text:
Roles of IFN-γ and γδ T Cells in Protective Immunity Against Blood-Stage Malaria

Malaria is caused by infection with Plasmodium parasites. Various studies with knockout mice have indicated that IFN-γ plays essential roles in protective immunity against blood-stage Plasmodium infection. However, after Plasmodium infection, increased IFN-γ production by various types of cells is involved not only in protective immunity, but also in immunopathology. Recent reports have shown that IFN-γ acts as a pro-inflammatory cytokine to induce not only the activation of macrophages, but also the generation of uncommon myelolymphoid progenitor cells after Plasmodium infection. However, the effects of IFN-γ on hematopoietic stem cells and progenitor cells are unclear. Therefore, the regulation of hematopoiesis by IFN-γ during Plasmodium infection remains to be clarified. Although there are conflicting reports concerning the significance of γδ T cells in protective immunity against Plasmodium infection, γδ T cells may respond to infection and produce IFN-γ as innate immune cells in the early phase of blood-stage malaria. Our recent studies have shown that γδ T cells express CD40 ligand and produce IFN-γ after Plasmodium infection, resulting in the enhancement of dendritic cell activation as part of the immune response to eliminate Plasmodium parasites. These data suggest that the function of γδ T cells is similar to that of NK cells. Although several reports suggest that γδ T cells have the potential to act as memory cells for various infections, it remains to be determined whether memory γδ T cells are generated by Plasmodium infection and whether memory γδ T cells can contribute to the host defense against re-infection with Plasmodium. Here, we summarize and discuss the effects of IFN-γ and the various functions of γδ T cells in blood-stage Plasmodium infection.

Malaria is one of the most serious public health problems worldwide, and the malaria-endemic areas include both tropical and subtropical regions. Plasmodium, the malaria-causing protozoan parasite, has a complex life cycle. Mammalian hosts, such as humans, monkeys, and mice, are inoculated with sporozoites into the host dermis of skin via a bite by a Plasmodium-infected Anopheles mosquito (1). Sporozoites then enter blood stream for invasion into hepatocytes and develop into merozoites with replication (liver-stage). Merozoites parasitize red blood cells (RBCs), replicate, and then rupture the RBCs, allowing for new infection of additional normal RBCs (blood-stage). In contrast to asymptomatic liver-stage malaria, blood-stage malaria is the symptomatic phase. Thus, protective immunity against blood-stage malaria is important for reducing the severity of disease (Figure 1).
Infection with Plasmodium falciparum causes the most severe malaria in humans. The predominant symptoms are anemia, splenomegaly, and fever. Cerebral malaria, liver dysfunction, acute renal failure, acidosis, hypoglycemia, respiratory distress, and edema are also observed as complications in malaria patients, although these symptoms do not always appear. Certain Plasmodium species can specifically infect rodents and cause malaria. A rodent malaria model is very useful not only for the development of anti-malarial drugs and vaccines, but also in research studies into the protective and pathologic immune responses during malaria. The lethality of the Plasmodium infection is dependent upon combinations of Plasmodium species and inbred mouse strains. Here, we review experimental studies on Plasmodium infections in the mouse and human studies with P. falciparum, focusing on protective immunity against Plasmodium infections.
γδ T cells work as innate lymphocytes, the first line of defense against infectious pathogens. On the other hand, αβ T cells, such as CD4 + helper T cells and CD8 + killer T cells, are typically FIGURE 1 | Life cycle of Plasmodium parasites. A bite from a Plasmodium-infected Anopheles mosquito inoculates human hosts with sporozoites in the dermis of skin. The sporozoites move into the bloodstream, and parasitize hepatocytes. Invaded sporozoites develop into merozoites with replication (liver-stage). Then, merozoites are released from hepatocytes.
Merozoites parasitize red blood cells (RBCs), replicate, and then rupture the RBCs, leading to new infection of normal RBCs (nRBCs; blood-stage). Merozoites have several developmental phases: the ring form (early trophozoite), trophozoite, and schizont. Furthermore, some merozoites mature into female and male gametocytes. related to adaptive immunity. γδ T cells play critical roles in protective immune responses against protozoan parasites, bacteria, and viruses that are associated with various infectious diseases (26-32). This review focuses on the protective abilities of γδ T cells and IFN-γ in the response against malaria infection.

IFN-γ MEDIATES PROTECTIVE IMMUNITY AGAINST BLOOD-STAGE PLASMODIUM PARASITES
Mice (on a C57BL/6 or CBA background) that are genetically IFN-γ-deficient or IFN-γ receptor (IFN-γR)-deficient or that are treated with anti-IFN-γ antibody and infected with blood-stage P. berghei are unable to control the infecting parasite (33-35). In the cases of infection with blood-stage P. chabaudi and P. yoelii parasites, genetically IFN-γ-deficient or IFN-γ receptor (IFN-γR)deficient mice or anti-IFN-γ antibody-treated mice on a C57BL/6 or CBA background show delayed elimination of the parasites (36-39). These experimental malaria models demonstrate that IFN-γ is a key pro-inflammatory cytokine for controlling bloodstage Plasmodium parasites (Table 1). IFN-γ is produced by many cell types and involved in many steps of immune responses. αβ T cells, NK cells, NKT cells, and γδ T cells have been shown to produce IFN-γ after infection with Plasmodium parasites. The contributions of producers of IFN-γ to protective immunity against Plasmodium parasites are complicated (Figure 2). Thus, more detailed experiments using IFN-γ signaling-deficient models should be performed to determine the mechanism(s) underlying the involvement of IFN-γ in immune protection against Plasmodium parasites.

IFN-γ PRODUCERS AND THEIR ACTIVATION IN PLASMODIUM INFECTION
CD4 + αβ T CELLS AND CD8 + αβ T CELLS Given that MHC class II-deficient mice on a C57BL/6 background are unable to control P. yoelii-and P. chabaudi-infected RBCs (iRBCs), CD4 + αβ T cells respond to Plasmodium-iRBCs after priming with malarial antigens in an MHC class II context on DCs (40). CD4 + αβ T cells strongly increase their ability to produce IFN-γ after infection with P. berghei (32). High proportions of CD4 + αβ T cells from P. falciparum-infected human subjects respond to iRBCs and produce IFN-γ, compared to CD4 + αβ T cells from naïve human subjects (4). Moreover, some studies have shown that CD4 + αβ T cell-depleted C57BL/6 or CBA mice treated with anti-CD4 antibody are unable to control blood-stage P. chabaudi or P. berghei (32, 33, 41, 42). These lines of evidence Frontiers in Immunology | Immunotherapies and Vaccines suggest that CD4 + αβ T cells are major sources of IFN-γ and are required for protective immunity against Plasmodium infection, resulting in complete clearance of the parasites. In contrast, the results for Plasmodium-infected mice with CD8 + αβ T-cell depletion are comparable to those for control mice (33, 41, 42). Moreover, CD8 + αβ T cells from highly immunized mice may play critical roles in protective immunity against infection with a lethal P. yoelii strain in C57BL/6 mice (41-43), although CD8 + T cells are not required for the resolution of primary infection with P. chabaudi or P. yoelii parasites in C57BL/6 mice. Interestingly, involvement of perforin molecules is only partial in protective immunity involving CD8 + αβ T cells. IFN-γ and macrophages are key molecular and cellular factors in protective immunity involving CD8 + αβ T cells (43). These data suggest that the role of the CD8 + αβ T cells is redundant in relation to the role of CD4 + αβ T cells in protective immunity in repeatedly immunized mice. A repeat infection with Plasmodium parasites may induce CD4 + αβ T-cell exhaustion (44). Cytokine production by T cells is inhibited by this exhaustion via the signaling of inhibitory receptors, such as PD-1 and LAG3. Since CD4 + αβ T cells are still needed to exert protective immunity against re-infection with the Plasmodium parasites, only partial exhaustion of CD4 + αβ T cells may occur. Therefore, both CD4 + αβ T cells and CD8 + αβ T cells may need to produce adequate quantities of IFN-γ to ensure clearance of the Plasmodium parasites.

DENDRITIC CELLS
Several reports have shown that IFN-γ is also produced by DCs (8-10). The IFN-γ-producing DCs are important for priming lymphocytes (10), although it remains to be determined whether IFN-γ production by DCs enhances protective immunity against Plasmodium parasites, and whether stimulation of Toll-like receptors (TLRs) by parasite components induces IFN-γ production by DCs (45-48).

γδ T CELLS AND NK CELLS
γδ T cells and NK cells are considered to be important IFN-γ producers in blood-stage malaria infections and to be associated with the control of malarial parasites (2, 3, 5). In the early stages of malaria infection, γδ T cells directly recognize the pathogen through MHC-independent mechanisms that involve the γδTCR, and high levels of IFN-γ production and proliferation are induced (49-51). Several reports have suggested that proliferation of γδ T cells depends on IL-2 (52, 53). Human and murine γδ T cells usually express TLRs, and the expression levels of TLRs are enhanced by γδTCR stimulation (54)(55)(56). Therefore, TLR is another candidate receptor for γδ T-cell responses to malaria antigens (46, 57). Although studies have suggested that γδ T cells have the potential to react to malarial antigens via TLRs, TLR signaling in myeloid cells may be more important for the induction of protective immunity against malaria (58). A study of experimental P. falciparum infection showed that, in contrast to γδ T cells, NK cells were minor IFN-γ producers in response to iRBCs before and after P. falciparum infection (5). A longitudinal study of children in Papua New Guinea indicated that IFN-γ-producing responses of malaria antigen-specific γδ T cells, but not those of NK cells, were important for protective immunity against P. falciparum infection (59). In contrast, several in vitro www.frontiersin.org FIGURE 2 | IFN-γ producers and their activation. CD4 + and CD8 + αβ T cells, NK cells, and γδ T cells have been shown to produce IFN-γ after infection with Plasmodium parasites. Innate immune cells can directly recognize Plasmodium antigens. γδ T cells can recognize phosphoantigens by MHC II-independent pathways via TCRγδ, resulting in the activation of DCs and IFN-γ production. Whether γδ T cells can recognize malarial antigens via TLR is unknown. DCs phagocytose iRBCs and recognize malarial DNA and hemozoin or glycosylphosphatidylinositol (GPI) anchors via TLRs, resulting in the activation of DCs. DCs are thought to have the potential to produce IFN-γ. However, it remains unclear as to whether IFN-γ production from DCs is induced by malarial antigens. Activated DCs produce IL-12 and present antigens to αβ T cells. αβ T cells are strongly activated by IL-12 and antigen presentation. IL-12 also activates NK cells. The malarial antigen-recognition receptor on NK cells remains to be determined. Activation of αβ T cells leads to high IFN-γ production. Furthermore, activated αβ T cells produce IL-2, resulting in the proliferation of γδ T cells and NK cells and the αβ T cells themselves.
culture studies have shown that NK cells rapidly induce IFN-γ production in response to P. falciparum-iRBCs via IL-12 and IL-18 signaling. These reports suggest that NK cells have the potential to produce IFN-γ in response to P. falciparum.

IFN-γ INDUCES PATHOLOGIC EFFECTS IN SEVERE MALARIA
Interferon-γ is not only a key factor in protection against Plasmodium infection, but also a pathogenicity factor in severe malaria symptoms (33, 38, 60, 61, 94). It is well-known that P. berghei ANKA infection leads to the development of experimental cerebral malaria (ECM), and P. berghei NK65 infection induces liver injury. Those P. berghei-infected mice are useful models for studies of severe malarial symptoms. IFN-γR-deficient mice on a C57BL/6 or 129 background do not develop ECM. Furthermore, P. berghei NK65-infected mice on a C57BL/6 or CBA background treated with anti-IFN-γ antibody live longer than those treated with control-IgG. These reports suggest that IFN-γ is important for the development of severe malarial symptoms (Table 1) (33,34,38,94). ECM also does not develop in P. berghei ANKA-infected mice after depletion of CD4 + T cells or CD8 + T cells (62). Cytotoxic CD8 + αβ T cells, which express perforin or granzyme B, have important roles in the development of ECM (63,64). Although both CD4 + αβ T and CD8 + αβ T cells have a similar potential to produce IFN-γ, CD8 + αβ T cells are not an important IFN-γ producer for the development of ECM (61). IFN-γ-producing CD4 + αβ T cells promote recruitment of cytotoxic CD8 + αβ T cells to the brain; in contrast, CD4 + αβ T cells dot not accumulate markedly in the brain (60). Because depletion of NK cells can prevent ECM after infection with P. berghei ANKA, NK cells are also associated with IFN-γ responses in the development of ECM. NK cells activate CD8α + DCs that prime CD8 + αβ T cells after infection with P. berghei ANKA (65). Furthermore, depletion of γδ T cells can also prevent ECM after infection with P. berghei ANKA. Therefore γδ T cells would be also associated with IFN-γ responses in the development of ECM (66). IFN-γ has both protective and pathologic effects on the immune response to Plasmodium infections. Therefore, simply inducing a reduction in blood IFN-γ levels by treatment with an antibody or drug may not be an effective way to treat cerebral malaria. Further studies focused on the regulation of CD8 + αβ T-cell activation by DCs are needed to develop a preventative therapy for cerebral malaria.

DOES IFN-γ INDUCE EFFECTIVE HEMATOPOIESIS IN THE HOST?
IFN-γ is an important mediator of hematopoietic stem cell and progenitor cell activation during bacterial infections (67)(68)(69). Furthermore, rodent Plasmodium infection induces the generation of uncommon myelolymphoid progenitor cells, which express IL7-R + and c-Kit hi via IFN-γ signaling. These myelolymphoid progenitor cells can differentiate into both myeloid cells and lymphoid cells (70). Host protection from malaria depends on hematopoietic differentiation (i.e., hematopoiesis) to supply many types of differentiated cells. First, differentiation of erythroid progenitor cells is necessary to avoid the development of severe anemia. Second, differentiation of myeloid progenitor cells is necessary to supply a high number of phagocytic cells for the clearance of Plasmodium parasites. Hematopoiesis is a fundamental process for"curing" malaria and other infections. Moreover, several studies have shown IFN-γ-induced inhibition of some steps of hematopoiesis (71)(72)(73). Considering important roles of IFN-γ in protective immunity against Plasmodium parasites, IFN-γ would be a key factor for regulation of effective hematopoiesis in malaria. There are many cell types of IFN-γ producers in Plasmodium infection as mentioned above. Therefore, some of the IFN-γ producers in Plasmodium infection may play crucial role for regulation of hematopoiesis by the effect of IFN-γ or a combination of IFN-γ and other factors, which are produced from the IFN-γ-producing cells.

VARIETIES OF γδ T-CELL FUNCTION
γδ T cells play various roles, including in protective immunity against pathogens, in the curing of injured tissue, tumor surveillance, and as a bridge between innate and adaptive immunity. These multiple functions of γδ T cells are thought to be due to their abilities to produce various cytokines and chemokines. Such abilities are broadly restricted by the Vγ and Vδ repertoires of γδ T cells. The distribution of γδ T-cell subsets differs depending on their resident tissue (74). To examine the physiologic role(s) in the immune response against Plasmodium parasites, γδ T-cell subsets should be compared carefully between mice and humans based on their abilities, such as cytokine production or ligands for activation.

γδ T-CELL-RELATED PROTECTIVE IMMUNITY AGAINST BLOOD-STAGE PLASMODIUM INFECTION
Although previous reports of in vitro and in vivo experiments have suggested that γδ T cells are associated with protective immune responses during malaria infection, the functions of γδ T cells in the spleen remain largely unknown (75)(76)(77)(78)(79). We recently reported on the mechanism of γδ T-cell-related protective immunity against Plasmodium parasites using the rodent malaria parasite P. berghei XAT (32, 80) (Figure 3). P. berghei XAT is an attenuated strain derived from the lethal P. berghei NK65 strain (32, 81). P. berghei XAT was developed for investigations of vaccines and protective immune responses against Plasmodium parasites owing to its potent ability to induce immune memory, even against lethal P. berghei NK65 strains. Previous studies have reported the essential cytokines and immune cells required for the clearance of P. berghei XAT. Since γδ T-cell-deficient mice on a C57BL/6 background are unable to control P. berghei XAT infection, γδ T cells are essential for protective immunity against the parasites (32). Other studies using P. chabaudi parasites have also suggested that γδ T cells are related to, but not essential for, protective immunity against the parasites (76, 78). Thus, this P. berghei XAT strain is useful for investigating the mechanism(s) of γδ T-cell-related protective immunity against Plasmodium parasites. In general, the immunologic functions of γδ T cells are largely similar to those of NK cells. The difference in the need for γδ T cells between protective immunity against the two parasite strains may be influenced by the contribution of NK cells to protective immunity.
Although malaria infections in humans, monkeys, and mice lead to increased numbers of γδ T cells in the blood and spleen, the γδ T-cell population is still minor in relation to the total number of lymphocytes (82). It may be possible to explain why such a minor population of lymphocytes plays such key roles in protective immunity against malaria parasites. First, γδ T cells can directly recognize malarial parasites, making them major producers of IFN-γ in response to malaria antigens during the early phase of infection. In contrast, NK cells are reciprocally regulated by DCs. Thus, DC activation is needed first to activate NK cells in malaria infection (3, 83). NK cells are unexpectedly minor producers of IFN-γ in response to malarial antigens in human blood in experimental infections with P. falciparum parasites (59). Nevertheless, NK cells also become key players in protective immunity against malarial parasites, as has been shown in many studies (2, 3, 83), although NK cells are not required for the control of P. berghei XAT parasites (35). Second, γδ T cells have the ability to interact readily with other central immune players, such as DCs. Our recent report showed that about 30% of splenic γδ T cells www.frontiersin.org were localized in the vicinity of DCs even under naïve conditions. A more than twofold increase in the percentage of splenic γδ T cells that adhered to DCs was observed in the early phase of infection (32). The molecular mechanism of this adhesive ability of γδ T cells to DCs remains to be determined. As γδ T cells can produce chemokines, some γδ T-cell-produced chemokines may attract DCs and also αβ T cells to help antigen presentation by DCs (84,85). Furthermore, our study provided evidence that γδ T cells boost DC activation for protective immunity against P. berghei XAT parasites via CD40 ligand expression on γδ T cells. CD40L-CD40 signaling induces the expression of MHC II and costimulatory factors, such as CD40, CD80, and CD86 (32). Such signaling to DCs would be synergistically activated by the uptake of Plasmodium antigens (86).

MEMORY PHENOTYPE OF γδ T CELLS IN MALARIA
For long-standing protective immunity against pathogens, memory lymphocytes maintain phenotypes to respond against pathogens and live for a long period after infections or vaccination with antigens (87,88). In general, memory T cells are converted from naïve T cells through three phases (expansion, contraction, and memory phases; Figure 4). In the expansion phase, effector or killer T cells activate and proliferate after pathologic infection. Soon after pathogen clearance, the numbers of expanded antigen-specific T cells are reduced by induction of apoptosis in the contraction phase. Then, a fraction of the remaining antigen-specific T cells become memory T cells, resulting in long-lasting and rapid protective immunity against re-infection with pathogens (memory phase). Memory plasma cells and B cells maintain the potential to generate antigen-specific antibodies, and memory CD4 + T cells and CD8 + T cells maintain rapid helper and killer responses to antigens, resulting in rapid elimination of re-infecting pathogens. In malaria, those memory lymphocytes would also exist in human subjects who have been infected with Plasmodium parasites previously (4, 5). However, there is still controversy over the duration of memory lymphocyte maintenance (89,90). Previous studies have shown that human γδ T cells generate rapid and amplified responses to a secondary challenge of bacteria and viruses, such as mycobacteria and cytomegalovirus (91,92). These data suggest that human γδ T cells have the ability to develop memory cells for protection against re-infection. On the other hand, a recent report suggested that human γδ T cells develop effector memory cells after infection with P. falciparum parasites (5).
Some details about memory γδ T cells in malaria remain to be uncovered (Figure 4). The first point is the importance of memory γδ T cells in malaria. Although the numbers of malaria antigen-specific γδ T cells certainly increase in the year after primary infection, whether the memory γδ T cells are important for effective protection against re-infection with Plasmodium parasites remains to be determined. The second point is the developmental process of memory γδ T cells. γδ T cells function not only in adaptive immunity but also in innate immunity. Thus, the process for the development of memory γδ T cells may differ from that of general memory T cells in malaria or in other infections. The third point is the localization of memory γδ T cells. A recent report showed that memory T cells in lymph nodes and peripheral tissues FIGURE 4 | Memory γδ T cells. Naïve T cells are activated by pathologic infections and differentiate into effector or killer T cells. Activated T cells clonally expand in response to particular antigens (expansion phase). After pathogen clearance, high proportions of the expanded antigen-specific T cells undergo apoptosis (contraction phase). Some population of the surviving antigen-specific T cells is maintained as memory T cells (memory phase). γδ T cells also expand after infection with pathogens, including Plasmodium parasites. High-level proliferation of γδ T cells is involved in generating IL-2 from T H 1 cells or the γδ T cells themselves. It remains to be determined whether antigen-specific γδ T cells shift into a contraction phase and then a memory phase.
translocate to bone marrow for the long term (93). It would be difficult to examine whether this hypothesis applies to memory γδ T cells, and other memory lymphocytes, in human subjects. Thus, rodent malaria models are needed for examining the location of memory γδ T cells.

CONCLUDING REMARKS
Many studies have demonstrated that IFN-γ is responsible for protective immunity against Plasmodium parasites. The critical cellular source(s) of IFN-γ in malaria patients remains to be determined. γδ T cells are not only a major candidate as the key IFN-γ producer, but are also critical modulators of protective immunity against Plasmodium parasites. We should continue to investigate the roles of γδ T cells in host defenses against Plasmodium parasites. Thus, it is important to investigate whether the abilities of γδ T cells to produce IFN-γ and mediate immune responses against malarial antigens persist as memory cells. Mechanisms of immune responses in lymphoid tissues, especially in the spleen, Frontiers in Immunology | Immunotherapies and Vaccines to blood-stage malaria are gradually being uncovered by studies using rodent malaria models. To investigate further the relationship between IFN-γ and γδ T cells in immune responses against malaria, we should make more effort to study the issues using not only rodent malaria models, but also humanized mice that can be infected with P. falciparum parasites. To make this a reality, we first need to refine the humanized mice. Based on studies of γδ Tcell-related protective immunity, we may be able to develop novel strategies for a malaria vaccine.

ACKNOWLEDGMENTS
This work was supported by the Moritani Scholarship Foundation, the Japan Prize Foundation to Shin-Ichi Inoue. This work was also supported by Grants-in-Aid for Young Scientists (B) to Shin-Ichi Inoue (No. 23790462) and Mamoru Niikura (No. 24790405) and a Grant-in-Aid for Scientific Research (C) to Fumie Kobayashi (No. 23590493) from the Japan Society for the Promotion of Science (JSPS). We thank Mrs. Natsuki Yokota for her support of our work.


extfieldsofstudy: Biology Medicine

text:
Profiling Pre-MicroRNA and Mature MicroRNA Expressions Using a Single Microarray and Avoiding Separate Sample Preparation

Mature microRNA is a crucial component in the gene expression regulation network. At the same time, microRNA gene expression and procession is regulated in a precise and collaborated way. Pre-microRNAs mediate products during the microRNA transcription process, they can provide hints of microRNA gene expression regulation or can serve as alternative biomarkers. To date, little effort has been devoted to pre-microRNA expression profiling. In this study, three human and three mouse microRNA profile data sets, based on the Affymetrix miRNA 2.0 array, have been re-analyzed for both mature and pre-microRNA signals as a primary test of parallel mature/pre-microRNA expression profiling on a single platform. The results not only demonstrated a glimpse of pre-microRNA expression in human and mouse, but also the relationship of microRNA expressions between pre- and mature forms. The study also showed a possible application of currently available microRNA microarrays in profiling pre-microRNA expression in a time and cost effective manner.

Introduction
MicroRNAs (miRNA) are short conservative endogenous non-coding RNAs with a length of around 22 nucleotides, which have diverse functions [1]. Since its discovery in 1993 [2], miRNA has OPEN ACCESS been recognized as one of the key players in the transcript regulatory network of eukaryotes [3]. Despite intensive scientific research, many miRNAs still need to be explored or validated in different biological or pathological contexts.

Microarray Application in miRNA Expression Profiling
Diverse techniques are available for the purpose of miRNA expression profiling, e.g., Northern blotting [12], dot blotting [13], primer extension analysis [14], RT-qPCR [15] and next generation sequencing (NGS) [16]. RT-qPCR, microarray and NGS are the three most state-of-the-art methods applied in miRNA profiling [17]. RT-qPCR offers the most sensitive profiling signal with a reasonable cost. With a considerate design, RT-qPCR can also achieve high throughput miRNA profiling. The recent progress of NGS has led to rapid cost reduction, which lowered the impact of NGS application in miRNA profiling. Nonetheless, microarrays with immobilized DNA probes on solid substrate are still the choice of many laboratories with a moderate budget [18]. Hence, microarrays are widely applied also in miRNA profiling [19]. One of the challenges of miRNA expression profiling is to distinguish between mature miRNAs and their precursors and to detect the miRNA expression signal with high specificity. This problem can be solved with a precise and genuine probe design on microarray platforms.
Much more attention has been dedicated to mature miRNA expression profiling, although pri-miRNA and pre-miRNA are important intermediates during miRNA biogenesis [20]. Pre-miRNAs have also been acknowledged as useful disease biomarkers [21,22]. RT-qPCR has been, so far, the gold standard for pre-miRNA expression evaluation [23]. Recently, a global pre-miRNA landscape has also been successfully provided by applying a deep sequencing technique [24]. Since pre-miRNAs and mature miRNAs can both be detected in cytoplasm, we demonstrated in this paper the possibility of exploring the expression of mature and pre-miRNAs on a single microarray platform, simultaneously.

Experimental Section
Affymetrix GeneChip ® miRNA 2.0 arrays (Affymetrix, Santa Clara, California, USA) contain probes that interrogate the mature miRNA and pre-miRNA of a wide spectrum of species. In this study, profiling data of human and mouse samples (GSE39015 [25], GSE42915 and GSE33809 [26] focused on human samples; GSE33413 [27], GSE32352 [28] and GSE36257 [29] profiled mouse samples) on Affymetrix GeneChip miRNA 2.0 arrays (see Table 1) were retrieved from the Gene Expression Omnibus (GEO) database [30]. Raw data were normalized with the robust multi-array average (RMA). Present calls were made by using the detection above background (DABG) algorithm with a p-value cutoff of 0.05. Pre-miRNA and mature miRNA probes were identified according to annotations provided by Affymetrix. A paired t-test was applied on the median expression of miRNAs and pre-miRNAs for significancy calculations (see Figure 1).  The correlation of pre-and mature miRNA expressions were studied under two different aspects: (i) for study, i, (i = a GEO study re-analyzed in this manuscript: GSE39015, GSE42915, GSE33809, GSE33413, GSE32352 and GSE36257), the expression values of pre-miRNAs can be described as V i,pre = {v 1,pre; v 2,pre; …; v j,pre |j = the array number in study i} and the expression values of mature miRNAs as V i,mature = {v 1,mature; v 2,mature; …; v j,mature |j = the array number in study i}. Pearson correlation coefficients were pairwise calculated between V i,pre and V i,mature for all arrays in studies, i, and then presented as a bar plot (see Figure 4). (ii) The expression matrix, M, of pre-miRNA and mature miRNA in study, i, was defined as M i,pre = {m 1,pre; m 2,pre; …; m k,pre |k = the number of pre-miRNAs on one array} and M i,mature = {m 1,mature; m 2,mature; …; m k,mature |k = the number of mature miRNAs on one array}. Correlation between M i,pre and M i,mature was also calculated by pairwise Pearson correlation coefficients. The distribution of pairwise correlation coefficients of all miRNAs in every study were shown as a histogram ( Figure 5). Statistical analysis were completed with basic functions in R [31].

Results and Discussion
GeneChip ® miRNA 2.0 has 4,592 probe sets for human and 1,412 probe sets for mouse. Seven hundred sixty seven pre-miRNAs and 919 mature miRNAs are annotated for human samples. For the mouse samples, 510 pre-miRNAs and 601 mature miRNAs are available on the platform. Eight hundred thirty six pre-miRNA/mature-miRNA pairs can be mapped in human miRNA annotation, while there are 579 according to the mouse annotation.

Mature miRNAs Are More Abundantly Detected
Pre-miRNAs showed lower expression in these data sets than their matured products. Boxplots of expression values of pre-and mature miRNAs in three human and three mouse studies, respectively, are shown (Figure 1). In all murine studies, median expressions of pre-miRNA are significantly lower than those of mature miRNA, wherein one of the studies exhibits a minor difference (GSE32352). For human samples, the differences are not so pronounced; however, they are also significant for two of the three studies analyzed (GSE42915, GSE33809). At the same time, the upper quartiles of mature miRNAs distribution are located more in a higher expression value region. It is possible that mature miRNA is more enriched by sample preparation. Besides, the high stability observed by mature miRNAs could also contribute to their higher expression signal [32]. Moreover, the rapid processing of the intermediate product pre-miRNA might have an impact on the fewer copy number of pre-miRNA compared to the stable accumulating signal of mature miRNA. The lower expression value of pre-miRNA compared to its corresponding mature miRNA was also reported in another study using the RT-qPCR method [33]. Independent to the variety of individual samples, three data sets that used the same RNA isolation kit (GSE34413, GSE36257 and GSE42915) showed a similar pattern in expression value distribution. This observation indicates that sample preparation, especially RNA isolation, is a crucial factor for successful miRNA profiling.

Much More Mature miRNAs than Pre-miRNA Were Detected as Present
The general higher expression level of mature miRNA was also reflected in the detection call of present miRNAs. Present calls were made for pre-miRNA and mature miRNAs compared to background signals. Mature miRNAs have almost doubled present calls with respect to pre-miRNAs in all data sets (Figure 2).
Most of the pre-miRNAs and mature miRNAs share the same present/absent calls. That means, present pre-miRNAs have present mature counterparts, and absent pre-miRNAs have also absent mature corresponding miRNAs (Figure 3, red bars). However, there are also present pre-miRNAs, which have absent mature miRNA, and vice versa (Figure 3, blue and black bars). miRNAs that were detected as present in mature form, but as absent in pre-miRNA form, could support the hypothesis that mature miRNAs are more stable than their precursor counterparts. As demonstrated, there was a few number of miRNAs that were present in their premature form, but absent in their mature form. This phenomenon indicates a possible miRNA transcription regulation by degrading mature miRNA [34]. The reduction of specific mature miRNAs compared to their pre-miRNAs was also reported in human colorectal neoplasia by Northern analysis [35].

Correlation between Pre-miRNA Expression and Mature miRNA Expression
The expression of mature miRNA and pre-miRNA measured on the same arrays showed a positive interdependency (Figure 4). Notwithstanding different expression levels, a positive correlation did exist between expression of pre-and mature miRNAs on the same array. This is consistent with the results reported previously [36]. Correlation coefficients of pre-and mature miRNA on the same array in one data set were consistent, while the values clearly varied between different data sets. For example, GSE32352 demonstrated a much lower correlation coefficient between pre-and mature miRNA expression. GSE34413, GSE36257 and GSE42915 showed similar correlation coefficients. While in the studies, GSE34413, GSE36257 and GSE42915, the same RNA isolation kit (TRIzol from Invitrogen) was used; in the GSE32352 study, the mirVana RNA isolation kit (Ambion) was used. For this reason, we believe that the observed correlation coefficients were strongly affected by the choice of sample preparation. The influence of the kind of probe labeling can be excluded, because all probes were labeled with the same kit (FlashTag biotin HSR kit from Genisphere). Correlation coefficients were also calculated for the expression profile of every mature miRNA to its corresponding pre-miRNA across the arrays in the data sets. These coefficients support the evidence of the dependency between pre-miRNA and mature miRNA expression regulation, which differs according to different tissue sources and/or treatments. Correlation coefficients for all miRNAs in each study have been calculated between the pre-miRNA expression pattern and the mature miRNA expression pattern ( Figure 5). In all data sets analyzed in this paper, correlation coefficients were widely distributed between −1 and 1. In particular, not only positive coefficients were observed, but also extreme negative ones. That means that upregulation of mature miRNA can be observed in a study even when the corresponding pre-miRNA is downregulated. This result indicates that during maturation of pre-miRNA to mature miRNA, complex factors are involved in regulation, depending on the kind of treatment, tissue-specific development or different biological/pathological contexts.

Conclusions
Six publicly available miRNA profiling raw data sets generated from human and mouse samples on Affymetrix GeneChip ® miRNA 2.0 array platforms were re-analyzed in this study. Pre-miRNA and mature miRNA expression signals were retrieved and normalized to gain comparable signals. miRNA precursors exhibited a lower expression level in most of the analyzed data sets. Present calls of individual mature miRNAs and pre-miRNAs showed a great part consistency, with minor discrepancies. On the same array, mature miRNA expressions are positively correlated to pre-miRNA expressions. The distribution of expression values of pre-miRNA and mature miRNA, as well as the correlation coefficients between them, seem to be influenced by the RNA isolation methods applied in the studies. Because of the sample size limitation and the lack of other experimental verifications, we are not able to judge the pros and cons of RNA isolation methods for miRNA profiling at this point. The expression regulation patterns of mature miRNAs showed no clear positive correlation to the expression regulation patterns of the corresponding pre-miRNAs. Therefore, we believe in the existence of regulation factors of miRNA maturation according to treatment, tissue type and biological/pathological contexts involved in individual studies. The results presented in this paper require definitely further research and verification with more data sets and with other techniques, like RT-qPCR. Nevertheless, our study demonstrated the possibility of profiling pre-miRNA and mature miRNA simultaneously without separate sample preparation. Compared to methods like RT-qPCR, the microarray platform applied in this paper has the advantage of data normalization without the bias caused by specific reference RNA(s). At the same time, we can obtain a genome-wide overview for pre-miRNA and miRNA expression. An additional advantage of this platform is that only one simple labeling procedure is sufficient for both pre-miRNA and mature miRNA profiling.


text:
A microfluidic phenotype analysis system reveals function of sensory and dopaminergic neuron signaling in C. elegans electrotactic swimming behavior

The nematode (worm) C. elegans is a leading multicellular animal model to study neuronal-basis of behavior. Worms respond to a wide range of stimuli and exhibit characteristic movement patterns. Here we describe the use of a microfluidics setup to probe neuronal activity that relies on the innate response of C. elegans to swim toward the cathode in the presence of a DC electric field (termed “electrotaxis”). Using this setup, we examined mutants affecting sensory and dopaminergic neurons and found that their electrotactic responses were defective. Such animals moved with reduced speed (35–80% slower than controls) with intermittent pauses, abnormal turning and slower body bends. A similar phenotype was observed in worms treated with neurotoxins 6-OHDA (6- hydroxy dopamine), MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) and rotenone (20–60% slower). We also found that neurotoxin effects could be suppressed by pre-exposing worms to a known neuroprotective compound acetaminophen. Collectively, these results show that microfluidic electrotaxis can identify alterations in dopamine and amphid neuronal signaling based on swimming responses of C. elegans. Further characterization has revealed that the electrotactic swimming response is highly sensitive and reliable in detecting neuronal abnormalities. Thus, our microfluidics setup could be used to dissect neuronal function and toxin-induced neurodegeneration. Among other applications, the setup promises to facilitate genetic and chemical screenings to identify factors that mediate neuronal signaling and neuroprotection.

Introduction
The survival and functioning of living organisms depend on their ability to continuously monitor their surrounding environment. This process is mediated by the nervous system that detects environmental stimuli and depending upon the context and the experience of the animal generates an appropriate response. Understanding how neurons respond to stimuli and initiate a specific behavioral outcome is complicated in vertebrates due to the presence of a very large number of neurons and complex interconnections. Invertebrate animal models, such as the nematode (worm) C. elegans, offer a simpler nervous system and a wide range of experimental techniques to dissect the neuronal basis of behavior. The adult C. elegans hermaphrodite contains 302 neurons, whose morphology and inter-connections are well established. 1,2 The animal has a short life cycle (2.5-3 d) making it possible to study developmental processes relatively quickly. In addition, it can be manipulated in the laboratory using a number The nematode (worm) C. elegans is a leading multicellular animal model to study neuronal-basis of behavior. Worms respond to a wide range of stimuli and exhibit characteristic movement patterns. Here we describe the use of a microfluidics setup to probe neuronal activity that relies on the innate response of C. elegans to swim toward the cathode in the presence of a DC electric field (termed "electrotaxis"). Using this setup, we examined mutants affecting sensory and dopaminergic neurons and found that their electrotactic responses were defective. Such animals moved with reduced speed (35-80% slower than controls) with intermittent pauses, abnormal turning and slower body bends. A similar phenotype was observed in worms treated with neurotoxins 6-OHDA (6-hydroxy dopamine), MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) and rotenone (20-60% slower). We also found that neurotoxin effects could be suppressed by pre-exposing worms to a known neuroprotective compound acetaminophen. Collectively, these results show that microfluidic electrotaxis can identify alterations in dopamine and amphid neuronal signaling based on swimming responses of C. elegans. Further characterization has revealed that the electrotactic swimming response is highly sensitive and reliable in detecting neuronal abnormalities. Thus, our microfluidics setup could be used to dissect neuronal function and toxin-induced neurodegeneration. Among other applications, the setup promises to facilitate genetic and chemical screenings to identify factors that mediate neuronal signaling and neuroprotection.
of powerful genetic and genomic tools. These features have greatly facilitated the study of neuronal cell fate specification and cell signaling.
The sensory neurons in C. elegans that mediate odor detection (chemosensory) and physical contacts (mechanosensory) have been studied in significant detail. Many of these have ciliated dendrites that are directly or indirectly exposed to the external environment and facilitate detection of environmental stimuli by activating signal transduction pathways. The signaling process is mediated by several proteins including G-protein coupled receptors (GPCRs) and transient receptor potential (TRP) channels. [3][4][5] In hermaphrodites eight of the mechanosensory neurons (two pairs of CEPs and one pair of ADE in the head region and one pair of PDE in the posterior region) produce dopamine (DA) neurotransmitter. 6 All of these neurons have ciliated endings that are embedded in the cuticle and their processes extend to the nose tip (CEPs) and along the anterior and posterior lateral midlines (ADE and PDE, respectively). 5 The dopaminergic neurons pesticide). These toxins cause degeneration of DA neurons similar to that shown in mammalian models. [26][27][28] A common mode of their action appears to be the inhibition of mitochondrial respiratory chain and generation of reactive oxygen species (ROS). 29 We found that toxin-treated worms had abnormal swimming behavior. A comparison with plate-based behavioral assays revealed that the microfluidics assay is highly sensitive in detecting movement defects. The phenotypic analyses of worms were performed by quantifying parameters that eliminated subjectivity and bias. Lastly, we investigated the role of a neuroprotective compound acetaminophen and found that it suppresses the effect of toxins in the channel assay. Together, these results demonstrate that the microfluidic electrotaxis is a sensitive assay to study neuronal signaling and toxin-induced neuronal damage, as well as to identify neuroprotective drugs in C. elegans.

Results
The microfluidic device used in our electrotaxis assay ( Fig. 1) provides an environment in which the electric field streamlines are confined in the axial direction of the channel. This results in a uniform stable field that stimulates worms to move along the channel length. Furthermore, the narrow diameter (300 μm) ensures motion in a near straight line fashion. We have earlier shown that this setup allows worms to swim toward the cathode when exposed to a low voltage DC electric field. 20 The electrotactic swimming response of C. elegans is robust, continuous and occurs at a characteristic speed. Changing the direction of the electric field causes the worm to stop transiently, turn toward the cathode and resume motion ( Fig. 1D and E). Therefore, any deviation from these characteristics can be considered as an abnormality. As described in Materials and Methods, we characterized the electrotaxis behavior of worms using four parameters, namely electrotaxis speed, turn time, body bend frequency and electrotaxis time index (ETI).
Initially, we investigated the behavior of wild-type N2 worms in the channel in the absence of an electric field. The animals showed random swimming and turning activities with a mean body bend frequency of 1.3 (n = 6) (Video 1). Due to multiple reversals, they failed to cover long distance in any one direction. Thus, the electrical stimulus is required to propel worms in a directed manner.
The electrotactic swimming of C. elegans in the channel depends on its intact neuronal and muscular systems. 20 Consequently, defects in any of these components could alter the swimming behavior. While the electrosensory defect may result from the inability of sensory neurons to receive and process electrical signals, general locomotion defects could arise due to problems in the neuromuscular system that controls motor responses such as speed and amplitude of motion. Hence, electrosensory mutants will have difficulty in sensing the direction of the electric field. Such animals are expected to swim toward either pole while reversing direction frequently. However, the locomotory mutants should recognize the electric field polarity and move specifically toward cathode, albeit at a reduced pace. Our analysis of the neuronal mutants in following sections agrees with these modulate different behavioral responses of the animal such as foraging, locomotion rate, egg laying, defecation and swimming in a liquid environment. [7][8][9][10][11] The traditional assays to measure behavioral activities of C. elegans involve qualitative and quantitative analyses of phenotypes using manual and semi-automated approaches. These methods are slow, labor-intensive and prone to errors. Consequently, they represent a significant bottleneck in analyzing a large number of animals in a rapid and unbiased manner. Recent advances in microfluidics research has made it possible to perform many of the routine and laborious experiments in C. elegans automatically, precisely and in a high-throughput manner. [12][13][14][15] Microfluidics offers unparalleled control over manipulation of worms because of its ability to precisely control microscopic volumes of fluids inside minute chambers and channels that are comparable to the size of worms. This allows efficient handling of worms and confers greater control over confinement, treatment and observation at a high resolution. Furthermore, microfluidics deals with tiny volumes of liquid resulting in reduced cost of media and chemical usage. Microfluidics devices have a wide variety of applications in research involving C. elegans. These include growth studies, mutant screening, nanosurgery, neuronal imaging and movement analysis. [16][17][18][19] We recently demonstrated that low voltage direct current (DC) and pulsed DC electric fields inside a microfluidic channel cause C. elegans to swim toward cathode (termed "electrotaxis"). 20,21 The electrotactic responses of some of the nematodes, including C. elegans, were examined earlier using an open gel surface Petri plate setup. [22][23][24] It was noted that although animals generally were attracted toward the cathode, they did not travel directly toward it along the imaginary axis from anode to the cathode. Instead, they were found to travel at an angle with respect to this line. The angle of motion was proportional to the strength of the electric field making the response more complex to analyze. 24 Unlike the open gel surface setup, the microchannel environment streamlines the electric field along its axis and stimulates worms to swim in a straight line with a characteristic sinusoidal pattern. This simplifies the movement analysis of animals by allowing precise quantification of parameters such as speed, body bend frequency and reversals. Furthermore, the electrotaxis response is instant, reversible and highly robust. As a result, abnormalities in neuronal signaling can be identified fairly quickly and reliably. Finally, microfluidics is amenable to parallelization and automation thereby accelerating the pace of discovery.
The precise role of electrotaxis behavior in worms is currently unknown but research involving plant and insect parasitic nematodes has suggested that it could facilitate host finding. 23,25 Studies in C. elegans have revealed that such a response in an open gel surface environment is mediated by amphid neurons. 24 In this study, we examined the electrotactic swimming behavior of worms in a microfluidic channel device using neuronal mutants and found that in addition to amphid sensory neurons dopaminergic neurons are also involved in this process. The role of dopaminergic neurons was further investigated by exposing worms to three different neurotoxins, 6-OHDA (6-hydroxy dopamine), MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) and rotenone (a the animals either took a very long time to turn (average turn time 49.2 sec, n = 16, compared with 10.3 sec, n = 12 for control) (Fig. 2) or failed to turn at all (16%, n = 19). In agreement with these findings, the ETI of lin-11 was greatly reduced (p < 0.0001) (Fig. 3). Taken together, these results demonstrate that osm-5 and lin-11 are required for the electrosensory response of C. elegans in the microfluidic channel assay.
Defects in dopamine signaling affect electrotaxis of animals. DA signaling is involved in modulating the locomotion of C. elegans in response to environmental stimuli. 7 Therefore, we examined the electrotaxis swimming response of DA pathway mutants in our microfluidic channel setup. Animals having mutation in possibilities and demonstrates that the microfluidics setup can be used to identify and characterize new genes mediating electrotaxis behavior.
Amphid sensory neurons mediate electrosensory responses in a microfluidic channel device. Although the biological basis and the mechanism of electrotaxis is poorly understood, a subset of neurons in the anterior ganglion were found to respond to the electric field stimulus in an open gel surface Petri dish setup. 24 We used osm-5 mutants to examine the role of sensory neurons in our channel assay. The osm-5 gene encodes an intraflagellar transport protein that is homologous to human IFT88 and is required for cilia formation in sensory neurons including the amphids. 30 The osm-5 animals appeared very active and in the absence of the electric field exhibited swimming behavior similar to wild-type N2 (body bend frequency of 1.8 Hz, n = 6; see above for N2 data) (Fig. S1). However, in the presence of the electric field, the animals showed severely defective electrotactic response. The average speed of osm-5 was nearly 60% lower than the wild-type N2 (n = 11) (Fig. 2). Whereas N2 worms swam straight toward cathode without pausing (Video 2), the osm-5 worms stopped and reversed direction many times (number of reversals for each animal ranged between 3-20, n = 11) (Video 3). Few of them became immobile after slight initial swimming and did not recover (18%, n = 11) (Video 4). In addition to spontaneous turning and lack of motion, osm-5 worms also exhibited intermittent pauses, abnormal body postures and swimming in wrong direction (i.e., toward anode) indicating that they lacked a sense of direction. Consequently, the turn time of animals was highly variable (Fig. 2). To further demonstrate the electrotaxis defect in osm-5 animals, we computed the time of all cathode-directed swimming events and determined ETI (see Materials and Methods). As expected, the ETI of osm-5 was greatly reduced compared to N2 (p < 0.0001) (Fig. 3). As a control, we also tested a nonneuronal mutant him-8(e1479) that produces increased frequency of males due to defects in X-chromosome segregation. 31 The speed, turn time and ETI of him-8 were comparable to N2 (Figs. 2 and 3).
We examined another neuronal mutant lin-11 that is weakly uncoordinated and exhibits chemosensory and thermosensory defects. 32-34 lin-11 is a founding member of the LIM Homeobox family of transcription factors. 35 It is expressed in a subset of neurons in the head ganglion (including sensory neurons ADF and ADL, interneurons AIZ, RIC, AVG, AVA and AVE, and chemosensory neuron ASG) and is necessary for their differentiation. 32,33 We found that lin-11(n389)null mutants 35 had severely defective electrotactic response. Some animals showed no reaction to the stimulus (17%, n = 23) (Videos 5 and 6) whereas others moved in the channel but with multiple pauses and significantly reduced speed (76 μm/sec, n = 19, compared with N2 control: 141 μm/sec, n = 12) (Fig. 2). Additionally, lin-11 worms were also defective in sensing the direction of the electric field. Upon switching the field polarity, is not completely abolished (40% residual DA compared with wild type 38 ). Because dat-1 mutants show reduced speed but no impact on sensing the electric field polarity, it suggests that DA signaling modulates locomotion without affecting the electrosensory response of animals. This conclusion is supported by the normal ETI response of dat-1 animals (p = 0.1253) (Fig. 3).
In addition to mutants, we also used three different chemical compounds 6-OHDA, MPTP and rotenone that are toxic to DA neurons. Previous work in vertebrates has shown that these chemicals cause degeneration of DA neurons in the substantia nigra region of the brain. 39,40 6-OHDA is preferentially taken up by DA neurons via the DAT transporter. Once inside the DA neuron, it causes multiple reactions including inactivation of the mitochondrial respiratory chain leading to an increase in ROS level. 29,41,42 the cat-2 gene, the tyrosine hydroxylase required for DA biosynthesis, 36 showed normal response (average speed 296 μm/sec and turn time 4.1 sec, n = 10 for the e1121 hypomorph); however, dopamine transporter mutants (dat-1(ok157)) showed a defective phenotype (average speed 189 μm/sec and turn time 26.1 sec, n = 10) compared with control animals (Fig. 4). Since DAT-1 mediates the reuptake of DA from the synaptic cleft back into the presynaptic terminal, 37 it is possible that in the absence of DAT-1 function, extracellular DA alters the activity of certain post-synaptic neurons, thereby causing reduced electrotaxis speed in the microfluidic channel. Additionally, DA could stimulate certain dopamine receptors on motor neurons resulting in a slower speed of animals. The lack of electrotaxis phenotype in cat-2(e1121) animals may be attributed to the fact that DA level  In summary, our experiments involving DA pathway mutants and neurotoxin-treated worms demonstrate the sensitivity of the microfluidic electrotaxis assay in detecting abnormalities in DA signaling. Quantification of swimming defects allowed us to compare phenotypes in different conditions as well as between different sets of animals. Together, these results showed that a reduction in DA signaling contributes to abnormalities in electrotaxis swimming behavior in the channel assay. We conclude that our microfluidics setup can be used to identify and study factors affecting DA signaling and neurodegeneration in worms.
Plate-based phenotypic analysis of mutants and toxintreated animals. In addition to the electrotaxis phenotype, we also examined gross morphology and behavior of animals on Petri plates. This was done to investigate whether movement defects are specific to the microfluidic device or observed on open agar gel surface as well. For this, we measured the speed of osm-5, dat-1 and lin-11 mutants by following their tracks on bacterial lawns (see Materials and Methods). The average speed of dat-1 and osm-5 was 162 μm/sec (n = 23) and 183 μm/sec (n = 18), respectively, which is not significantly different from the wild-type N2 (190 μm/sec, n = 19) and him-8 (188 μm/sec, n = 9) (Fig. 6A). However, lin-11 animals showed a lower speed compared with N2 (40 h stage, average speed 53 μm/sec, n = 16, N2: 77 μm/sec, n = 14) (Fig. 6A). This agrees well with earlier studies showing that lin-11 animals are weakly uncoordinated. 34 Overall, our results provide support to the conclusion that the microfluidic electrotaxis is a sensitive method to detect abnormalities in the swimming response of animals. Furthermore, they reveal that the lack of electrotaxis in some lin-11 animals and spontaneous reversals in osm-5 are unique to the channel environment and not observed in plate-based assay.
Next, we examined plate-level responses and cellular defects in toxin-treated worms. In general, worms were healthy and In the case of MPTP, it is metabolized into an active toxic product MPP + that enters DA neurons through the DAT-1 transporter. MPP + induces inhibition of mitochondrial respiratory enzyme complex I and an increase in ROS production. 43,44 Exposure to rotenone in rat and Drosophila melanogaster models has shown to cause apoptosis and oxidative damage of DA neurons. 39,45 C. elegans DA neurons are equally sensitive to the above three neurotoxins and undergo degeneration upon exposure. 26,27 To carry out the electrotaxis assay on worms exposed to 6-OHDA, MPTP and rotenone, we first optimized chemical exposure conditions (see Materials and Methods). Toxin-treated worms were placed inside the channel without any pre-selection and their electrotactic responses were analyzed. We found that animals had defects in swimming behavior. The phenotypes included slower speed, intermittent pauses and reduced sensitivity. Exposure to 6-OHDA (either 1 h or 4 h at 100 μM concentration) caused a significant reduction in the speed (40-60% slower, 126-174 μm/sec average speed) of animals (n = 12 for 1 h condition and 15 for 4 h condition) without altering the turn time (Fig. 5). Besides reduced speed, we also observed other defects in movement. Frequently, animals showed incoherent electrotaxis characterized by active sinusoidal motion followed by periods of slow responses or a lack of activity (Videos 7 and 8). Partial paralysis was also observed where the posterior half of the body was rigid such that the worm appeared to drag itself while moving (Video 9). In addition, we also detected phenotypes such as sudden freeze (Video 10), tremor (Video 11) and a lack of motion (Video 12). Not all phenotypes were observed in every animal and, furthermore, these were specific to microfluidic channel environment. Animals grown on Petri plates did not show any such phenotype. In few cases, we also measured body bend frequency and found it to be lower in exposed worms compared with controls (average frequency 0.2 Hz for 6-OHDA 4 h exposure, n = 11 animals and 1.8 Hz for N2, n = 10 animals).
Treatments of MPTP and rotenone for 8 h caused similar defects (roughly 40% slower speed in each case, MPTP: 178 μm/sec, rotenone: 160 μm/sec; n = 12 and 13 worms, respectively), albeit the reduction in speed was somewhat less compared with 6-OHDA (see above, Fig. 5). This is different from the 6-OHDA exposure that caused maximum defect in as little as 1 h (see above). The turn time was also affected (up to 2-fold slower for each toxin; MPTP: 19 sec; rotenone: 15 sec) (Fig. 5). Consistent with slow electrotaxis swimming speed, the body bend frequency of animals was also reduced (MPTP 8 h: 1.0 Hz, n = 3, compared with 1.6 Hz, n = 4 for N2 control; rotenone, data not shown). affects the electrotaxis behavior of worms, then the phenotype could be suppressed by protecting DA neurons. To examine this possibility, we tested a known neuroprotective compound acetaminophen. Studies in rats and C. elegans have shown that acetaminophen has a protective effect against MPP + , 6-OHDA and glutamate toxicity in DA neurons. [46][47][48] We found that pre-treatment of acetaminophen conferred significant protection on DA neurons against all three neurotoxins. The pre-exposed worms were phenotypically normal and had significantly faster speed compared with neurotoxin-treated worms (Fig. 8). However, the turn time of animals showed no improvement (data not shown). Because acetaminophen protects DA neurons, 46,48 these results further support the involvement of DA signaling in controlling the electrotaxis behavior of animals. Additionally, our findings show that the microfluidic channel-based assay can be used as a screening tool to identify new chemicals with neuroprotective properties.

Discussion
Microfluidic electrotaxis as a sensitive and reliable assay to detect neuronal activity. We have used a new microfluidics electrotaxis assay to examine neuronal signaling in worms. This unique method allows us to indirectly control the motion inside the channel. In the absence of an electric field, worms show random swimming behavior and cannot be guided in a desired direction. However, when exposed to the field, they move toward cathode in a stereotypic manner and with a constant speed. 20 Previous findings 20,24 and results presented in this paper demonstrate that this process depends on the function of neurons fertile with no obvious morphological defect at any stage. Except for 4 h 6-OHDA-exposure condition that caused weak uncoordinated movement (Unc) in roughly 40% of the population, in all other cases, animals moved well and were fairly active (Fig.  6B). In one case, 6-OHDA 1 h, we also determined the chemotaxis response to NaCl and found it to be similar to untreated N2 control animals (Fig. 6C). The number of animals examined for plate-based assays are: 6-OHDA -605 for 1 h and 420 for 4 h, and N2 control -220; MPTP -502 and control -192; Rotenone -353 and control -163. For chemotaxis assays the numbers are: 6-OHDA 1 h -169 (NaCI) and 34 (water); control -101 (NaCI) and 33 (water).
To correlate microfluidic behavioral defects in toxin-treated worms with DA neuronal function, the morphology of neurons was also investigated. For this, we used a dat-1 p ::YFP transgenic strain (bhEx120) in which YFP expression is observed in DA neuronal cell bodies as well as their projections (Fig. 7A). 26,37 The synchronized L1 stage bhEx120 animals were exposed to 100 μM 6-OHDA for 4 h and subsequently grown on a standard NG agar plate seeded with OP50 bacteria. The DA neurons were examined in adults. We found an age-dependent increase in neurodegeneration in toxin-treated animals (18%, n = 81 on day 3 and 26%, n = 42 on day 6) (Fig. 7B-D). The dendritic processes of CEPs showed variable degeneration such that YFP fluorescence had spotty appearance (Fig. 7B). In some cases, the entire dendritic processes were missing (Fig. 7C). These phenotypes are similar to that reported earlier 26 and are consistent with the electrotaxis defect in worms that we observed.
Acetaminophen confers neuroprotection against toxins in the microfluidics assay. If toxin-induced neuronal damage  Figure 2 for an explanation of the plot structure and other details. Control refers to unexposed wild-type N2 animals. In all cases, the speed of exposed worms is significantly different from the control. A similar phenotype is also observed for turn time of animals.
www.landesbioscience.com Worm e24558-7 swimming of different parts of their body relative to one another. This lack of synchrony translates into an overall gait abnormality that is characterized by short, staggered movements. We did not observe such a phenotype on the plate level. 6-OHDA-induced gait problems have been observed previously in the rat model but most importantly, this phenomenon relates closely with the shuffling gait that is a classical symptom of human Parkinson disease. 49 The execution of synchronized voluntary movement requires not only proprioceptive feedback from peripheral receptors but also higher-level supraspinal processing that allows for kinesthesia. 50 Defects in kinesthetic ability in toxin-treated worms could explain the etiology behind the short, staggered movements observed during electrotaxis. The gait abnormality observed in toxin-treated worms may result from a loss of proprioceptive ability due to the loss of dopaminergic neurons. Studies have shown that TRPN mechanosensitive ion channels are implicated in nematode proprioception. 51,52 Therefore, in the future it will be useful to examine TRPN function in the electrotaxis behavior. The sudden freeze, tremor and partial paralysis phenotypes of toxin-treated worms in the channel assay may be reminiscent of bradykinesia in Parkinsonian subjects. In C. elegans, sinusoidal movement requires the out-of-phase contraction of dorsal and ventral musculature. 53 In Parkinson patients, D1 and D2 class of DA receptors are proposed as major contributing factors to the development of bradykinesia. 54 The corresponding and muscles. The electrotaxis response is highly stereotypic, reproducible and can be quantified. 20,21 This makes it possible to assess precisely the impact of alterations on worm's behavior by observing its swimming characteristics.
We have shown that the normal electrotaxis response relies on the intact electrosensory system. In the case of osm-5 (human IFT88 homolog) and lin-11 (LIM Homeobox family) mutants that affect amphid neurons, animals showed significantly reduced speed and frequently failed to detect the electric field polarity. While the involvement of amphid neurons and osm-5 in mediating electrosensory behavior on open gel surface was previously reported, 24 our work has revealed novel turning and paralysis phenotypes in osm-5 and lin-11 animals in the microfluidic device assay.
We have provided the evidence for the role of DA neuron signaling in modulating electric fieldinduced swimming responses. This is based on results that DA transporter mutant dat-1 has a reduced activity as judged by slower speed and higher turn time of animals. A similar phenotype was also observed in worms exposed to DA-specific neurotoxins (6-OHDA, MPTP and rotenone). The low doses of toxins (25-700 μM range, for a maximum duration of 8 h) caused electrotaxis defects in the channel without seriously impacting the growth and viability of animals. Thus, the microfluidic platform can be used as an effective and non-invasive tool to detect neuronal abnormalities in worms.
The effect of neurotoxins on C. elegans electrotaxis. Previous studies and our own work have shown that neurotoxins 6-OHDA, MPTP and rotenone cause movement and other abnormalities in C. elegans 27,28 (see Materials and Methods). Some of the phenotypes, such as lethality, could be non-specific and may result from exposure to high doses of toxins. Because DA neurons are not required for survival, the viability defect could result from disruption of other cellular processes. Consistent with this, we found that reducing toxin doses eliminated lethality. Because worms exposed to low doses of toxins appear generally healthy and active on plates, a sensitive assay is needed to monitor DA signaling defects. While one could directly visualize neurons using a GFP reporter, such an approach is slow and subjective. Therefore, it is unlikely to be a sensitive measure of DA neuronal activity. Our work establishes microfluidic electrotaxis as a rapid and sensitive assay to monitor movement and its neuronal basis in worms. The quantification of movement parameters allows comparison between different groups of animals in a reliable manner and eliminates any bias associated with manual counting and judgment.
Aside from the reliable quantitative analysis, our assay provides a unique opportunity to investigate some of the phenotypes caused by DA neuronal loss. During electrotaxis experiments, we observed that toxin-treated worm often failed to coordinate the viability and specific cellular and molecular markers. The existing methods of phenotypic analyses are slow, tedious and prone to human errors. The microfluidic system described here allows for objective and quantitative analysis of worm behavior following drug exposure. With some modifications, including development of automated video capturing and data analysis tools, the system can be used to screen for chemicals affecting neuronal function in a high-throughput manner. As an example, we tested the effect of acetaminophen, a compound with known neuroprotective response in C. elegans. 48 The results showed that acetaminophen protects worms from the toxic effect of 6-OHDA as judged by the normal electrotaxis response in the channel assay. Therefore, our microfluidic electrotaxis system holds promise in chemical screening and identification of potential therapeutic compounds with neuroprotective properties.
Synchronized worms were used for all the assays and were prepared by bleach treatment. 20 Briefly, gravid hermaphrodites were treated with a solution containing commercial bleach and 4 N NaOH (3:2 ratio). The dead worms were washed with M9 buffer and incubated at room temperature for 24 h to allow fertilized embryos to hatch into L1 larvae.
Except for lin-11(n389) animals, all other plate-based and electrotaxis assays were done with 69 h young adults. This stage was chosen based on our finding that almost all synchronized wild-type L1s, when placed on NG-agar plates, reach to adulthood by 69 h at 20°C (97% adult and remaining younger stages, n = 1,002). The lin-11 mutants were tested at 40 h stage because of their egg laying-defective (Egl) phenotype. 57 Molecular biology and transgenics. The dat-1 p ::YFP plasmid pGLC72 was made by amplifying a 710 bp fragment of dat-1 5' genomic region using primers GL563 (5'-AGGAAGCTTCCA GTTTTCACTAAAACGACTCATACACTTCTC-3') and GL564 (5'-ATGGGTACCGGCACCAACTGCATGGCT AAAAATTGTTGAG-3'). The resulting PCR product was digested with HindIII and KpnI and subcloned into pPD136.64 (Fire lab vector, www.addgene.com). pGLC72 was injected into unc-119(ed4) animals to generate stable transgenic lines.
Toxin treatments and optimizations. All toxin treatments were done with synchronized L1 populations. Worms were exposed to toxins for different time periods with mild shaking on a rocking platform. Following exposure, tubes were briefly centrifuged and worm pellets were washed once with M9 buffer. Worms were transferred to NG-agar culture plates.  55 Therefore, it is conceivable that reduced dopamine in toxin-treated worms might affect DOP receptor function resulting in abnormal biphasic activity of muscles. Analysis of mutants affecting these and two other DA receptors (dop-4, D1 class; dop-2, D2 class) in the microfluidics setup should reveal their function in mediating electrotaxis.
Electrotaxis-based behavioral assay as a tool to screen for neuroprotective compounds. Traditional chemical screening using C. elegans involves feeding worms with compounds of interest present in food (e.g., in a 96-well microtiter plate). The subsequent analysis of animals involves monitoring their growth, dat-1 and him-8 and 40 h (post-L1) larvae for lin-11. The corresponding stages of N2 worms were used as controls. Worms were placed, one at a time, on a fresh 1-d-old thin bacterial lawn. Following acclimatization for 5 min. the animal's movement was observed for 30 sec. The beginning and end positions of tracks were marked and the image was captured using a digital camera (Point Grey, FL3-GE-13S2C) attached to Leica S8APO microscope. The movement speed was calculated by NIH ImageJ software (rsbweb.nih.gov/ij/).
Chemotaxis assay was performed as described earlier. 59,60 NaCl 100 mM solution was used as a chemo-attractant. 6-OHDAtreated animals were washed thoroughly and placed at the center of the agar plate. The plate contained a drop of NaCl at one end and water control at the other end. The assay was run for 1 h. After this, animals toward water and NaCl spots were counted and chemotaxis index (CI) was calculated.
Electrotaxis assays and data analysis. The experimental setup of microfluidic electrotaxis and fabrication of channels have been described earlier 20 (Fig. 1). Briefly, soft lithography technique was used to fabricate the microchannel (5 cm long and 300 μm wide) in polydimethylsiloxane (PDMS) (Fig. 1A). The DC electric field strength was set at 3 V/cm. Worms were loaded into the channel using a syringe pump and brought in the field of view. The electric field was activated and each animal was allowed to travel a minimum distance of 5,000 μm in one direction (toward cathode) ( Fig. 1B and C). The electric field was reversed when the worm reached the end of the channel thereby causing it to turn and swim in the opposite direction ( Fig. 1D and E). It should be mentioned here that the movement of the worm inside the microfluidic channel during electrotaxis assay is not affected by the electrokinetic flow. 20 Electrotaxis responses of worms were recorded using a Nikon Coolpix digital camera (P5100) attached to a Leica stereomicroscope. Each data set typically consisted of 10-25 animals. For each animal, the response was monitored for 2-9 min duration and up to 45,000 μm total distance (in both directions). NIH ImageJ was used to analyze the captured videos.
Four swimming parameters, electrotaxis speed, turn time, body bend frequency and electrotaxis time index (ETI), were quantified manually. The electrotaxis speed is an average response of animals 6-OHDA solution is sensitive to light therefore it was kept in the dark.
We modified toxin exposure protocols by lowering the concentration and exposure time. This was necessary because plate-based assays in the past used high doses of chemicals resulting in pleotropic defects such as delayed growth and lethality. 26,27,58 This precluded us from carrying out electrotaxis experiments. Additionally, we were concerned about non-specific effects due to prolonged exposures to high doses of chemicals that could affect neurons other than those involved in DA signaling. For example, L1 animals treated with 5 mM 6-OHDA for 30 min (one of the lowest concentrations reported) showed extreme sluggishness, uncoordinated (Unc) movement, protruding vulva, growth arrest and early larval lethality 58 (data not shown). We were unable to examine such worms in the channel because they were practically immobile and unresponsive to the electric field stimulus. Lowering the 6-OHDA concentration by 50-fold (100 μM) improved the overall health of worms, thereby allowing us to carry out electrotaxis assays.
We also optimized the MPTP and rotenone treatment protocols. It was reported earlier that worms grown in the presence of 1.4 mM MPTP for 3 d (starting L1 larval stage) were uncoordinated and extremely slow growing. 27 In the case of rotenone, a dose of 25 μM for 4 d caused lethality. 28 We found that reducing the rotenone exposure (25 μM rotenone for 12 h) allowed animals to survive but affected their growth and movement. However, none of the above toxin conditions could be used in the channel assay since worms were too sick to move and did not respond to the electric field stimulus. So, we further modified the toxin treatment conditions and found that animals exposed to 700 μM MPTP or 25 μM rotenone up to 8 h were generally healthy on plates and could be used to perform electrotaxis experiments.
Neuroprotection assay. The 10 mM stock solution of acetaminophen (Sigma Aldrich, A7085) was prepared fresh and diluted to a final concentration of 100 μM at the time of the assay. Synchronized L1 stage animals were incubated for 24 h in 100 μM drug containing M9 buffer. They were washed once with M9 and placed in toxin-containing solution for 1 h (100 μM 6-OHDA) or 8 h (700 μM MPTP and 25 μM rotenone). After an additional wash, the animals were transferred to NG-agar plates. The electrotaxis assays were performed on 69 h adults.
Microscopy. Worms were mounted on glass slides containing agar pads and observed using Zeiss AxioImager D1 and Nikon Eclipse Nomarski fluorescence microscopes. Epifluorescence was visualized using a GFP filter (HQ485LP, Chroma Technology USA). Degeneration of DA neurons was examined in 6-OHDAtreated dat-1 p ::YFP transgenic worms. For this, L1 worms were exposed to 6-OHDA for 4 h, washed once with M9 and then plated on NG agar plates. Adults were examined for YFP fluorescence in dopaminergic neurons and their processes.
Plate-based assays. Well-fed synchronized worms (described above) were used to quantify their movement responses on agar plates. The stages were 69 h (post-L1) young adult for osm-5, Figure 8. Electrotactic responses of worms treated with toxins and acetaminophen (para-acetylaminophenol, APAP). Control refers to untreated wild-type N2 animals. Refer to Figure 2 for an explanation of the plot structure and other details. The speed of APAP-treated group is comparable to the control and higher than the corresponding toxin-treated group.
The statistical significance of data was evaluated using unpaired Student's t-test and non-parametric Mann Whitney test. The p values less than 0.05 were considered statistically significant.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Acknowledgments
We thank Devika Sharanya for generating dat-1 p ::YFP transgenic worms and members of BPG, PRS and RM laboratories for feedbacks. Some of the strains used in this study were obtained by Caenorhabditis Genetics Center that is supported by National Institutes of Health -National Center for Research Resources.

Supplemental Materials
Supplemental material may be found here: www.landesbioscience.com/journals/worm/article/24558/ that was obtained by dividing the total swimming distance by elapsed time. In this analysis, only movement toward cathode was used to calculate the distance. Any motion toward anode was ignored. The elapsed time is the duration of the assay. The turn time is the period to complete a U-turn following a switch in the electric field polarity. Up to three turning events were used to determine the average response of each animal. The body bend frequency is the average number of sine waves per second. This was obtained by dividing the total number of sine waves produced by an animal in an experiment by the duration of the assay (in seconds). Only those sine waves were counted that spanned at least half of the channel diameter. Finally, ETI is the measure of electrotaxis response. The ETI of an animal was calculated by dividing the sum of all cathode-directed swimming time by total time of assay. For a given genotype, a lower ETI indicates less cathodedirected motion of animals whereas higher ETI indicates greater amount of time spent in moving toward cathode.