Deltamethrin Increases Neurite Outgrowth in Cortical Neurons through Endogenous BDNF / TrkB Pathways

Deltamethrin (DM), a type II pyrethroid, robustly increases brain-derived neurotrophic factor (Bdnf) expression and has a neurotrophic effect in primary cultures of rat cortical neurons. In this study, we investigated the effect of DM on neurite morphology in cultured rat cortical neurons. DM significantly increased neurite outgrowth, but this increase was abolished when the BDNF scavenger tropomyosin receptor kinase B (TrkB)-Fc was added 10 min before the DM treatment. In contrast, the addition of TrkB-Fc 1 h after the treatment did not affect DM-induced neurite outgrowth. Our previous research has indicated that type II, but not type I, pyrethroids have the ability to induce Bdnf mRNA expression, but neither permethrin nor cypermethrin, which are type I and type II pyrethroids, respectively, affected neurite outgrowth in the current study. These results suggest that this effect is not due to increased Bdnf expression, and the effect is unique to DM. We previously demonstrated that calcineurin plays a role in the DM-mediated induction of Bdnf expression. However, the calcineurin inhibitor FK506 did not significantly affect DM-induced neurite outgrowth. DMinduced neurite outgrowth was abolished by U0126 and rapamycin, indicating the involvement of the mitogenactivated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) pathways. Taken together, these findings suggest that DM activates endogenous BDNF/TrkB-mediated MAPK and mTOR pathways, thereby increasing neurite outgrowth.

Pyrethroids have been associated with some toxicity because they target the nervous systems of both mammals and insects.The most common symptom of acute poisoning by type I pyrethroids, which are devoid of an α-cyano moiety, is tremor syndrome (T-syndrome), and that by type II pyrethroids, which have an α-cyano moiety, is choreoathetosis with salivation syndrome (CS-syndrome) (Verschoyle and Barnes, 1972;Barnes and Verschoyle, 1974;Verschoyle and Aldridge, 1980).Recent epidemiological studies have shown a link between environmental and occupational exposure to pyrethroids and abnormalities such as changes in sperm quality and reproductive hormones (Saillenfait et al., 2015).Furthermore, prenatal exposure to pyrethroids appears to be a risk factor for neurodevelopmental delay and autism spectrum disorders (Shelton et al., 2014).Therefore, it is important to elucidate the effects of pyrethroids on the nervous system.
We previously demonstrated that deltamethrin (DM), a type II pyrethroid, triggers a prolonged increase in intracellular calcium concentrations and strongly elevates brainderived neurotrophic factor (Bdnf) expression in primary cultures of rat cortical neurons (Ihara et al., 2012).Furthermore, DM has an antidepressant-like effect in mice (Takasaki et al., 2013).Although DM has a neurotrophic effect and increases neurite outgrowth in cultured neurons (Ihara et al., 2012), it remains unclear how DM affects neurite outgrowth.In the present study, we found that DM increases neurite outgrowth in cultured rat cortical neurons through tropomyosin receptor kinase B (TrkB)-mediated signaling pathways via endogenous BDNF, probably without de novo Bdnf transcription.Our present study would provide insight into one of the underlying molecular and cellular mechanisms that DM may be a risk factor for neurodevelopmental disorders, possibly mediated by disturbing neuronal network in the developing brain.

Cell culture
Primary cultures of cortical neurons were prepared from the cerebral cortices of 17-day-old Sprague-Dawley rat embryos (Japan SLC, Hamamatsu, Japan) as described previously (Imamura et al., 2006;Ihara et al., 2012).Maternal rat and embryos were deeply anesthetized by sodium pentobarbital to obtain rat embryonic brains.Maternal rat was euthanatized by carbon dioxide and rat embryos were done by decapitation under deep anesthesia.All animal care and experiments were approved by the Animal Experiment Committee of the University of Toyama (Approval number: A2011PHA-4) and were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Toyama.Cells were plated at 7.0×10 5 cells/well onto 18-mm coverslips.The coverslips were placed in 12-well plates (AGC ASAHI GLASS, Tokyo, Japan) coated with poly-D-lysine (Sigma-Aldrich).

Analysis of neurite outgrowth
To examine how DM affects neurite outgrowth, a green fluorescent protein (GFP) expression vector was transfected into cells at 4 days in vitro using the calcium phosphate co-precipitation method, as described previously (Ishikawa et al., 2010;Ihara et al., 2012).Two days after DNA transfection, the cells were treated with DM and/or other reagents, and immunostaining was performed with anti-GFP (MBL, Nagoya, Japan) and antimicrotubule-associated protein 2 (MAP2) antibodies (Sigma-Aldrich) to identify GFP-positive neurons.It has been previously reported that low molecular weight form of MAP2, MAP2C, is found in both dendrites and axons in immature developing neurons (Meichsner et al., 1993).Probably due to this reason, it was likely hard to distinguish dendrites and axons in this study.Therefore, we focused on GFP and MAP2 positive cells, and evaluated changes in morphology of all neurites.Changes in neurite morphology were evaluated by Sholl analysis (Fig. 1A), as described previously (Sholl, 1953;Ishikawa et al., 2010;Ihara et al., 2012).A series of concentric circles at 20-μm intervals were centered on the cell body and the number of intersections with GFP-positive processes was recorded as an index of neurite outgrowth.Total neurite length was also measured by tracing all of the neurites starting at the cell body and extending within a 400-μm-diameter circle using Image J software.All morphological changes were blindly examined.We analyzed morphologies of about 55 neurons in each experiment.All experiments were independently repeated at least three times (n=3-5).

Statistical analysis
All values are expressed as the mean±SEM.Statistical analyses were performed using repeated measure ANOVA with the Bonferroni/Dunn test (Sholl analysis) or one-way ANOVA with Scheffé' s F-test (neurite length).

DM increases neurite outgrowth in cultured rat cortical neurons
Treatment of cortical neurons with DM increased neurite outgrowth (Fig. 1B, C), as previously reported (Ihara et al., 2012).We evaluated the effect of DM on neurite outgrowth using Sholl analysis (Sholl, 1953;Ishikawa et al., 2010;Ihara et al., 2012) (Fig. 1A).DMSO, a solvent for DM, did not significantly affect neurite morphology (Fig. 1B).In contrast, neurite outgrowth significantly was enhanced 3 h after DM treatment, and the increases continued 24 h after treatment (Fig. 1C).However, neurite outgrowth was likely to decline 24 h after the treatment (Fig. 1B-D).We also examined the effect of DM on neurite length of cultured cortical neurons.Similarly, we observed that DM increased neurite length 6 h after the treatment in particular (Fig. 1E).On the other hand, the total number of crossings and neurite length also gradually increased in the absence of DM (Fig. 1D, E), possibly because of physiological neurite outgrowth during culture.Therefore, significant effect of DM on the total number of crossings and neurite length was not observed 12 and 24 h after the treatment (Fig. 1D, E).
We previously reported that DM robustly increased Bdnf mRNA expression levels in cultured rat cortical neurons (Ihara et al., 2012).Furthermore, the DM-induced neurite outgrowth was abolished when TrkB-Fc, which blocks BDNF function, was added prior to DM treatment (Ihara et al., 2012).This result indicated that endogenous BDNF is involved in DM-induced neurite outgrowth.Because type II, but not type I, pyrethroids have the ability to induce Bdnf mRNA expression in cultured rat cortical neurons (Imamura et al., 2006), we hypothesized that type II, but not type I, pyrethroids would increase neurite outgrowth.Therefore, we tested permethrin (PM), a type I pyrethroid, and cypermethrin (CM), a type II pyrethroid in our current assay.Unexpectedly, in contrast to DM, neither PM nor CM significantly affected neurite outgrowth (Fig. 2).These results suggest that, among the pyrethroids, DM has a unique ability to affect neurite outgrowth in cultured rat cortical neurons.

Effect of TrkB-Fc on DM-induced neurite outgrowth
Consistent with our previous study (Ihara et al., 2012), DM-induced neurite outgrowth was almost fully abolished when TrkB-Fc was added to the neuronal culture 10 min before DM treatment (Fig. 3A).In contrast, DM-induced neurite outgrowth was unaffected when TrkB-Fc was added 1 h after treatment (Fig. 3B).These results suggest that, although endogenous BDNF is necessary for DM-induced increase in neurite outgrowth, DM-induced endogenous BDNF signaling that contributes to DM-induced neurite outgrowth would occur only in the early phase (possibly within 1 h after the DM treatment).In support, transient but slight increases in phosphorylation of TrkB and extracellular signal-regulated kinase (ERK) 1/2 were likely observed 10 min after the DM treatment (Supplementary Fig. 1A).In addition, we found that the cellular levels of BDNF protein did not change 3 h after the DM treatment (Supplementary Fig. 1B).Taken together with the results showing that addition of TrkB-Fc after DM treatment did not affect DMinduced neurite outgrowth, the effect of DM may be mediated by pre-existing BDNF protein in cortical neurons.On the other hand, we have previously confirmed that sole addition of TrkB-Fc did not affect neurite morphology of cortical neurons (Ihara et al., 2012).

DM-induced neurite outgrowth is dependent on the MAPK and mTOR pathways
We previously found that the DM-induced increase in Bdnf mRNA levels was mediated by the ERK/mitogen-activated protein kinase (MAPK) and calcineurin pathways (Ihara et al., 2012).Among these pathways, calcineurin plays a particularly important role in the DM-mediated induction of Bdnf mRNA expression (Ihara et al., 2012).However, the calcineurin inhibitor FK506 did not significantly suppress DM-induced neurite outgrowth (Fig. 4).In contrast, U0126, a MAPK/ERK kinase 1/2 inhibitor, significantly suppressed the effect of DM (Fig. 4).These results suggest that the TrkB/MAPK pathway plays a key role in the DM-induced increase in neurite outgrowth.Because FK506 did not significantly affect neurite outgrowth, de novo Bdnf expression is unlikely to be necessary for the DM-triggered increase in neurite outgrowth.
Mammalian target of rapamycin (mTOR) has been repor-ted to regulate dendritic morphology (Kumar et al., 2005).Furthermore, BDNF has been shown to control local protein synthesis via mTOR (Takei et al., 2004).Therefore, we examined the effect of rapamycin on DM-induced neurite outgrowth.Although sole addition of rapamycin slightly, but not significantly, increased neurite outgrowth, the DMinduced neurite outgrowth was not observed in the presence of rapamycin (Fig. 5), suggesting that rapamycin blocked the DM-induced neurite outgrowth.These results indicate that DM regulates neurite outgrowth in cortical neurons through the ERK/MAPK and mTOR pathways via endogenous BDNF/TrkB activation.

Discussion
We have demonstrated the involvement of endogenous BDNF in DM-induced neurite outgrowth of cultured rat cortical neurons (Fig. 6).The results obtained using a series of inhibitors and other pyrethroids suggest that the enhancement of neurite outgrowth by DM would be dependent on endogenous BDNF/TrkB-mediated signaling but independent on de novo Bdnf expression.Although the main molecular targets of pyrethroids are VGSCs, PM and CM did not significantly affect neurite outgrowth.In previous research, DM-induced neurite outgrowth was abolished by nicardipine, indicating the involvement of L-type voltage-dependent calcium channels (L-VDCC) (Ihara et al., 2012).We also found previously that DM produced a prolonged increase in intracellular calcium concentrations (Ihara et al., 2012).CM induces calcium influx into neocortical neurons less potently than DM, while PM has no effect (Cao et al., 2011).These results suggest that the ability to induce calcium influx into neurons, probably via L-VDCC, is necessary for increasing neurite outgrowth.
Because an increase in calcium levels in neurons triggers regulated secretion of BDNF protein (Kuczewski et al., 2009), it is plausible that DM increases intracellular calcium concentration, resulting in increased secretion of BDNF.Although we tried to measure secreted endogenous BDNF protein in the culture medium, the BDNF protein level in the medium was nearly undetectable, possibly due to immature neurons.However, our preliminary data suggested only a slight increase in secreted BDNF protein after DM treatment (Our unpublished observations).We observed a slight and transient increase in phosphorylation of TrkB and its downstream kinase ERK 1/2 after the DM treatment.Therefore, it is possible that DM slightly increases endogenous BDNF release by inducing calcium influx into the neurons, and secreted BDNF then increases neurite outgrowth via TrkB/MAPK and mTOR pathways.This transient activation of endogenous BDNF/TrkB signaling under the DM treatment might be one of the reason why the effect of DM on neurite outgrowth was optimally observed 6 h after the treatment and the effect was likely to decline 24 h after the treatment.In any case, because of the potent neurotrophic effect of BDNF, a slight increase in BDNF secretion by DM is likely sufficient to enhance neurite outgrowth.However, de novo BDNF protein might also contribute to DM-induced neurite outgrowth, and its contribution should be further examined in future.
MAPK and mTOR have been shown to be involved in the regulation of dendritic morphology (Kumar et al., 2005;Jaworski et al., 2005), which is consistent with our present findings that U0126 and rapamycin significantly suppress DM-induced neurite outgrowth.Secreted BDNF regulates local dendritic growth in nearby neurons (Horch and Katz, 2002) and activates local translation in dendrites through mTOR pathways (Takei et al., 2004).These reports, together with our current results, suggest that DM enhances endogenous BDNF signaling, which locally activates translation machinery via MAPK and mTOR pathways, resulting in an increase in neurite outgrowth.Crosstalk between the MAPK and mTOR pathways has been reported to be involved in the regulation of cell survival, differentiation and proliferation in response to biological cues (Mendoza et al., 2011).Furthermore, functional interactions between the ERK/MAPK and mTOR pathways up-regulate the dendritic translation machinery in long-term potentiation (Tsokas et al., 2007).Consistent with these observations, the addition of U0126 or rapamycin almost completely suppressed the effect of DM in the present study, suggesting that the MAPK and mTOR pathways coordinately regulate neurite outgrowth triggered by the insecticide.
Because DM strongly increases Bdnf expression in neurons (Imamura et al., 2006;Ihara et al., 2012), DM is anticipated to have trophic effects on neurons.Indeed, we previously reported that DM significantly increases the viability of cultured rat cortical cells (Ihara et al., 2012).Furthermore, DM has an antidepressant-like effect, which is abolished by Trk inhibitor K252a (Takasaki et al., 2013).Therefore, DM may be beneficial for the adult brain.However, DM has been found to negatively affect brain function during development.For example, compared with young adult rats, preweanling and weanling rats exhibit markedly elevated brain DM concentrations after exposure to the insecticide (Kim et al., 2010).Developmental exposure to DM has been reported to be a risk factor for attentiondeficit/hyperactivity disorder (Richardson et al., 2015).In addition, childhood exposure to DM at low levels may be related to neurocognitive impairments by 6 years of age (Viel et al., 2015).These neurotoxic effects of DM in the young may be due to immaturity of the blood-brain barriers.
Taken together, we demonstrated that DM has an ability to increase neurite outgrowth of neurons, which is mediated by endogenous BDNF/TrkB-dependent MAPK and mTOR pathways.Our present findings, along with previous observations, suggest that DM disrupts the formation of neural networks, particularly in the developing brain.Despite its possible beneficial effects on the nervous system and in psychiatric disorders such as depression, DM exposure during development may result in an elevated risk of neurological disorders.

Fig. 1 .
Fig. 1.Effect of DM treatment on neurite outgrowth in rat cortical neurons.(A) Sholl analysis used in this study.To identify GFP-positive neurons, immunostaining was performed with anti-GFP and anti-MAP2 antibodies.Changes in neurite outgrowth were evaluated by Sholl analysis.A series of concentric circles at 20-μm intervals were centered on the cell body and the number of intersections with GFP-positive processes was recorded as an index of neurite outgrowth.(B, C) Representative images of cultured rat cortical neurons 6 h after treatment with DMSO (B) or 1 μM DM (C) (Scale bar=100 μm) and time-course of changes in the number of crossings 0, 3, 6, 12, or 24 h after treatment with DMSO (B) or 1 μM DM (C).*p<0.05 and **p<0.01versus 0 h, NS: not significant.(D, E) Time-course of changes in the total number of crossings (D) and the total length of neurites (E) in the absence or presence of DM. **p<0.01versus DM (-) at the same time points.The experiment was repeated independently three times.