-mediated immune control and its potential impairment in MS is important for a better understanding of the role of NK cells in MS pathogenesis and the mechanism of action of NK cell-modulating therapies.The aim of the current study was to characterize the role of NK cells in the pathogenesis of MS by investigating the presence, distribution, and function of NK cells in three different compartments [CNS, cerebrospinal fluid (CSF), and PB]. Furthermore, a potential deficit in NK-cell immune-regulatory function, its underlying molecular mechanism, and the impact of IL-2R modulation by daclizumab high-yield process (DAC HYP) were explored by studying PB mononuclear cells (PBMCs) derived from clinically stable therapy-naïve MS patients and MS patients receiving daclizumab treatment in comparison with those derived from healthy individuals.     

Single-neuron firing cascades underlie global spontaneous brain events

The resting brain consumes enormous energy and shows highly organized spontaneous activity. To investigate how this activity is manifest among single neurons, we analyzed spiking discharges of ∼10,000 isolated cells recorded from multiple cortical and subcortical regions of the mouse brain during immobile rest. We found that firing of a significant proportion (∼70%) of neurons conformed to a ubiquitous, temporally sequenced cascade of spiking that was synchronized with global events and elapsed over timescales of 5 to 10 s. Across the brain, two intermixed populations of neurons supported orthogonal cascades. The relative phases of these cascades determined, at each moment, the response magnitude evoked by an external visual stimulus. Furthermore, the spiking of individual neurons embedded in these cascades was time locked to physiological indicators of arousal, including local field potential power, pupil diameter, and hippocampal ripples. These findings demonstrate that the large-scale coordination of low-frequency spontaneous activity, which is commonly observed in brain imaging and linked to arousal, sensory processing, and memory, is underpinned by sequential, large-scale temporal cascades of neuronal spiking across the brain.

The brain at rest exhibits slow (<0.1 Hz) but highly organized spontaneous activity as measured by functional MRI (fMRI) (1, 2). Much research in this area has utilized the temporal coordination of these signals to assess the functional organization a large number of brain networks. In recent years, however, new attention has been directed to a less-studied aspect of this signal, namely the conspicuous and discrete spontaneous events that occur simultaneously across the brain (3–5). These global resting-state fMRI events appear to reflect transient arousal modulations at a timescale of ∼10 s (4, 6) and also to be closely related to activity among clusters of cholinergic projection neurons in the basal forebrain (4, 5).The nature of global brain events is of great interest, as is their spatiotemporal dynamics. Some evidence suggests they take the form of traveling waves, propagating coherently according to the principles of the cortical hierarchy (7, 8), and shaping functional connectivity measures important for assessing the healthy and diseased brain (7, 9, 10). Other work has linked such global activity to phenomena as varied as modulation of the autonomic nervous system (11–14), cleansing circulation of cerebrospinal fluid in the glymphatic system (15–19), and memory consolidation mediated by hippocampal sharp-wave ripples (20, 21). In general, the global activity measured through brain imaging appears coordinated over timescales of seconds with a range of other neural and physiological events (11, 12, 14, 21–23). In a few cases, the relationship between local and global neural events has been studied using simultaneous measurements. For example, brain-wide fMRI fluctuations and local field potential (LFP) power changes are locked to the issuance of hippocampal ripples (21, 24). However, very little is understood about the extent to which single neurons participate in the expression and coordination of global spontaneous events of the seconds timescale. To approach this topic, recent technological advances have made it possible to track and compare the spiking activity of a large number of isolated neurons recorded simultaneously across multiple brain areas.A few recent studies utilizing high-density neuronal recording (25) have accumulated initial evidence suggesting a close relationship between the brain state and neuronal population dynamics (26–28). A large proportion of neurons, regardless of their location, showed strong modulations in their discharging rate that were coordinated in time with physiological arousal measures (28), across thirsty and sated states (26), and during exploratory and nonexploratory behaviors (27). Nevertheless, these studies leave open the question of how neuronal population dynamics are organized at a finer timescale of seconds surrounding spontaneous global events during immobile rest, and whether and how such dynamics are coincident with arousal modulations, hippocampal ripples, and sensory excitability. To investigate this topic, we examine the spiking activity recorded from large neuronal populations of neurons in immobilized mice, focusing on their seconds-scale coordination with global events and with one another. We further studied the impact of this spontaneous spiking on the magnitude of visually evoked responses and its time locking with other physiological signals related to arousal, such as LFP changes, hippocampal ripples, and changes in pupil diameter.     

An image-computable model of how endogenous and exogenous attention differentially alter visual perception

Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: 1) the parafoveal advantage in segmentation, 2) the uniform improvements across eccentricity by endogenous attention, and 3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field.

Endogenous and exogenous spatial attention prioritize subsets of visual information and facilitate their processing without concurrent eye movements (1–3). Selection by endogenous attention is goal-driven and adapts to task demands, whereas exogenous attention transiently and automatically orients to salient stimuli (1–3). In most visual tasks, both types of attention typically improve visual perception similarly [e.g., acuity (4–6), visual search (7, 8), perceived contrast (9–11)]. Consequently, models of visual attention do not distinguish between endogenous and exogenous attention (e.g., refs. 12–19). However, stark differences also exist. Each attention type differentially modulates neural responses (20, 21) and fundamental properties of visual processing, including temporal resolution (22, 23), texture sensitivity (24), sensory tuning (25), contrast sensitivity (26), and spatial resolution (27–34).The effects of endogenous and exogenous attention are dissociable during texture segmentation, a visual task constrained by spatial resolution [reviews (1–3)]. Whereas endogenous attention optimizes spatial resolution to improve the detection of an attended texture (32–34), exogenous attention reflexively enhances resolution even when detrimental to perception (27–31, 34). Extant models of attention do not explain these well-established effects.Two main hypotheses have been proposed to explain how attention alters spatial resolution. Psychophysical studies ascribe attentional effects to modulations of spatial frequency (SF) sensitivity (30, 33). Neurophysiological (13, 35, 36) and neuroimaging (37, 38) studies bolster the idea that attention modifies spatial profiles of neural receptive fields (RFs) (2). Both hypotheses provide qualitative predictions of attentional effects but do not specify their underlying neural computations.Differences between endogenous and exogenous attention are well established in segmentation tasks and thus provide an ideal model system to uncover their separate roles in altering perception. Texture-based segmentation is a fundamental process of midlevel vision that isolates regions of local structure to extract figures from their background (39–41). Successful segmentation hinges on the overlap between the visual system’s spatial resolution and the levels of detail (i.e., SF) encompassed by the texture (39, 41, 42). Consequently, the ability to distinguish between adjacent textures varies as resolution declines toward the periphery (43–46). Each attention type differentially alters texture segmentation, demonstrating that their effects shape spatial resolution [reviews (1–3)].Current models of texture segmentation do not explain performance across eccentricity and the distinct modulations by attention. Conventional models treat segmentation as a feedforward process that encodes the elementary features of an image (e.g., SF and orientation), transforms them to reflect the local structure (e.g., regions of similarly oriented bars), and then pools across space to emphasize texture-defined contours (39, 41, 47). Few of these models account for variations in resolution across eccentricity (46, 48, 49) or endogenous (but not exogenous) attentional modulations (18, 50). All others postulate that segmentation is a “preattentive” (42) operation whose underlying neural processing is impervious to attention (39, 41, 46–49).Here, we develop a computational model in which feedforward processing and attentional gain contribute to segmentation performance. We augment a conventional model of texture processing (39, 41, 47). Our model varies with eccentricity and includes contextual modulation within local regions in the stimulus via normalization (51), a canonical neural computation (52). The defining characteristic of normalization is that an individual neuron is (divisively) suppressed by the summed activity of neighboring neurons responsive to different aspects of a stimulus. We model attention as multiplicative gains [attentional gain factors (15)] that vary with eccentricity and SF. Attention shifts sensitivity toward fine or coarse spatial scales depending on the range of SFs enhanced.Our model is image-computable, which allowed us to reproduce behavior directly from grayscale images used in psychophysical experiments (6, 26, 27, 29–33). The model explains three signatures of texture segmentation hitherto unexplained within a single computational framework (Fig. 1): 1) the central performance drop (CPD) (27–34, 43–46) (Fig. 1A), that is, the parafoveal advantage of segmentation over the fovea; 2) the improvements in the periphery and impairments at foveal locations induced by exogenous attention (27–32, 34) (Fig. 1B); and 3) the equivalent improvements across eccentricity by endogenous attention (32–34) (Fig. 1C).Open in a separate windowFig. 1.Signatures of texture segmentation. (A) CPD. Shaded region depicts the magnitude of the CPD. Identical axis labels are omitted in B and C. (B) Exogenous attention modulation. Exogenous attention improves segmentation performance in the periphery and impairs it near the fovea. (C) Endogenous attention modulation. Endogenous attention improves segmentation performance across eccentricity.Whereas our analyses focused on texture segmentation, our model is general and can be applied to other visual phenomena. We show that the model predicts the effects of attention on contrast sensitivity and acuity, i.e., in tasks in which both endogenous and exogenous attention have similar or differential effects on performance. To preview our results, model comparisons revealed that normalization is necessary to elicit the CPD and that separate profiles of gain enhancement across SF (26) generate the effects of exogenous and endogenous attention on texture segmentation. A preferential high-SF enhancement reproduces the impairments by exogenous attention due to a shift in visual sensitivity toward details too fine to distinguish the target at foveal locations. The transition from impairments to improvements in the periphery results from exogenous attentional gain gradually shifting to lower SFs that are more amenable for target detection. Improvements by endogenous attention result from a uniform enhancement of SFs that encompass the target, optimizing visual sensitivity for the attended stimulus across eccentricity.