Introduction

Neospora caninum is an intracellular parasite that naturally infects dogs and cattle1. N. caninum is closely related to Toxoplasma gondii and was reported for the first time in 19882. There is no evidence of N. caninum infectivity in humans, but serological evidence suggests that humans can be exposed to N. caninum3. N. caninum causes a variety of neurological symptoms such as pelvic limb paralysis and rigid hyperextension, which are particular to and often observed in dogs4,5,6. In cattle, N. caninum is one of the main causes of abortion and calves vertically infected with the parasites show neurological disorders1.

Activated microglia produce pro-inflammatory cytokines, nitric oxide (NO) and excitatory amino acids7 and thus play a central role in the innate immune response in the central nervous system (CNS). These microglial responses are also strongly related to neuronal disorders and neurodegeneration. Neuroinflammatory activation can lead to altered levels of neurotransmitters such as glutamate (Glu), dopamine (DA), 5-hydroxytryptamine (5-HT). Furthermore, expression of immediately early genes (IEG) is regulated by neuronal activity. c-Fos is a regulatory transcription factor which can indirectly influence cell function depending on the genes that they regulate8,9,10. Expression of activity-dependent cytoskeletal protein (Arc) is induced in response to synaptic activity and implicated in synaptic plasticity11.

Information about pathogenicity and immune response of neosporosis has been derived mainly from rodent models12. Mice infected with N. caninum develop various kinds of nervous symptoms such as wryneck, circular movement and paralytic gait13,14,15. Thus, mouse models seem to be suitable for analyses of brain pathology during N. caninum infection. However, few studies have been done to investigate the effects of N. caninum infection on neuronal disorder and levels of neurotransmitters. In our previous study, we investigated gene expression profiles and histopathological changes in the brain of BALB/c mice infected with N. caninum. Our findings indicated that N. caninum infection stimulated the immune response in the brain16, because BALB/c are susceptible to N. caninum and exhibit encephalitis caused by the infection15. Although natural hosts chronically infected with N. caninum are usually asymptomatic, the asymptomatic to symptomatic mechanisms of neosporosis onset is still unknown. C57BL/6 male mice are a suitable model for the asymptomatic condition because this strain is relatively resistant to intraperitoneal infection of N. caninum (strain Nc-1). Thus, to understand the onset mechanism of neosporosis, we examined sickness behaviour, parasite distribution, histopathological lesions, levels of neurotransmitters (monoamines and amino acids) and expression of IEGs using asymptomatic C57BL/6 male mice during subacute infection with N. caninum. Our findings provide insight into neosporosis associated with brain dysfunction via inflammation, dysregulation of neurotransmitters and downregulation of IEGs.

Results

Effect of N. caninum infection on mice behaviour

In our experimental model using C57BL/6 male mice, typical acute stage symptoms and neurological symptoms were not observed in the infected animals during the experimental period. However, infected mice showed differences in locomotor activity on the open field test (Fig. 1). Total distance travelled, average speed and rearing counts were significantly decreased in mice infected with N. caninum. This result suggests N. caninum infection may affect the brain function of asymptomatic mice.

Figure 1
figure 1

Effects of N. caninum infection on behaviour of mice.

Changes in the locomotor activity and exploration parameters at 30 days post infection (dpi): (a) total distance travelled (cm), (b) average speed (cm/sec) and (c) rearing counts (counts/5 min). Data were summarised from two independent experiments. To reserve the number of mice required in N. caninum-infected group, each experiment used uninfected, n = 8; N. caninum-infected mice, n = 12. Because one mouse died at 28 dpi in N. caninum-infected, the total numbers of uninfected mice were n = 16; N. caninum-infected mice, n = 23. Significant differences were determined by unpaired t tests (*p < 0.05, **p < 0.01, ****p < 0.0001).

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Histopathological lesions and parasite distribution in distinct brain regions

We analysed eight regions of the brain histopathologically (Fig. 2). Histopathological lesions, including perivascular cuff, mononuclear cellular meningitis, glial cell activation and focal necrosis were observed in the brains of infected mice. Although there were no significant differences between brain regions, the pathological score tended to be lower in the cerebellum compared with other regions (Fig. 2(b)). Next, parasite load in distinct brain regions was examined by quantitative PCR (Fig. 2(c)). N. caninum was observed in all brain regions examined, but there were no significant differences among the brain regions. This result indicates that N. caninum infection may induce wide-ranging histopathological lesions in the brains of asymptomatic mice.

Figure 2
figure 2

Histopathological lesions and parasite load in different brain regions of N. caninum-infected mice.

(a) Representative examples of histopathological lesions from brains of N. caninum-infected mice. score 1: slight meningitis, score 2: mild perivascular cuffs, score 3: moderate inflammatory cell infiltration, score 4: extensive necrosis with rarefaction. (b) Total pathological score for each brain region. Brain samples were collected at 45 days post infection (dpi). Histopathological lesions were scored as above. Minimum value = 0 and maximum value = 16. N. caninum-infected mice, n = 5 from one experiment. (c) Parasite number per 50 ng tissue DNA. Brain samples were collected at 54 dpi. N. caninum-infected mice, n = 12 from one experiment. Significant differences were analysed by one-way ANOVA with Tukey’s post hoc test, but there were no significant differences.

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Expression levels of interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) in brains of N. caninum-infected mice

We examined the expression levels of IFN-γ and TNF-α in five brain regions involved in neuroinflammation and CNS disorders mediated via activated microglia. Expression of IFN-γ was significantly upregulated in the cortex, hippocampus, striatum and thalamus of N. caninum-infected mice compared with uninfected mice (Fig. 3(a)). Additionally, the expression level of TNF-α was significantly upregulated in the cortex, amygdala, striatum and thalamus of the infected mice (Fig. 3(b)). However, there was no significant difference in the level of IFN-γ and TNF-α expression amongst brain regions. Although the expression level of TNF-α in hippocampus was low compared with the other regions, the mean TNF-α expression level in the hippocampus of infected mice was over 30 fold compared with that of uninfected animals. Increased expression of IFN-γ and TNF-α may be associated with histopathological lesions in brains of infected mice.

Figure 3
figure 3

Expression levels of IFN-γ and TNF-α in the brain of N. caninum-infected mice.

Brain samples of uninfected and N. caninum-infected mice were collected at 40 days post infection (dpi). Fold expression was determined by real-time PCR and calculated relative to expression levels of the IFN-γ and TNF-α in the hippocampus of uninfected mice. Significant differences in each brain region between uninfected and N. caninum-infected group were determined by unpaired t tests (*p < 0.05, **p < 0.01). Significant differences among brain regions were analysed by one-way ANOVA with Tukey’s post hoc test, but there were no significant differences. Data represent mean ± SEM. Control, n = 6; N. caninum-infected mice, n = 6. Data are representative of two independent experiments.

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Neurotransmitter levels in distinct brain regions of N. caninum-infected mice

Next, we measured neurotransmitters in the brain. Levels of several neurotransmitters were altered in distinct brain regions of infected mice compared with uninfected mice (Fig. 4). Glu levels in the cortex, caudoputamen and hippocampus of N. caninum infected mice were significantly higher than those of uninfected animals (Fig. 4(a)). Glycine (Gly) levels in the cortex, hippocampus, thalamus and amygdala of infected mice were higher than those of uninfected animals (Fig. 4(b)). Since Gly is known to be an inhibitory neurotransmitter in the hindbrain, the levels of Gly in the medulla oblongata and pons, midbrain and cerebellum were also analysed (Fig. 4(c)). Gly levels of infected mice were increased in these regions compared with uninfected animals. These results suggested that excess Gly neurotransmission in the brain occurred because of N. caninum infection. In comparison to uninfected mice, higher levels of gamma-aminobutyric acid (GABA) in the amygdala and lower levels in the cortex were observed in infected mice (Fig. 4(d)). In the case of the monoamine neurotransmitter DA, levels in the caudoputamen and amygdala were higher than those of uninfected animals. However, lower levels of DA in hippocampus and thalamus were seen in infected mice (Fig. 4(e)). In addition, higher 5-HT levels in the cortex, caudoputamen, amygdala and thalamus were observed in infected mice (Fig. 4(f)). Together, N. caninum infection may trigger a difference in neurotransmitter levels in several brain regions compared with uninfected mice.

Figure 4
figure 4

HPLC analysis of amino acids and monoamines.

(a,b,d–f) Brain samples were collected at 40 days post infection and analysed by HPLC for glutamate (Glu), glycine (Gly), gamma-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT). Data represent mean ± SEM. Control, n = 6; N. caninum-infected mice, n = 6. Data are representative of two independent experiments. (c) Brain samples were collected at 52 days post infection and analysed by HPLC for Gly in hindbrain areas. Data represent mean ± SEM. Control, n = 8; N. caninum-infected mice, n = 12. Significant differences in each brain region between uninfected and N. caninum-infected group were determined using unpaired t tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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Expression levels of c-Fos and Arc in distinct brain regions of N. caninum-infected mice

Finally, we examined expression levels of c-Fos and Arc because they have been used as markers for neuronal activation. Expression of c-Fos decreased in all regions of N. caninum-infected mice (Fig. 5(a)). Arc expression decreased in the cortex, caudoputamen, hippocampus and amygdala, but not in the thalamus of N. caninum-infected mice (Fig. 5(b)). These data indicate that N. caninum infection reduces expression of IEGs in the brain.

Figure 5
figure 5

Expression levels of c-Fos and Arc in the brain of N. caninum-infected mice.

Brain samples of uninfected and N. caninum-infected mice were collected at 40 days post infection. Fold expression was determined by real-time PCR and calculated relative to expression levels of c-Fos and Arc in the hippocampus of uninfected mice. Significant differences in each brain region between uninfected and N. caninum-infected group were determined by unpaired t tests (*p < 0.05, **p < 0.01, ***p < 0.001). Data represent mean ± SEM. Control, n = 6; N. caninum-infected mice, n = 6. Data are representative of two independent experiments.

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Discussion

Generally, N. caninum infection in dogs causes encephalomyelitis, polyradiculoneuritis, pelvic limb paralysis, rigid hyperextension and muscle atrophy4,5,6. N. caninum infection not only causes similar neurological signs in cattle but is also associated with abortions in cows4. Moreover, N. caninum-infected rodents develop various kinds of neurological signs such as wryneck, circular movement and paralytic gait13,14,15. However, the mechanism of onset of neosporosis is largely unknown. Because neurological signs appear asymptomatic and then become symptomatic, it is important to study the brain of N. caninum-infected asymptomatic animals. In this study, N. caninum-infected mice showed low activity and exploratory behaviour in a novel environment, 30 days post infection (dpi). To our knowledge, our study is the first to report the behavioral change of mice infected with N. caninum. The behavioral change of rodents infected with T. gondii, which is closely related to N. caninum, has been well studied in the past decade. The effects of T. gondii infection on rodent behavior vary with experimental design. Time post infection may affect the activity level of mice17. For example, hypoactivity of infected animals was found within 2 to 3 months post infection18. In contrast, hyperactivity of infected animals was observed at a later clinical stage (3–7 months post infection)19,20,21,22. In addition, another study did not find any significant difference in activity at 3 months post infection23,24. Consequently, hypoactivity of mice infected with T. gondii were observed at an acute to sub-acute stage. It has been suggested that hypoactivity of infected mice was due to acute illness. Hyperactivity of infected mice was thought to be a result of histopathological damage associated with an immune response and development of cysts in the brain23. In this study we carried out open field tests at 4 weeks post infection and showed mild decrease of locomotor activity in mice infected with N. caninum. Although N. caninum infected mice showed no typical clinical symptoms and loss of body weight, they expressed high levels of inflammatory cytokines and histopathological damage in the brain. Thus, our results suggested that at 4 weeks post infection, N. caninum-infected mice had less activity levels because of sustained inflammatory response and histological damage of the brain.

Several studies have indicated that the brain stem and cerebellum show high sensitivity to N. caninum infection25,26,27,28. However, there was no association between parasite density and the severity of the lesion in those regions16. In the present study using asymptomatic mice, both tissue damage and parasite load did not show tropism. These results suggested that tissue damage associated with N. caninum infection had no region specificity in the brain during chronic stage. Previously, we found that the frontal lobe and medulla oblongata were mainly affected in symptomatic BALB/c mice infected with N. caninum and some mice showed severe histopathological lesions in the cerebellum16. Therefore, lesion formation in the medulla oblongata and the cerebellum may be characteristic of neosporosis in mice. In the present study using asymptomatic C57BL/6 mice, lesion formation in the cerebellum was low compared with other regions, indicting the importance of histopathological lesions in the cerebellum for onset of neosporosis.

Our previous study showed that activation of microglia and increased expression level of inducible nitric oxide synthase were observed in the brains of mice infected with N. caninum16. In this study, expression of IFN-γ and TNF-α were highly upregulated in the brain of N. caninum-infected mice, suggesting neuroinflammation and CNS disorder via inflammation by immune cells. Generally, IFN-γ controls N. caninum proliferation in macrophage cultures29,30,31. Treatment of microglia and astrocytes with IFN-γ and TNF-α shows inhibition of parasite growth32. Moreover, IFN-γ and TNF-α inhibit N. caninum growth in a bovine cerebellar cell culture33. These responses induce neuronal disorder and neuronal cell death while they can eliminate parasites in tissues29,32,33,34. IFN-γ and TNF-α produced by immune cells disrupt mitochondrial ATP production and cause Glu excitotoxicity35,36. Excess Glu release induces neuronal cell death via N-methyl d-aspartate receptor35,36. Together, our results indicate chronic inflammation in the brain of asymptomatic C57BL/6 mice infected with N. caninum.

Pathogen infection into the brain can alter neurotransmission and cause dysfunction of this organ37. Some studies have suggested that T. gondii can alter DA metabolism and host behavior38,39. Stibbs showed that DA levels were 14% higher in mice with chronic infections than controls40. Gatokowska et al. suggested that an increase of 5-HT and the DA system might be responsible for low activity and exploratory behaviour of mice41. These results suggest differences in several neurotransmitter levels of mice infected with T. gondii. In this study, we examined changes in neurotransmitter levels in brain tissue following N. caninum infection for the first time. Our results showed that N. caninum infection induced marked change of excitatory and inhibitory neurotransmitter levels in wide brain areas and these responses may induce sickness behaviour in infected mice. The amount of Glu was increased in the cortex, caudoputamen and hippocampus of mice infected with N. caninum. Level of GABA, a major inhibitory neurotransmitter, was decreased in the cortex of infected mice suggesting that elevated Glu levels might be due to lack of effects of GABA. Our previous study showed activation of microglia in the brain of mice infected with N. caninum16. Activated microglia induce excess Glu and causes excitotoxicity to neuronal cells via the N-methyl-D-aspartate (NMDA) receptor35,36. Because GABA levels showed no difference in the caudoputamen and hippocampus between infected and uninfected mice, our result supports elevated Glu might be a result of an immune reaction mediated via activated microglia.

Release of IFN-γ leads to tryptophan degradation via indoleamine-2,3-dioxygenase and elevated 5-HT42. In contrast, our data showed increased level of 5-HT in almost all regions examined while the expression level of IFN-γ was highly upregulated in those areas, suggesting a possibility that tryptophan levels were increased in the brains of mice infected with N. caninum. It has been suggested that increased tryptophan levels are associated with infection and are often accompanied by 5-hydroxyindole acetic acid, a major metabolite of 5-HT37. In fact, T. gondii-infected male mice showed increased serotonergic activity 3 weeks post infection41. Interestingly, IL-1 and lipopolysaccharide (LPS) administration increase concentration of tryptophan and 5-HT throughout the brain37. In addition, LPS administration increased levels of DA and 5-HT43. Therefore, N. caninum infection induced marked change of monoamine levels in wide brain areas via long-lasting immune reactions.

Since noradrenergic and serotonergic systems function to inhibit active behavior37, elevated brain serotonergic neurons by N. caninum infection may contribute to behavioral changes. Furthermore, DA levels in the caudoputamen were increased in mice infected with N. caninum, while that in the thalamus was decreased. Both the caudoputamen and thalamus are part of the basal ganglia circuit which control voluntary motor movements44. Thus, dysregulation of dopaminergic neurons in these regions may contribute to behavioral changes associated with N. caninum infection.

Although differences in changes of neurotransmitter levels among brain regions were observed, further research is required to clarify the relationship between neurotransmitter level and parasite load and severity of brain lesions. In this study there were no data concerning the correlation between severity behavioral deficits and the amount of neurotransmitter change because mice tested in the open field were different from mice prepared for HPLC and qPCR analysis. However, we additionally analyzed the correlation between behavioral changes and parasite load (supplemental Table 1). We found that the number of parasites in the thalamus had a negative correlation with the total distance travelled and average speed in the open field test. In addition, the number of parasites in the caudoputamen showed a negative correlation with average speed in the open field test. The thalamus and caudoputamen have a connection to the motor cortex44. Thus, these results suggested that parasites in these regions might contribute to behavioral changes in N. caninum-infected mice. Furthermore, the number of parasites in the cerebellum had a negative correlation with rearing counts in the open field test. Normal cerebellum processing is necessary for motor control44. Therefore, our results suggested that parasite load in the cerebellum affect the total number of rearing counts.

Hind limb paralysis and rigid hyperextension are a characteristic symptom of N. caninum infection in dogs4,5,6. N. caninum-infected mice show a similar type of neurological symptom13,14,15. However, the mechanism of neurological symptom is almost unknown. Gly levels are altered by N. caninum infection in several brain regions. In addition, levels of Gly were increased in the hindbrain including pons and oblongata, midbrain and cerebellum of the infected mice. Gly is the major inhibitory neurotransmitter in the hindbrain and the spinal cord45. Increased Gly level in cerebrospinal fluid caused severe hyperkinesia in humans46. Thus, these results implicate that increased Gly levels in the progressive stage of neosporosis might cause hind limb paralysis.

The expression of IEGs is induced by neuronal activity. c-Fos is a regulatory transcription factor and a known marker of neuronal activity. Arc is an effector protein, a key protein implicated in synaptic plasticity and synapse strength and hence learning and memory47. In the present study, expression levels of c-Fos and Arc were decreased in N. caninum-infected mice, suggesting N. caninum infection downregulated gene expression associated with neuronal cell function. Thus, N. caninum infection may impair learning and memory capacity of its host. Because we confirmed the upregulation of IFN-γ and TNF-α expression and alteration of neurotransmitter levels in the brain of N. caninum-infected mice, downregulation of c-Fos and Arc expression in the brain may be triggered by neurological pathology following N. caninum infection. In conclusion, our results suggest that chronic neuroinflammation following N. caninum infection may cause neurological dysfunction and neuronal cell death, resulting in neosporosis.

Methods

Ethics Statement

This study was performed in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of Ministry of Educations, Culture Sports, Science and Technology, Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Obihiro University of Agriculture and Veterinary Medicine (Permit number 25–59, 25–60, 25–62). Mice were decapitated without anesthesia for brain sampling and all efforts were made to minimize animal suffering.

Mice

Male C57BL/6 mice (8 weeks old) were purchased from Clea Japan (Tokyo, Japan). Mice were housed (four to six mice/cage) in 12-h light (8:00–20:00) in the animal faculty of the National Research Center for Protozoan Diseases at Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. All mice were treated using the guiding principles for the care and use of research animals endorsed by the Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. All animal experiments began after 1 week of habituation.

Preparation of N. caninum tachyzoites and parasite infection of mice

N. caninum (strain Nc-1) parasites were maintained in monkey kidney adherent epithelial cells (Vero cells) cultured in Eagle’s minimum essential medium (Sigma, St. Louis, MO, USA) containing 8% heat-inactivated fetal bovine serum (FBS). For the purification of the tachyzoites, the parasites and host-cell debris were washed in cold phosphate-buffered saline (PBS) and the final pellet was resuspended in cold PBS and passed through a 27-gauge needle and a 5.0-μm pore filter (Millipore, Bedford, MA, USA). N. caninum were intraperitoneally inoculated (1 × 105 tachyzoites) into mice (9 weeks old). To check clinical condition, daily body weight measurements were taken for 30 days after infection.

Open field test

To investigate activity and exploratory behaviour of N. caninum-infected mice, mice were tested one at a time. Exploration in open field, a circular area with a diameter of 50 cm (Muromachi, Tokyo, Japan), was recorded for 5 min using a video tracking system (Comp Act VAS ver. 3.0×, Muromachi). Total travelled distance, average speed and rearing counts were measured. Behavioural experiments were performed at 30 dpi and commenced at 7:00 am, under 300 lux light intensity.

Brain sampling

For histopathological analysis, five infected mice were sacrificed at 45 dpi and their brains were rapidly removed. The brains were perfused with 4% paraformaldehyde solution. For quantitative PCR of the parasites, two uninfected mice and twelve infected mice were sacrificed at 54 dpi. Their brains were divided into eight different areas: cortex, hippocampus, caudoputamen, amygdala, thalamus, hypothalamus, midbrain and cerebellum. The samples to be used for DNA extraction and quantitative PCR of parasites were stored at −20 °C until analysis. For quantitative reverse-transcription-PCR (qRT-PCR) and high-performance liquid chromatography (HPLC) analysis, six uninfected mice and six infected mice were sacrificed at 40 dpi. The half brains were divided into five regions: cortex, hippocampus, caudoputamen, amygdala and thalamus and were then frozen at −80 °C until use. The sample from the right and left brain was used for qRT-PCR and HPLC, respectively. To measure Gly levels in the hindbrain regions, eight uninfected mice and twelve infected mice were sacrificed at 54 dpi. The brain samples were divided into three hindbrain regions, cerebellum, midbrain and medulla oblongata plus pons and were then frozen at −80 °C until analysis.

Histopathological analysis

After fixation with 4% paraformaldehyde solution, brain samples were cut coronally, embedded in paraffin wax, sectioned at 4 μm and then stained with hematoxylin and eosin. Pathological lesion severity was scored by the following scheme: 0, no lesion; 1, slight lesion; 2, mild lesion; 3, moderate lesion; and 4, severe lesion. An example of the scoring is shown in Fig. 2(a). Total scores (from 0 to 4) for four types of lesions (meningitis, perivascular cuffs, inflammatory cells including glial cells, macrophages and lymphocyte infiltration and necrosis) in each region by a severe degree were determined. Pathological scores were calculated by adding each score for different lesions, with total scores ranging from 0 to 16. The total score for each region was used for data analysis.

DNA extraction and quantitative PCR for the detection of N. caninum DNA

DNA was isolated from each brain region and parasite counts were analysed by real-time PCR using the Nc5 gene, as described previously48. The primers complementary to the Nc5 gene of N. caninum: the forward primer spanning nucleotides 248–257 (5′-ACT GGA GGC ACG CTG AAC AC-3′) and the reverse primer spanning nucleotides 303–323 (5′-AAC AAT GCT TCG CAA GAG GAA-3′). PCR was performed using an ABI prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA) and amplification monitored using the SYBR Green method (Applied Biosystems). A standard curve was constructed with tenfold serial dilutions of N. caninum DNA extracted from 1 × 105 parasites. The curve ranged from 10,000 to 0.01 parasites. Parasite number was calculated by plotting Ct values on the standard curve.

qRT-PCR

Total RNA was extracted from mouse brain using TRI reagent (Sigma). First-strand cDNA was synthesised from 0.4 μg total RNA by SuperScript® First-Strand Synthesis System for RT-PCR (Invitrogen, Mount Waverley, Australia). Real-time PCR was performed according to Applied Biosystems (Applied Biosystems) and amplification monitored using SYBR Green (Applied Biosystems). IEG primers have been described previously49: Arc, 5′-GGA GGG AGG TCT ACC GTC-3′ and 5′-CCC CCA CAC CTA CAG AGA CA-3′; c-fos, 5′- CGA AGG GAA CGG AAT AAG-3′ and 5′-CTC TGG GAA GCC AAG GTC-3′. IFN-γ, 5′-GAG GAA CTG GCA AAA GGA TG-3′ and 5′-TGA GCT CAT TGA ATG CTT GG-3′. TNF-α, 5′-GGC AGG TCT ACT TTG GAG TCA TTG C -3′ and 5′-ACA TTC GAG GCT CCA GTG AA -3′. The housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was amplified in parallel (5′-TGT GTC CGT GGA TCT GA-3′ and 5′-CCT GCT TCA CCA CCT TCT TGA T-3′) and used as an internal standard. The optimal reference gene was selected based on the Cotton EST database (http://www.leonxie.com). Expression levels of each gene relative to GAPDH were calculated using the 2ΔCt method (User Bulletin no. 2; Perkin-Elmer, Boston, MA, USA). Fold expression levels among brain regions were calculated relative to expression levels of the corresponding gene in the hippocampus of uninfected mice.

HPLC

Each brain sample was homogenised using a bio-masher (Funakoshi, Tokyo, Japan) and then 300 μL/10 mg tissue of 0.2 M perchloric acid (containing 100 μM EDTA-2Na) was added. Both isoproterenol HCL (monoamine internal standard) and homoserine (amino acid internal standard) (both Sigma) were added. Homogenates were placed on ice for 30 min and then centrifuged at 20,000 × g for 15 min at 0 °C. Supernatants were mixed with 1 M sodium acetate to adjust the pH to 3.0 and filtered by Ultra free MC (Millipore). Final products were injected into the HPLC system HTEC-500 (electrochemical detector; Eicom, Kyoto, Japan), equipped with a SC-5ODS column for monoamines or a SA-5ODS column for amino acids. Chromatographs were analysed using PowerChrom software version 2.5 (eDAQ Pty Ltd., Densitone East, Australia).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Statistical differences between two groups were analysed using two-tailed unpaired t tests. With three groups or more, statistical differences were determined using one-way ANOVA followed by Tukey’s multiple comparison test. P values < 0.05 represent statistical differences.

Additional Information

How to cite this article: Ihara, F. et al. Changes in neurotransmitter levels and expression of immediate early genes in brain of mice infected with Neospora caninum. Sci. Rep. 6, 23052; doi: 10.1038/srep23052 (2016).