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The association of latent toxoplasmosis and level of serum testosterone in humans

Objectives Latent toxoplasmosis modifies various hormones and behaviors in infected hosts and possibly involves in etiology of different neurologic and psychiatric disorders. The aim of the current study was to assess possible associations between latent toxoplasmosis and testosterone concentration in Toxoplasma infected and free subjects. Briefly, 18–49 year-old participated in the study. After collected blood samples, sera were analyzed for the detection of anti-Toxoplasma IgG antibody. Totally, 76 positive sera were selected as study group (38 from men and 38 from women) and a same number of negative sera as control group. Results Comparison of testosterone concentrations and control groups showed that testosterone concentration in study group was higher than that in control group with statistically significant difference (P = 0.024 and P = 0.043 for men and women, respectively). Significant differences were found in testosterone concentrations and anti-Toxoplasma IgG antibody levels in study and control groups (P < 0.05). Toxoplasmosis can affect the mean concentration of serum testosterone in human. Alteration of testosterone during latent toxoplasmosis can result in alterations in behavioral, physiologic and immunological parameters in long time.

Introduction
Toxoplasma gondii is one of the most common parasitic protozoans in humans which cause toxoplasmosis [1]. The prevalence of toxoplasmosis varies from 20 to 80% in different parts of the world. Humans become infected through the oral route by the consumption of raw or undercooked meat contaminated with tissue cysts and other food products, water or vegetables contaminated with oocysts. Congenital infection can occur via vertical transmission of T. gondii tachyzoites from pregnant mother to developing fetus during the primary infection that could be life threatening for the fetus [2]. Therefore, the accurate diagnosis of acute maternal toxoplasmosis in pregnant women is critical [3]. Latent toxoplasmosis is clinically asymptomatic in immunocompetent hosts. However, the infection is usually long-lasting characterized by the presence of Toxoplasma cysts, typically in nervous and muscular tissues. Furthermore, the infection mostly results in a lifetime protective immunity (humoral and cellular) to reinfection, presenting low levels of anti-Toxoplasma IgG in serum of infected individuals [4]. Latent toxoplasmosis is known to induce various hormonal and behavioral changes in infected humans and animals and may be involved in etiology of different neurologic and psychiatric disorders [5][6][7]. Infected mice and rats have been shown to suffer from impaired motor neuron performance and coordination, deficit learning and reduced avoidance of open spaces and predators [8][9][10][11][12]. These are believed to be evolutionary mechanisms to increase the chance of hosts being eaten by felines [13]. Furthermore, latent toxoplasmosis increases chance of giving birth to males in humans and mice [14,15]. Patients with changed testosterone levels may experience physical symptoms such as dermal hyper reactions including irritation, erythema, hirsutism and acne as well as abnormal growth of muscles, kidney failure and psychological deficits such as mood swings, depression and anxiety [16]. An effect of latent toxoplasmosis on serum testosterone changes is still being discussed by researchers. Published data have shown increased and decreased testosterone levels associated with T. gondii seropositivity in humans [17][18][19]. In the current study, effects of latent toxoplasmosis on serum testosterone were assessed in men and women.

Samples and patients
In this case-control study,  year-old men and women with no clinical complications were participated. Blood samples were collected in clinical laboratories in Tehran, May-September 2013. Information sheets were prepared and demographic questionnaires completed for the participants. Then, 3 ml of whole blood were collected and sera were tested for the detection of anti-Toxoplasma IgG antibody. In total, 76 positive sera were selected as study group (equally from men and women) and further 76 negative as control group.

Serological tests
Enzyme-linked immunosorbent assay (ELISA) was used to detection of anti-Toxoplasma IgG antibody in blood sera. The cut off values of optical densities (OD) were calculated according to a protocol by Hillyer et al. [20]. The OD of each sample was compared with cut off and recorded.

Antigen preparation
Antigen was prepared as previously described [21]. The RH strain of T. gondii was obtained from the Department of Parasitology, Tehran University of Medical Sciences, Tehran, Iran. Briefly, tachyzoites of T. gondii, RH strain were inoculated intraperitoneally into BALB/c mice. After 48-72 h, tachyzoites were collected using peritoneum washing with sterile normal saline (pH 7.2). Tachyzoites were washed with phosphate-buffered saline (PBS pH 7.4) for three times, sonicated in PBS (pH 7.4) and centrifuged at 12,000 g for 1 h at 4 °C. Then, supernatants were collected and protein density was assessed using Bradford method. Animal experiments were done according to Committee for the Update of the Guide for the Care and Use of Laboratory Animals and approved by the Ethical Committee of Tehran University of Medical Sciences for the use of laboratory animals.

Detection of anti-Toxoplasma IgG antibody using ELISA technique
The 96-well microplates (Nunk, Germany) were coated with 5 µg/ml of soluble antigen of T. gondii RH strain in carbonate-bicarbonate buffer (pH 9.6) and stored at 4 °C. Plates were washed for three times with PBST (PBS, 0.05% tween 20) and sera were diluted 1:200 in PBST and 100 µl from diluted sera was added to each well of microplate. After incubation for 1 h at 37 °C and three times of washing, 100 µl of anti-human IgG conjugated with hourseradish peroxidase (HRP) (Dako, Denmark) diluted 1:500 in PBST and added to each well. After incubating and washing, 100 µl of substrate of ortho-phenylenediamine (OPD) (Sigma-Aldrich, USA) was added to wells. The catalytic enzyme was stopped by adding 50 µl of 20% sulfuric acid at a specific time and the absorbance was measured at 490 nm using automated ELISA reader (BIOTEC LX800, USA). Furthermore, all samples were approved for the determination of anti-Toxoplasma IgG using commercial kits (Trinity Biotech Captia, New York, USA) according to the manufacturer's instructions.

Testosterone assessment
Concentration of testosterone was assessed at 37 °C using Roche Cobas ® e 411 Immunoassay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. In the first incubation step, 20 μl of the sample were incubated with a biotinylated monoclonal testosterone-specific antibody and 2-bromoestradiol (to release testosterone). In the second step, streptavidincoated microparticles and a ruthenylated testosterone derivative were added to the mixture. The reaction mixture was transferred to a measuring cell and the microparticles were magnetically captured on the surface of an electrode. Chemiluminescence was measured using photomultiplier and the concentration of testosterone was calculated using calibration curve [22]. Interpretation of the testosterone concentration was based on the manufacturer's recommendation as follows: normal range for men, 2.49-8.36 ng/ml; and for women, 0.084-0.481 ng/ ml. Experiments were carried out in triplicate, and the mean was calculated for each sample.

Statistical analysis
Statistical analyses were carried out using SPSS Software v.16. Data were analyzed using multiple univariate analyses of variance (ANOVA) and Chi square test. Pearson product-moment correlations were used between optical density of ELISA and concentration of testosterone. Comparison of quantitative variants between two groups was assessed by student t test. Data description was carried out by calculating frequencies and 95% confidence intervals. Differences were considered as significant when P ≤ 0.05.

Results
Results of anti-Toxoplasma IgG antibody detection and serum testosterone concentration in infected and non-infected subjects are shown in Table 1. Differences in mean concentrations of testosterone were reported between infected and non-infected subjects (P < 0.05) as testosterone concentration was significantly higher in IgG-positive group than that in IgG-negative one. In infected subjects, 13.2 and 26.3% of men and women had high concentrations of serum testosterone, respectively. The mean concentration of serum testosterone was higher in men and women infected by T. gondii and statistically significant (P = 0.02 and P = 0.04, respectively), compared to that in control group. Furthermore, correlation between the mean OD of ELISA and concentration of testosterone was significant in infected men and women with values of 0.007 and 0.004, respectively. No statistically significant association was found between IgG titers and testosterone levels in men and women in Toxoplasma seropositivity group in comparison with control group (P > 0.05).

Discussion
Parasite-induced changes to the host endocrine system provide a possible mechanism of altering host behaviors. Significant sex differences have been reported regarding host changes in response to T. gondii infection [19]. Testosterone is an important influencing factor in behavior and personality in both sexes. As shown in majority of the studies, increased testosterone was associated with antisocial, aggression and dominance behaviors [23][24][25]. Altered testosterone levels have been observed in T. gondii infections; however the literatures lack consensuses. Evidence suggests that testosterone activation may cause sexual arousal directed towards feline odor in some rodents [26]. Interestingly, castrated male rats do not exhibit loss of fear phenotype, suggesting that testosterone plays a direct role in this behavior [27]. Results of the current study have shown that mean concentrations of serum testosterone are significantly higher in men and women infected by toxoplasmosis, compared to that in control group. Increased concentration of testosterone during latent toxoplasmosis can result in inducted behavioral alterations and immunosuppressive effects characterized by lower cellular immunity [25,28]. Administration of exogenous testosterone can reduce fear in humans and rodents [29]. It is possible that men with increased levels of testosterone have greater chance of Toxoplasma infection either due to impaired immunity or changed behavior and personality profile. For instance, personal tendency to disregard rules of the society can result in lower hygienic standards and hence increase risk of contact with sources of infection [30][31][32][33]. The underlying mechanism for these behavioral alterations are usually thought to be variations in neurotransmitter functions and more specifically due to high levels of dopamine. In addition, there are indications that enhanced testosterone levels play an important role in behavioral abnormalities [34].
Several studies have shown that, direct and indirect evidence exist on increased testosterone in Toxoplasma infected human and animals [35][36][37][38][39]. T. gondii produces high concentrations of testosterone in infected hosts and enhanced mRNA expression of luteinizing hormone receptor (LHR), which regulate the synthesis of testosterone in testes on Leydig cells [27]. The Toxoplasma infected men are about 3 cm taller than Toxoplasma free men and having further muscles and dominant faces [38,39]. Toxoplasma infected men and women have a lower second to fourth digit length ratio in the left hand (2D:4D ratio) and are more likely to give birth to boys than girls [14,40]. The findings of current study is in accordance with James hypothesis (2010) that many parasites and pathogens could change the concentration of steroid hormone. He has demonstrated that infected hosts, often with shifted sex ratio, increase number of males in generations [40]. Testosterone is a hormone which is responsible for the growth of secondary male sexual characteristics. An alternative hypothesis explaining Toxoplasma associated sex ratio shift suggests that the phenomenon is caused by the higher possibility of survival of more immunogenic male embryos by inducing immunosuppression mechanisms [30]. Indeed, both hypotheses may be compatible since the proximate mechanism of immunosuppression remains unknown and may involve the parasite-induced shift in steroid hormones.
The results of present study do not agree with Flegr et al. showed that Toxoplasma infected men had a higher concentration of testosterone while women had a lower concentration of the hormone, compared to control group [19]. Furthermore, Flegr suggested that the personality profiles of infected men and women are different and the opposite direction of the testosterone shift in men compared to women can explain the observed gender specificity of behavioral changes in people infected with Toxoplasma parasite. They have concluded that infected women are warm-hearted, conscientious, outgoing, persistent, and moralistic while infected men are more likely to disregard rules and were more expedient, suspicious, jealous, and dogmatic [19]. Contrary to our results, a controversial study by Kankova et al. has shown a decrease testosterone levels (total testosterone and free testosterone) in female and male laboratory mice infected by virulent strains of Toxoplasma at a latent phase, compared to uninfected controls. This controversy may be seen due to the different parasite strain, which differs in virulence and epidemiological occurrence [41]. Therefore, the parasite genotype seems to be an important parameter influencing the clinical infection in humans [42]. It is possible that the physiological reaction to Toxoplasma infection qualitatively differs between mice and humans. Other reports show that reduced serum and testicular testosterone levels was found in male rats infected by high doses of T. gondii, RH strain compared to controls [43]. Similarly, Oktenli et al. have demonstrated that concentration of follicle stimulating hormone (FSH), luteinizing hormone (LH), free testosterone (FT) and total testosterone (TT) were significantly lower than controls in serum of male patients during acute toxoplasmosis [44]. Results of the current study showed that the mean concentration of serum testosterone was higher in men and women infected by toxoplasmosis, compared to that in control group. Of various mechanisms described in T. gondii for behavioral alterations, increased testosterone seems to play a significant role. Alterations of testosterone during latent toxoplasmosis can affect several behavioral, physiologic and immunological parameters in a long time.

Limitations
In this study the association of latent toxoplasmosis and psychological disorders was not tested in patients. It is suggested to further investigation of direct correlation between latent toxoplasmosis and psychological disorders in animal and human models.

Author contributions
HK designed the experiments and provided important advice for the experiments and financial support. NZ collected the samples and performed the experiments. MM and SS analyzed and interpreted the data and contributed to manuscript preparation. NZ drafted the original manuscript. HK, SS and MM reviewed and revised the manuscript. All authors read and approved the final manuscript.


extfieldsofstudy: Chemistry Medicine

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Rpb9-deficient cells are defective in DNA damage response and require histone H3 acetylation for survival

Rpb9 is a non-essential subunit of RNA polymerase II that is involved in DNA transcription and repair. In budding yeast, deletion of RPB9 causes several phenotypes such as slow growth and temperature sensitivity. We found that simultaneous mutation of multiple N-terminal lysines within histone H3 was lethal in rpb9Δ cells. Our results indicate that hypoacetylation of H3 leads to inefficient repair of DNA double-strand breaks, while activation of the DNA damage checkpoint regulators γH2A and Rad53 is suppressed in Rpb9-deficient cells. Combination of H3 hypoacetylation with the loss of Rpb9 leads to genomic instability, aberrant segregation of chromosomes in mitosis, and eventually to cell death. These results indicate that H3 acetylation becomes essential for efficient DNA repair and cell survival if a DNA damage checkpoint is defective.

. Depletion of Rpb9 is lethal in H3 K9,14,23R cells. Anchor-away strains with Rpb9 depletion system along with wild-type strain (AKY796) and rpb9Δ mutant (AKY1037) were spotted in 10-fold dilutions onto YPD plates containing DMSO (control) or 1µg/ml rapamycin (for Rpb9 depletion). Plates were photographed after 2 days of incubation. Genetic backgrounds of the strains are summarized in the table. FRB tag (fused to C-terminus of Rpb9 protein) and RPL13A-2xFKBP12 are essential components of the anchor-away system; tor1-1 mutation is required for bypass from rapamycin-induced cell cycle arrest.  Figure S2. 10-fold serial dilutions of Rpb9 anchor-away strains along with RAD53 and rad53Δ strains expressing wt H3 or the H3 K9,14,23R mutant were spotted onto synthetic compleyte (SC) plates containing DMSO (+Rpb9) or rapamycin (-Rpb9). Cells were treated with ionizing radiation (50-150 Gy), or plated on media containing 30 µg/ml camptothecin (CPT). Strains AKY1162 and AKY1190 express rpb9-FRB protein that can be removed from cell nucleus by anchor-away technique. rad52Δ strain (AKY1518) is sensitive to various DNA damaging agents and it was used as a positive control for DNA damage induction. Plates were photographed after 3 days of growth at 30°C. Figure S3. Western blot analysis of H2A (A and B) and Rad53 (C and D) phosphorylation in response to MMS treatment in Rpb9-depleted cells. Rpb9 anchor-away strains with wt or K9,14,23R mutant H3 were incubated with DMSO (+Rpb9) or rapamycin (-Rpb9) for 6 hours before 0.01% MMS was added to the cells and samples were taken at indicated time-points. Western blot analysis of H3 (E and F) is shown as a loading control. A positive sample from unrelated experiment was loaded as marker (lane "M" on blots). Glucose + rapamycin (or DMSO) (3h, 6h, 20h) 1.

Experimental scheme:
HO endonuclease Glucose Galactose

HO cut site
Yeast cells express HO endonuclease under the control of galactose-inducible promoter. In glucose-or raffinose-containing medium, expression of HO endonuclease is repressed. When the cells are shifted to galactose-containing medium, expression of HO is induced and the nuclease makes a DSB at its recognition site in the MAT locus.
The formed DNA break is repaired primarily by homologous recombination (HR) using the gene cassettes in HMLα or HMRa loci as donors. As long as the HO endonuclease is expressed, the locus is continuously cut and repaired. During the course of the experiment, the intactness of the MAT locus is detected by PCR. When cells are grown in galactose-containing medium, the majority of cells contain a DSB in HO recognition site and amplification of the MAT locus by PCR is strongly reduced. When HO expression is shut-down in glucose-containing medium, the locus is repaired and can be detected by PCR. Depending on the donor sequence used for DSB repair, the lenght of the PCR product can be either 1.0 kb (MATa) or 1.1 kb (MATα).
1. Cells are pre-grown in raffinose-containing medium. Expression of HO endonuclease is OFF.
2. Expression of HO nuclease is induced in galactose-containing medium for 1.5 hours to induce DSBs in the MAT locus.
3. Rapamycin (or DMSO) is added to growth medium to induce depletion of Rpb9 protein from the cell nucleus. Cells are grown for further 1.5 hours. Expression of HO endonuclease is ON. 4. Expression of HO endonuclease is shut-off by transferring the cells into glucose-containing medium. DNA samples are collected at different time-points to estimate the efficiency of MAT locus repair in the presence or absence of Rpb9 protein in the cell nucleus (DMSO and rapamycin samples, respectively). Figure S4. Overview of the DSB repair assay. In all strains the galactose-inducible HO endonuclease is integrated into ade3 locus and Rpb9 protein can be removed from cell nucleus by the anchor-away technique. HO cuts its recognition site in the MAT locus in chromosome III. The site is repaired mainly by homologous recombination using the HMLα or HMRa loci in the same chromosome as donor sequences. Location of the chromosome III centromere relative to the other loci is indicated as a blue circle. Red arrow above the MAT locus indicates the HO cut site and black bars under the MAT locus indicate the region amplified by PCR to estimate the intactness of the locus. Figure S5. (A and B) Agarose gel electrophoresis analysis of HO cutting and repair. PCR products were obtained from cells before HO induction (Raf), 3 hours after HO induction (Gal) and 3, 6 or 20 hours after repression of HO (Glc). PCR product of the VPS13 locus was used as an internal control. Thermo Scientific GeneRuler 1 kb DNA ladder (M) was used. (B) Different PCR cycles (19,21,26) were tested to make sure that the PCR product formation was in linear range.


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Contralateral Suppression of DPOAEs in Mice after Ouabain Treatment

Medial olivocochlear (MOC) efferent feedback is suggested to protect the ear from acoustic injury and to increase its ability to discriminate sounds against a noisy background. We investigated whether type II spiral ganglion neurons participate in the contralateral suppression of the MOC reflex. The application of ouabain to the round window of the mouse cochlea selectively induced the apoptosis of the type I spiral ganglion neurons, left the peripherin-immunopositive type II spiral ganglion neurons intact, and did not affect outer hairs, as evidenced by the maintenance of the distorted product otoacoustic emissions (DPOAEs). With the ouabain treatment, the threshold of the auditory brainstem response increased significantly and the amplitude of wave I decreased significantly in the ouabain-treated ears, consistent with the loss of type I neurons. Contralateral suppression was measured as reduction in the amplitude of the 2f1−f2 DPOAEs when noise was presented to the opposite ear. Despite the loss of all the type I spiral ganglion neurons, virtually, the amplitude of the contralateral suppression was not significantly different from the control when the suppressor noise was delivered to the treated cochlea. These results are consistent with the type II spiral ganglion neurons providing the sensory input driving contralateral suppression of the MOC reflex.

Introduction
In the cochlea, the neurons of the spiral ganglion emit peripheral processes that extend to the organ of Corti and have central processes that project to the cochlear nuclei via the auditory nerve. There are two types of spiral ganglion neurons (SGNs): (1) myelinated type I spiral ganglion cells, which innervate the inner hair cells and represent 90%-95% of the population; (2) unmyelinated type II ganglion cells, which innervate the outer hair cells (OHCs) and represent 5%-10% of the population. The scarcity and small caliber axons of the type II SGNs make them difficult to study. In consequence, there has been little experimental research into the physiology of the type II spiral SGNs [1].
To magnify low-intensity sounds and compress the dynamic intensity range, cochlear outer hair cells amplify the basilar membrane vibrations in a nonlinear, intensitydependent pattern. Instantaneous waveform distortion is produced in this process, generating new sound frequencies that are absent from the original stimulation. These distortion products can be detected in the ear canal as otoacoustic emissions. The "cochlear amplifier" can be evaluated by measuring the distortion product otoacoustic emissions (DPOAEs) [2].
Medial olivocochlear (MOC) fibers projecting to the cochlea originate on both sides of the medial portion of the superior olivary complex, where they form synapses with the outer hair cells (OHCs). By hyperpolarizing the OHCs, the MOC efferents inhibit the electromotility of the OHCs, thereby reducing the gain of the cochlear amplifier, which manifests as a reduction in the DPOAEs. The MOC reflex includes both contralateral suppression and ipsilateral suppression. The contralateral suppression is commonly detected with a suppressor sound contralateral to the DPOAE test ear [3]. With an intact olivocochlear bundle, the amplitude of DPOAEs decrease quickly after the contralateral noise is turned on. After this "rapid onset adaptation," the response returns to a value similar to that before the contralateral noise [3]. MOC efferent feedback is suggested to protect the ear from acoustic injury and to increase its ability to discriminate sounds against a noisy background [3].
However, the sensory input that drives the MOC efferent reflex has not been identified. A recent study [4], based on a peripherin (Prph) knockout mouse model, proposed that the type II SGNs drive the MOC reflex. Peripherin is strongly expressed in type II (but not type I) SGNs [5]. In the transgenic mouse model, the outer spiral bundle of type II SGNs is largely absent in Prph −/− cochleae [4]. The study found that both contralateral suppression and ipsilateral suppression were almost totally lost in the Prph −/− ears compared with Prph +/+ ears. On the contrary, another report suggested that type II SGNs are not the sensory limb of the cochlear efferent reflex [6]. That study showed that peripherin is also expressed in MOC fibers and that the inactivation of the MOC reflex observed in the peripherin knockout mice could be interpreted as the loss of the MOC function [6]. Therefore, in this study, an ouabain-treated mouse model was used, in which the apoptosis of the type I SGNs was induced, while the type II SGNs remained intact [7][8][9], to explore whether the type II SGNs play a role in the MOC reflex.

Materials and Methods
2.1. Animal Groups and Surgery. The cold method was used to prepare poloxamer 407 gels [10]. Poloxamer 407 was slowly added to cold distilled water to prepare a 20% (weight/weight) stock solution. The ouabain solution was prepared by dissolving ouabain powder in distilled water to produce a 10 mM stock solution. The stock solution was diluted with cold distilled water to produce a solution containing 18% (weight/weight) poloxamer 407 and 2 mM ouabain. This formulation is in a fluid state at room temperature and in the gel state at the body temperature of mice. The gel promotes the prolonged release of ouabain.
The experiments were performed on eight 4-week-old male C57BL/6 mice. The animals were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally (i.p.)). One-third of the initial dose of anesthetic was given when needed. A posteroinferior skin incision was made in the retroauricular area of the right ear. To expose the bulla, the nearby muscles and facial nerve were separated. A small opening was made in the bulla to expose the round window. A 2.5 μl Hamilton syringe was used to apply the ouabain gel described above (1.6 μl) to the round window membrane. Sham operations were conducted in another group of 4-week-old C57BL/6 mice as controls (n = 8). The bulla was covered with bone wax, and a nonabsorbable suture was used to close the incision. The animal was placed on a homeothermic blanket for recovery. Electrophysiological tests of cochlear function were conducted 2 weeks after the surgeries. All the procedures were approved by the Institutional Animal Care and Use Committee of the Eye and ENT Hospital of Fudan University, China.

Cochlear Function
Tests. An auditory-evoked potential and DPOAE workstation (TDT system 3 with RX6 and RX6-2 signal processors; Tucker Davis Technologies, Fort Lauderdale, FL, USA) was used to conduct the cochlear function tests, with the BioSig32 software. The auditory brainstem responses (ABRs) and DPOAEs were recorded as per previous study [4]. The amplitude of the 2f 1 −f 2 (cubic) distortion products and the surrounding noise floor were recorded. The mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). The threshold for ABR was determined as the lowest intensity at which a repeatable ABR waveform could be identified. The wave I component was identified and the peak-to-peak amplitude computed by off-line analysis of stored waveforms. In the DPOAE recordings, the frequency ratio f 2 /f 1 was 1.2 and the intensity of f 1 and f 2 was the same, increasing together in 5 dB steps (from 20 dB SPL to 80 dB SPL). The amplitude of the 2f 1 −f 2 distortion products and the surrounding noise floor were recorded. The DPOAE thresholds were determined based on when the cubic (2f 1 -f 2 ) distortion product reached 5 dB above the noise floor, as the tone intensity increased, as previously described [4].
The contralateral suppression was measured after DPOAE test without dismounting the DPOAE-measuring probe from the untreated ear. The parameter setting for contralateral suppression was optimized based on prior studies [11,12]. The right ears (after surgeries) of ouabaintreated mice were exposed to 76 dB SPL, 13-20 kHz broadband suppressor noise (continuous for 15 s, closed field), whereas the DPOAEs were elicited in the left ears (without surgeries) with 60 dB SPL, 16 kHz primary tones. Shamoperated mice without ouabain treatment served as controls. Three measurements were averaged (4/s) for each recording. The DPOAEs were monitored before and after the suppression noise to obtain the baseline DPOAE measurement. The amplitude of the 2f 1 −f 2 distortion products relative to the noise floor was recorded before, during, and after the noise stimulation. The broadband noise was generated with the Cool Edit Pro software (Adobe Systems, San Jose, CA, USA). The stimulus was delivered as closed field using MF1 Multi-Field Magnetic Speakers (Tucker-Davis Technologies), with a customized coupler. To address the effect of the cross talk to contralateral suppression, we measured contra-noise effects before and after mechanical destruction of the contralateral cochlea (right ears) by opening the cochlear basal turn (n = 6). As shown in the Supplemental Figure 1, the destruction completely eliminated the contralateral suppression.
2.3. Immunofluorescence Staining. Immediately after the dissection of the cochlea, 4% paraformaldehyde in phosphatebuffered saline (PBS) was perfused through the round window and oval window. The cochlea was immersed in the same solution for 2 h at 4°C. For frozen sectioning, the cochlea was decalcified (0.1 M ethylenediaminetetraacetic acid (EDTA)) and cryoprotected in 30% sucrose at 4°C overnight prior to embedding in optimal cutting temperature compound (OCT). Then, the sections were made and mounted on slides for immunofluorescence staining. Immunostaining began with blocking buffer (10% donkey serum in PBS) for 2 h at room temperature. The sections were then incubated at 4°C overnight in a combination of the following primary antibodies: (1) mouse anti-tubulin β3 (Tuj1, BioLegend), diluted 1 : 300; (2) rabbit antiperipherin (Prph, Abcam), diluted 1 : 500. The samples were then incubated at 4°C overnight with a species-appropriate secondary antibody (Alexa Fluor 488-labeled anti-mouse IgG antibody, Alexa Fluor 594-labeled anti-rabbit IgG antibody). The images were captured using fluorescence microscope (NIKON ECLIPSE Ni U, Nikon Instruments Inc., Japan), and Photoshop CS5 software (Adobe Systems, San Jose, CA, USA) was used to adjust contrast and brightness of images.

SGN Counting.
Tubulin β3-(Tuj1-) positive and peripherin-(Prph-) positive neurons in 12 serial midmodiolar sections of the cochlear (120 μm thickness in total) were counted [13]. The sectional area of Rosenthal's canal was calculated from the images with the Photoshop CS5 software (Adobe Systems, San Jose, CA, USA) as the literature [13] described; an image of a standard slide was used to calibrate the scale, which was converted from pixels to micrometers; the outline of Rosenthal's canal was circumscribed in every section; the total number of pixels for Rosenthal's canal was calculated and converted into square micrometers. In Rosenthal's canal, Tuj1-positive and Prph-negative cells were defined as type I SGNs; Tuj1-positive and Prph-positive cells were defined as type II SGNs. The total number of each type of SGN was then divided by the two-dimensional area to obtain the density of SGNs per square millimeter [14].

Statistical
Analysis. Data are expressed as the population mean ± standard errors of the means (SEM) except for the data in Figure 1(b). The statistical analysis was performed with SPSS 11.5 (SPAA Inc., Chicago, IL, USA). Two-way ANOVA was used to compare the thresholds of ABR and DPOAE and the amplitudes of the 2f 1 −f 2 distortion products at 16 kHz and the amplitude of wave I between the ouabaintreated and control groups [4]. Two-way ANOVA was also used to compare contralateral suppression between the ouabain-treated and control groups [4]. One-way ANOVA was used to compare the peak amplitudes of the rapid contralateral suppression between the ouabain-treated and control groups and to compare the numbers of type I and type II SGNs between the ouabain-treated and control groups.

Cochlear Function Tests.
To explore whether type II spiral ganglion neurons participate in the contralateral suppression of the MOC reflex, ouabain was applied to the right cochlea at the round window to eliminate the type I spiral ganglion neurons. This application of ouabain selectively induced the apoptosis of the type I spiral ganglion neurons, but left the type II spiral ganglion neurons intact as reported previously [8]. The sham-operated mice without ouabain treatment were used as controls. Cochlear function was tested after the ouabain treatment. Two weeks after the application of ouabain, the ABR threshold increased significantly compared with that in the control ear (P < 0 01 at 8, 12, 16, 20, and 24 kHz; Figure 2(a)). The ABR thresholds increased by 30-45 dB SPL at all frequencies after ouabain treatment. Besides, the amplitude of wave I at 16 kHz decreased significantly compared with the control group (P < 0 01 at 60, 70, and 80 dB SPL, Figure 2(b)). However, ouabain treatment had no significant effect on the threshold and amplitude of DPOAEs at 16 kHz compared with the control ears (Figures 2(c) and 2(d)), indicating that the cochlear outer hair cells remained intact 2 weeks after the application of ouabain.

Cochlear Histopathology.
We immunostained frozen sections of the whole cochleae for two marker proteins: (1) tubulin β3 (Tuj1), which is expressed in both type I and type II SGNs, and (2) peripherin (Prph), a type III intermediate filament, whose immunoreactivity is restricted to the soma and processes of type II SGNs in the mature cochlea. Figures 3(a) and 3(d) showed that after ouabain treatment, there was a nearly complete elimination of type I SGCs. Figures 3(b) and 3(e) show that nearly all of peripherin-positive type II SGCs remained, which were detected in both the control cochleae and the experimental cochleae 2 weeks after treatment with ouabain.

SGN Counting.
The type I SGNs (Tuj1-positive and Prph-negative) and type II SGNs (Tuj1-positive and Prphpositive) were counted in 12 midmodiolar sections of the ouabain-treated and control cochleae. In the control group, the average densities of type I and type II SGNs were 1898 ± 103/mm 2 and 202 ± 58/mm 2 , respectively. After ouabain treatment, about 99% of the type I SGNs were eliminated and the density of the remaining type I neurons was 10 ± 6/mm 2 (P < 0 01). Meanwhile, the average density of surviving type II SGNs was 190 ± 62/mm 2 . The density of type II SGNs has no significant difference between the control and ouabain treatment groups (P = 0 97). These data indicate that there was no loss of type II neurons and almost total loss of type I neurons after the application of ouabain. These results also suggest that about 9.59% ± 0.39% of the SGNs in the normal mouse ear were type II.

Contralateral Suppression in Mice of Control and
Ouabain-Treated Groups. The functional effect of the MOC reflex, evident as contralateral suppression, was evaluated as a reduction in 2f 1 −f 2 DPOAEs induced by suppressor noise in the ear opposite to that which the DPOAE was measured in. With an intact olivocochlear bundle, the amplitude of distortion products decreased quickly after the contralateral noise was turned on. Thus, wideband noise was delivered to the ouabain-treated ears (right ears) and the DPOAEs were recorded in the left ears as the previous study reported [15]. Sham-operated mice without ouabain treatment served as controls. Both in the control and ouabain-treated groups, suppression was obvious from the onset of the contralateral noise, and the maximal reduction in the DPOAEs occurred within 1 s (Figure 1(a)). The suppression displayed near-complete adaptation after the noise had been presented for 10 s. The ouabain-treated ears showed similar contralateral suppression time course to that in the control group (Figure 1(a), P = 0 35). The amplitude of the peak suppression in the ouabain-treated ears (2.88 ± 0.46 dB, n = 8) did not differ significantly from that in the control group (2.86 ± 0.46 dB, n = 8, Figure 1(b), P = 0 77). The peak suppression in both the control and ouabain-treated ears occurred within 1 s after the onset of the contralateral noise. The control and ouabain-treated ears showed similar rates of shift in their DPOAEs.

Discussion
The afferents of the MOC reflex must originate from either the type I SGNs and/or the type II SGNs. A recent report [4] proposed that the type II neurons drive the MOC reflex.
A peripherin knockout mouse model lacking type II innervation of the cochlea was used in that study. Peripherin is strongly expressed in type II but not type I SGNs [5]. The contralateral and ipsilateral suppression of the MOC reflex was nearly eliminated in the Prph −/− ears compared with that in the Prph +/+ ears. On the contrary, another report suggested that peripherin is also expressed in some MOC fibers.
The loss of MOC function in the peripherin knockout mice can be interpreted as the inactivation of contralateral suppression and ipsilateral suppression [6]. Besides the type II neurons, the function of other peripheral neurons is probably affected in Prph −/− mice because peripherin is widely expressed in the peripheral nervous system. To study the function of type II neurons, an animal model that separates type I and type II neurons without affecting other neurons is required. Therefore, in this study, we used a mouse model in which ouabain was applied unilaterally to the cochlear round window, which induced the apoptosis of type I SGNs but left the type II SGNs intact in the ouabain-treated ear.
The MOC fibers in the other ear (in which DPOAE were elicited) were not affected by ouabain. Contralateral suppression was used to evaluate the inhibition of cochlear amplification by the MOC reflex. Several previous studies [7,8,16,17] have shown that the application of ouabain to the round window causes the nearly complete elimination of the type I SGNs but leaves the type II SGNs intact, with no obvious loss of or damage to the hair cells. The results of our electrophysiological tests of cochlear function and cochlear histopathology in the ouabain-treated ears are consistent with those of the previous studies. Ouabain treatment significantly elevated the ABR threshold and decreased the amplitude of wave I, with no significant shift in the DPOAE threshold or amplitude ( Figure 2). Moreover, the immunofluorescence staining further confirmed that the application of ouabain selectively causes the death of type I neurons, without damaging type II neurons ( Figure 3). After ouabain exposure, the amplitude of contralateral suppression was not significantly different from that in the control group. Because the application of ouabain to the round window of the cochlea induces the apoptosis of type I SGNs but leaves the type II SGNs intact [7][8][9]16], the pathway from the inner hair cells and their type I SGNs to the brain is almost silenced in the ouabain-treated ears. Therefore, the responses of these ears to sound are attributed to an alternative mechanism of auditory sensing in which the outer hair cells and type II SGNs probably participate. The afferents of the MOC suppression reflex must either be type I SGNs and/or type II SGNs. Our results support the hypothesis that type II SGNs exclusively drive the MOC contralateral suppression reflex.
Recent studies suggest that type II SGNs act as cochlear nociceptors, functioning only when the OHCs are damaged [18,19]. Cochlear type II afferents and somatosensory C-fiber nociceptors share anatomical features, physiological properties, and protein expression [19]. Maison et al. [6] proposes that this finding contradicts the hypothesis that the type II SGNs drive the MOC contralateral suppression reflex, because nociceptors respond to traumatically high sound levels, whereas cochlear efferents respond near the hearing threshold. However, Flores et al. speculates that instead of pain, auditory nociception might elicit an axon reflex, an autonomic reaction, or an efferent response, like MOC reflex, to protect the inner ear from further damage [19].
There is only a 10-20 dB difference between the thresholds of MOC neurons and type I neurons [20], and type II neurons must be similarly sensitive to sound if they act as the afferents of this reflex. Recent electrophysiological experiments in isolated rat organs of Corti support this hypothesis. The integration of synaptic input from multiple OHCs by type II SGNs was identified [21]. The length constants of type II SGNs imply that synaptic inputs can sum effectively through the processes of the type II neurons [21]. Experimental records and the computational model both imply that the extended dendrites of the type II neurons can integrate these inputs. It is speculated that the small synaptic inputs elicited by the neurotransmitters released by individual OHCs will sum approximately linearly across the many tens of OHCs innervated by each of the type II SGN neurites. In the computation model, six synchronous synaptic inputs are required to generate a spike. Because the probability of transmitter release by OHCs is low, the simultaneous stimulation of 24 OHCs is required for action potential in the model of type II SGNs. This number is in the range of OHCs connected to type II neurons, estimated in the previous studies [22].
Some studies have reported that type II SGNs are insensitive to sound [23][24][25]. However, the antidromic stimulation used in those studies may not reflect the physical electrical features of type II SGNs. Moreover, the sample sizes in those studies were small, with only 1, 19, and 8 long-latency neurons studied by Robertson [23], Brown [24], and Robertson et al. [25], respectively. According to Robertson et al. [25], in both Robertson's [23] and Brown's [24] studies, it is possible that during the process of recording the SGNs, the opening of the cochlea may have selectively affected the OHCs and thus changed the response of the type II neurons. Robertson et al. and Brown were also unable to successfully fill all the neurons with horseradish peroxidase. Consequently, they could not ensure that the neurons were actually type II SGNs [25]. Another line of evidence inconsistent with our observation is that the antidromic response latencies of type II neurons to brainstem shocks are 6-7 ms and are therefore longer than the sound-evoked latencies of MOC efferents, which are 4.5 ms [26]. However, the point of antidromic activation of the central processes can greatly affect latency. For example, the stimulating electrode used by Brown was inserted into the cochlear nucleus, yet the electrode used by Robertson was set on the auditory nerve [25]. As a result, the mean latency for the long-latency neurons recorded by Brown was nearly five times greater than that recorded by Robertson. Our results support the hypothesis that type II SGNs drive the MOC contralateral suppression reflex. However, the ouabain-exposed mouse model has limitations. Although the morphological evidence showed that there were nearly no type I SGNs in 12 consecutive midmodiolar sections of the ouabain-exposed cochlea, it is possible that the few remaining type I SGNs were able to drive the MOC contralateral suppression reflex. An animal model in which the type II neurons can be manipulated selectively is required for the further exploration of their physiological functions.

Conclusions
After ouabain exposure, the amplitude of the contralateral suppression was not significantly different from that of the control group. Almost no type I spiral ganglion neurons remained in the ouabain-treated cochleae. Thus, the type II spiral ganglion neurons are almost certainly the afferents responsible for contralateral suppression. Our study helps resolve the controversy about whether type II afferents are driving medial olivocochlear (MOC) efferent fibers [4] or not [6].

Conflicts of Interest
The authors declare that they have no conflicts of interest.


s2fieldsofstudy: Biology Psychology Medicine

text:
Propionate Protects Haloperidol-Induced Neurite Lesions Mediated by Neuropeptide Y

Haloperidol is a commonly used antipsychotic drug for treating schizophrenia. Clinical imaging studies have found that haloperidol can cause volume loss of human brain tissue, which is supported by animal studies showing that haloperidol reduces the number of synaptic spines. The mechanism remains unknown. Gut microbiota metabolites, short chain fatty acids including propionate, are reported to have neuroprotective effect and influence gene expression. This study aims to investigate the effect and mechanism of propionate in the protection of neurite lesion induced by haloperidol. This study showed that 10 μM haloperidol (clinical relevant dose) impaired neurite length in human blastoma SH-SY5Y cells, which were confirmed by using primary mouse striatal spiny neurons. We found that haloperidol impaired neurite length were accompanied by a decreased neuropeptide Y (NPY) expression, but no effect on GSK3β signaling. Importantly, this project research found that propionate was capable of protecting against haloperidol-induced neurite lesions and preventing NPY reduction. To confirm this finding, we used specific siRNAs targeting NPY which blocked the protective effect of propionate on haloperidol-induced neurite lesions. Furthermore, since NPY is regulated by the nuclear transcription factor CREB, we measured pCREB that was decreased by haloperidol and was normalized by propionate. Therefore, propionate has a protective effect against pCREB-NPY mediated haloperidol-induced neurite lesions.

INTRODUCTION
Antipsychotic drugs are the primary therapeutic agents used to treat schizophrenia and its allied mental disorders (Huang and Song, 2018). Among them, haloperidol is the firstgeneration antipsychotic drug and widely used to treat schizophrenia patients (Tardy et al., 2014). Haloperidol acts on the dopamine D2 receptors (D2R) and controls psychotic symptoms including hallucinations, delusions, and aggressiveness (Jafari et al., 2012;Dold et al., 2015). However, it can also cause various side effects including extrapyramidal syndrome, tardive dyskinesia, and cerebrovascular events (Hufner et al., 2015). In brain morphology, chronic application of haloperidol has been reported to reduce brain volume (Ho et al., 2011). A number of studies have suggested that chronic or accumulative haloperidol administration can decrease synaptic spines and induce apoptosis (Frost et al., 2010;Nandra and Agius, 2012). A chronic application of haloperidol induces neurite lesion reported in human, animal, and cell-based studies (Kelley et al., 1997;Dorph-Petersen et al., 2005;Critchlow et al., 2006;Ho et al., 2011). A meta-analysis revealed that higher daily haloperidol intake in patients resulted in greater cortical gray matter reduction (Z, −2.31, p = 0.02) (Vita et al., 2015). Animal study shows that macaque monkeys treated with haloperidol for 17 to 27 months have a reduced brain weight by 8-11%, and these reductions were consistent across a number of brain areas (Dorph-Petersen et al., 2005). Another study shows that chronic administration of haloperidol at ∼0.35 mg/kg at 2-week intervals for 1 year significantly reduces neuronal cytoskeleton and spine-associated proteins in the cortices of rhesus monkey, where are rich in dopamine innervation and are implicated in the psychopathology of schizophrenia (Lidow et al., 2001). Therefore, there is an urgent need to search for a way to protect antipsychotic drug-induced neurite lesion.
Our previous study shows that haloperidol decreases neuropeptide Y (NPY) mRNA expression in the rat brain after haloperidol treatment (Huang et al., 2006). NPY is highly co-expressed in GABAergic neurons and is found to be a modulator of the neuroplasticity, neurotransmission, and memory (Gotzsche and Woldbye, 2016). Given these evidence, we have investigated whether or not NPY was involved in haloperidol-induced neurite lesion.
Short chain fatty acid (SCFA) including acetate, propionate, and butyrate are the metabolites produced by gut microbiome fermentation on dietary fiber. SCFA can enter the circulation via monocarboxylate transporters, cross the blood-brain barrier, and thereby enter the central nervous system (Pierre and Pellerin, 2005;Kekuda et al., 2013). More and more evidence show that SCFA regulate cell metabolism (Canfora et al., 2015), neurotransmitter synthesis and release (DeCastro et al., 2005;Shah et al., 2006), epigenetics (Yamawaki et al., 2012), and immune function (Correa-Oliveira et al., 2016). In particular, propionate and butyrate act as the histone deacetylases inhibitors (HDACi). HDACi regulates brain gene expression, improving the healthy state of patients suffering from Parkinson's disease, depression, and schizophrenia (Galland, 2014). However, the neurite protective, at high concentrations, propionate has also been reported to induce autism-like behavioral changes in rats (Macfabe, 2012). Collectively, these reports suggest that propionate may play an important role in neural function. Our study investigated whether or not propionate may be used to prevent haloperidol-induced neurite lesions. Furthermore, we have investigated the CREB-NPY signaling pathway in mediating the neurite protective effect of propionate in haloperidol-induced neurite lesion.

Cell Culture and Treatments
The undifferentiated human SH-SY5Y neuroblastoma cell line were grown in Dulbecco's modified Eagle's medium (DMEM)-F12 supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (FBS) from Bovogen Biologicals (Victoria, Australia). For differentiation, cells were seeded in culture plates coated with MaxGel TM ECM (E0282, Sigma Aldrich, Syndey). In the following day, media was removed and replaced with 10 µM retinoic acid (RA, R2625; Sigma-Aldrich) in DMEM-F12 with 1% FBS. Haloperidol (MP Biomedicals, Solon, OH) was dissolved in 100% dimethyl sulfoxide (Sigma-Aldrich). Sodium propionate was purchased from Sigma-Aldrich. Cells were treated with media containing either haloperidol or haloperidol with different concentrations of propionate. The neurite length was acquired in real time every 6 h for 24 h using IncucyteZoom Machine and analyzed with the Neuro Track software (Sartorius, Michigan).

Primary Striatal Neuronal Culture
Cultured striatal neurons were harvested from postnatal days 0 to 3 of C57Bl mice. Briefly, striatal neurons were gently dissociated with a plastic pipette after digestion with 0.5% trypsin (GIBCO, Los Angeles) at 37 • C for 30 min. Neurons were cultured in neurobasal medium (GIBCO) containing B27 supplement (GIBCO) and 20 mM glutamine (Sigma Aldrich). After 24 h of culture, 5-fluoro-2 -deoxyuridine (Sigma Aldrich) was added at a final concentration of 10 µM to repress the growth of glial cells. Cultures were maintained at 37 • C in a humidified 5% CO 2 incubator for 7 days (DIV7) prior to treatments. All experimental procedures for primary cell culture were approved by the Animal Ethics Committee, University of Wollongong, Australia, and complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Immunofluorescence Assay
Primary striatal neurons were grown to approximately 70% confluence on glass coverslips and treated with either negative control, haloperidol, haloperidol + propionate, or propionate for 24 h before being fixed in 4% formaldehyde for 15 min. Neurons were washed in PBS, and permeabilized with 0.3% Triton X-100 in PBS for 10 min. After blocking with 5% normal donkey serum for 1 h at room temperature, primary antibodies of MAP2 (M4403-2ML, Sigma-Aldrich), NPY, or GAD67 (MAB5406, Millipore, Bedford) were applied in 1% donkey serum in PBS at 4 • C overnight. This was followed by incubation in a secondary antibody Alexa Fluor 488-conjugated donkey antimouse IgG (Invitrogen, Carlsbad, CA) or Alexa Fluor 488conjugated donkey anti-rabbit IgG (Invitrogen, Sydney), Alexa Fluor TM 647-conjugated donkey anti-mouse IgG (Invitrogen), and Alexa Fluor 568 Phalloidin (A12380, Invitrogen) at room temperature for 2 h.
For SH-SY5Y cells, cells were seeded in the ibidi glass bottom dish (ibidi GmbH, Germany), treated with either haloperidol, haloperidol + propionate, propionate, or nil control in differentiated medium for 24 h, and then the above steps were followed by immunofluorescence assays. We applied the primary antibodies including pCREB (sc-101662, Santa Cruz Biotechnology) and MAP2 to cells at 4 • C overnight, followed by incubation in a secondary antibody cocktail of Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen) and Alexa Fluor 647-conjugated donkey anti-mouse IgG (Invitrogen) at room temperature for 2 h. Cells were viewed using 40× or 63× oil immersion objective on a DMI6500B confocal microscope (Leica, Mannheim, Germany). The neurite length and protein expression were measured using the ImageJ Software.

Spine Morphology
Primary striatal neurons were cultured for 14 days (DIV14) and were used for spine morphology study. The procedure was similar to the Immunofluorescence assay. After blocking, neurons were incubated with Alexa Fluor TM 568 Phalloidin (A12380, Invitrogen) for 1 h and washed with PBS. Neurons were viewed using a 63× oil immersion objective on a DMI6500B confocal microscope (Leica, Mannheim, Germany). The number of synaptic spines was measured using ImageJ Software.

Statistics
SPSS program (version 21; Chicago, IL, United States) was used for statistical analysis. One-way analysis of variance (ANOVA) followed by post hoc Tukey's tests was performed for multiple comparisons. Data were expressed as mean ± SEM, and p < 0.05 value was considered statistically significant.

Haloperidol Inhibited Neurite Outgrowth
In order to investigate the effects of haloperidol on neurite morphology, RA-induced differentiated SH-SY5Y cells were incubated with various concentrations of haloperidol (0, 1, 10, 50 µM) for 24 h. The neurite length was measured in realtime. Haloperidol treatment significantly reduced the neurite outgrowth in a dose-response manner [F(3,11) = 145.401, p < 0.001, Figure 1A]. The neurite length was significantly shorter compared with control group after 1, 10, and 50 µM haloperidol treatments (all p < 0.001). As it is known that the plasma concentration of haloperidol-treated patients is between 2 and 10 ng/ml (Volavka et al., 1995), we used 3.8 ng/ml or 10 µM concentration of haloperidol. We repeated the results with 10 µM haloperidol and confirmed its effect on neurite inhibition ( Figure 1B). To confirm these observations, we applied the same concentration of haloperidol in primary striatal neurons. We found that haloperidol treatment at 10 µM for 24 h significantly reduced neurite length visualized by MAP2 staining (p < 0.001, Figures 1C,D).

Haloperidol-Induced Neurite Lesions Did Not Alter GSK3β Signaling but Decreased NPY Expression
We examined GSK3β signaling since this signaling pathway is involved in neurogenesis and synaptic plasticity (Cole, 2013). No changes were detected in pGSK3β and β-catenin expression after 10 µM haloperidol treatment (p > 0.05, Figures 2A-C). However, we found a significant reduction of NPY expression after 10 µM haloperidol treatment in SH-SY5Y cells (p < 0.01, Figures 2D,E). The results suggested that NPY was associated with neurite reduction induced by haloperidol.

Propionate Prevented Neurite Lesion-Induced by Haloperidol
Previous studies suggest that propionate may play important roles in neurodevelopment. We tested whether or not propionate could prevent haloperidol-induced neurite deficit. We treated SH-SY5Y cells with sodium propionate at concentrations of 0, 10, 50, and 100 µM (Fasting plasma concentration in humans is about 24 µM.) for 30 min prior to 10 µM haloperidol treatment. We observed that propionate prevented neurite impairment induced by haloperidol [F(4,11) = 5.357, p = 0.012, Figure 3A]. Specifically, 100 µM propionate completely prevented neurite lesions induced by haloperidol (p < 0.05). Furthermore, we investigated if propionate could prevent neurite lesion in primary mouse striatal neurons. Our results showed that propionate prevented neurite lesion induced by haloperidol [F(3,20) = 11.656, p < 0.001, Figures 3B,D]. Post hoc analysis showed that the neurite lesions was prevented by 100 µM propionate ( Figure 3B, p < 0.01). It has been reported that haloperidol can decrease the number of synaptic spines in the rat striatum (Kelley et al., 1997). We quantified the number of basilar synaptic spines. We found that propionate prevented the loss of synaptic spines-induced by haloperidol [F(3,20) = 9.994, p < 0.001, Figures 3C,D].

Propionate Prevented NPY Reduction Induced by Haloperidol
Since we observed that the neurons with neurite deficits induced by haloperidol have decreased NPY, we examined if propionate could prevent the reduced NPY. We found that propionate completely prevented NPY reduction induced by haloperidol in SH-SY5Y cells [F(3,8) = 17.502, p = 0.001, Figure 4A]. This result supported our above discovery that NPY is involved in haloperidol-induced neurite lesions and propionate prevented neurite lesions were involved in the regulation of NPY. Furthermore, we used primary mouse striatal neurons to validate our finding. Again, we found that haloperidol significantly reduced NPY, which could be prevented by propionate in Further characterization was performed using glutamic acid decarboxylase 67 (GAD67) antibody staining for GABA synthesis (Chattopadhyaya et al., 2007). We showed these haloperidolresponding striatal neurons contained both NPY and GABA ( Figure 4C).
Furthermore, to confirm the role of NPY in propionateinduced neuroprotection against haloperidol-induced neurite lesions, we used specific siRNA to knock down NPY in SH-SY5Y cells (Figures 5A-C). In this case, propionate was no longer able to protect the neurite lesion-induced by haloperidol [F(3,8) = 11.788, p = 0.003, Figure 5A]. As shown that the neurite lesions induced by haloperidol (p < 0.01) was protected by 100 µM propionate (p < 0.05), while siNPY abolished the neural protective effects of propionate. Western blotting results showed that the NPY was reduced in NPY-siRNA treated cells compared with the cells without NPY-siRNA treatment (p < 0.05, Figures 5B,C). Similarly, propionate prevented neurite lesion in primary mouse striatal neurons but not after NPY-siRNA treatment [F(3,20) = 29.663, p < 0.001, Figures 5D,E]. These results indicated that propionate protective effect was mediated by NPY.

CREB Phosphorylation Was Involved in the Neuronal Protective Effect of Propionate
The cAMP responsive element binding protein (CREB) is a ubiquitous transcription factor located in CRE promoter regions, which modulates the transcription of genes with cAMP responsive elements (CRE). Since it is known that CREB gene transcription factor regulates NPY expression in neurons, we investigated possible correlations between phosphorylated CREB in our propionate treated cells. As expected, we observed that haloperidol decreased CREB phosphorylation, which was prevented by propionate; however, propionate alone did not alter CREB phosphorylation [F(3,20) = 14.741, p < 0.001, Figures 6A,B]. These data suggested that propionate could prevent the down-regulation of CREB phosphorylation induced by haloperidol, which in turn prevented down-regulation of NPY and neurite lesions (Figure 7).

DISCUSSION
Antipsychotic drugs are widely used in treating schizophrenia and allied disorders. However, antipsychotic drug treatment Mean ± SEM, * p < 0.05, * * p < 0.01, * * * p < 0.001. could result in neurite lesion in brain areas, rich in dopamine D2R (Huang and Song, 2018). This is because D2R plays an important role in neurite growth and synaptogenesis (Jia et al., 2013) and virtually all antipsychotic drugs have D2R antagonist properties (Jafari et al., 2012;Huang and Song, 2018). Haloperidol is a typical antipsychotic drug having strong D2R antagonist property. Plasma concentrations of patients treated with haloperidol are between 2 to 10 ng/ml (Volavka et al., 1995). This study used a clinical relevant dose 3.8 ng/ml (equivalent to 10 µM; molecular weight of haloperidol is 376 g/mol), which resulted in neurite lesions in both SH-SY5Y and primary striatal neurons. A previous study has shown that 0.1 µM haloperidol treatment decreases the dendritic spine density as well as spinesenriched proteins of rat hippocampal neurons (Critchlow et al., 2006). Similarly, chronic haloperidol treatment (1.5 mg/kg daily similar to human dose) results in 58% reduction of spine density in rat striatum (Kelley et al., 1997). These data provide strong evidence that clinically used doses of haloperidol may cause neurite lesion.
Propionate is a short chain fatty acid which is a product of gut microbiome fermentation of dietary fiber (Neis et al., 2018). Short chain fatty acids can affect brain function and behavior (Selkrig et al., 2014). Our study showed that propionate prevented neurite lesions-induced by haloperidol. Previous study has suggested that propionate can have significant effects on brain neurites. For example, children with autistic spectrum disorders are characterized by elevated concentrations of propionate, which may link to exaggerated neural synaptic spine formation (Penzes et al., 2011;Frye et al., 2015). Introducing propionate into brain ventricle could induce autism-like behavioral changes in rats (MacFabe et al., 2007(MacFabe et al., , 2011. Our study showed that propionate protected the neurite and synaptic spine lesion induced by haloperidol supporting the effect of propionate on promoting neurite outgrowth. NPY is highly expressed in striatal GABAergic neurons and regulates GABA and glutamate release potentially contributing to neuroprotection, learning, and memory (Gotzsche and Woldbye, 2016). The present study showed that haloperidol decreased NPY expression in both human blastoma SH-SY5Y and primary striatal neurons. This is in consistent with our early in vivo study, which we have demonstrated that haloperidol decreases NPY expression in the rat amygdala and hippocampus (Huang et al., 2006). Other studies have shown that haloperidol decreases NPY in the striatum, but increases in the hypothalamus (Gruber and Mathe, 2000). The present study showed that propionate prevented haloperidol-induced NPY reduction in SH-SY5Y and striatal neurons. When we used NPY-siRNA to decrease NPY activity, we found that the protective effect of propionate against haloperidol-induced lesion in neurites was eliminated. Collectively, these results support the idea that the NPY pathway was involved in the prevention of haloperidol-induced neurite lesion by propionate.
The mechanism underlying haloperidol decreasing NPY expression is not clear. One possibility could be via CREB signaling. CREB is a transcriptional coactivator involved in the regulation of synaptic plasticity and long-term memory through activation of gene transcription (Vecsey et al., 2007). It is known that the CREB pathway regulates NPY expression (Wand, 2005). It has also been demonstrated that D2R regulates CREB. For example, D2R agonist quinpirole stimulates CREB phosphorylation by activating protein kinase C and Ca 2+ /calmodulin-dependent protein kinase (Yan et al., 1999).
In addition, GSK3β signaling pathway is involved in neurogenesis and synaptic plasticity (Cole, 2013). Previous studies have reported that haloperidol does not change the GSK3β pathway in SH-SY5Y cells and in primary hippocampal neurons (Park et al., 2009(Park et al., , 2011. In agreement with their studies, we did not find a change of GSK3β signaling after haloperidol treatment. Our study showed that haloperidol reduced CERB phosphorylation, which was prevented by propionate. Therefore, it is suggested that haloperidol may act on other D2R protein kinase dependent pathway to inhibit pCREB rather than the pGSK3β signaling pathway as per previous observations (Borroto-Escuela et al., 2013;Fuxe et al., 2014).
It is known that short chain fatty acids are ligands for G-protein coupled receptors GPR41 and GPR43 (Tan et al., 2014), which have no other known ligands (Tazoe et al., 2008). Until now, no GPR41 or GPR43 receptors have been reported in the brain. On the other hand, it is known that short chain fatty acids can directly enter brain and interact with neurons (Erny et al., 2015;Stilling et al., 2016). Our study showed that propionate prevented haloperidol-induced neurite lesions in a dose-dependent manner. The concentration of propionate is between 14 to 19 mM in human feces (Schwiertz et al., 2010) and 19.4 to 28.5 µM in human fasting plasma, which could vary depending on the detection method used or a person's dietary profile (De Filippo et al., 2010). It is possible that increasing propionate by either delivering highly concentrated propionate capsule or providing selected dietary fiber may provide a possible protective effect against neurite lesion.

CONCLUSION
Our study showed that haloperidol reduced neural pCREB-NPY signaling, which was involved in neurite and synaptic spine lesions. Propionate prevented haloperidol-induced neurite lesions via increased pCREB-NPY signaling in vitro. Further study needs to be performed to examine if propionate could protect neurite lesion induced by haloperidol in vivo.

AUTHOR CONTRIBUTIONS
XH, MH, PZ, and YX provided substantial contributions to the conception or design of the work. MH performed the acquisition and analysis of data for the work. XH, MH, ZB, YY, RT, AJ, and KZ were involved in the interpretation of data and manuscript preparation for important intellectual content. All authors have read the paper and agreed to be authors on the paper.