Differential dopamine-mediated effects in the 5-lipoxygenase deficient mice

doi:10.21203/rs.3.rs-4009886/v1

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Differential dopamine-mediated effects in the 5-lipoxygenase deficient mice

https://doi.org/10.21203/rs.3.rs-4009886/v1

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published 04 Oct, 2024

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The role of 5-lipoxygenase/leukotrienes on the central nervous system has been considered in both physiological end pathological states. Investigating the interaction between 5-lipoxygenase/leukotrienes and the dopaminergic system may provide better understanding of dopamine-related pathologies. This study aimed to investigate motor and non-motor dopamine-related responses in 5-lipoxygenase/leukotrienes-deficient mice. Pharmacological challenges of the dopaminergic system included amphetamine, apomorphine and reserpine treatment along with their respective effects on prepulse inhibition (PPI) response, general motor activity, and oral involuntary movements. Reserpine-treated mice were also investigated for their striatal glial markers’ expression (GFAP and Iba-1). 5-lipoxygenase/leukotrienes-deficient mice exhibited increased spontaneous locomotor activity, including horizontal and vertical exploratory activity, and stereotyped-like behavior compared to wild-type mice. This condition was attenuated by acute apomorphine treatment. Despite the absence of altered basal responses in the PPI there was a significant and selective decreased in susceptibility in amphetamine-induced PPI disruption in 5-lipoxygenase/leukotrienes-deficient mice. However, there was an increased vulnerability to reserpine-induced involuntary movements. There was no alteration in the basal expression of striatal GFAP and Iba-1 positive cells in 5-lipoxygenase/leukotrienes-deficient mice compared to wild-type mice. Reserpine treatment induced a significant increase in GFAP immunoreactivity in wild-type mice but this effect was absent in the 5-lipoxygenase deficient mice. The percentage of activated microglia was found to be significantly increased in reserpine-treated wild-type mice. This effect was absent in the 5-lipoxygenase/leukotrienes-deficient mice. Our results support the conception of a unique dopaminergic pathway phenotype in 5-lipoxygenase/leukotrienes-deficient mice. These findings suggest that leukotrienes may interfere with the orchestration of dopamine-mediated responses.

dopamine

5-lipoxygenase

leukotrienes

astroglial reaction

oral dyskinesia

reserpine

Eicosanoids (prostaglandins, leukotrienes and thromboxane) are the signaling molecules and inflammatory mediators that are generated in the pathway of arachidonic acid metabolism mediated by a family of distinct isozymes lipoxygenases (Serhan and Samuelsson, 1988). Specifically, 5-lipoxygenase catalyzes the formation of leukotriene A₄, which is subsequently converted to leukotriene B₄ (LTB₄) or to cysteinyl leukotrienes (CysLTS). LTB₄ is a highly potent chemoattractant factor, promotes enhanced expression of IL-12, IL-6, and TNF-alpha (Phillis et al. 2006; Tassoni et al. 2008). CysLTs (LTC₄, LTD₄, LTE₄) concur to the pathophysiology of asthma inducing infiltration of inflammatory cells bronchoconstriction, mucus production, and airflow obstruction (Funk and Chen, 2000). 5-lipoxygenase at high concentrations results in induction of large amounts of prostaglandin E2 (Phillis et al. 2006). Therefore, the 5-lipoxygenase/leukotrienes system actively participates in the inflammatory network (Funk and Chen, 2000).

There is evidence that 5-lipoxygenase is an important modulator in the development of age-related neuropathology (Yan et al. 2021). 5-Lipoxygenase has been associated with the development of neurological/neuropsychiatric disorders (Manev et al. 2006, 2011; Chou et al. 2013; Shen et al. 2022). Lipoxygenase isozymes are expressed in the brain, including the ventral midbrain (Bendani et al. 1995; Manev et al. 2001; Qu et al. 2000; Uz et al. 2001). In the CNS leukotrienes have been shown to regulate the hypothalamic-pituitary-adrenal axis (Hirai et al. 1985). Despite broad-spectrum of anti-inflammatory action glucocorticoids increase the transcriptional and protein levels of 5-lipoxygenase in the brain (Uz et al. 1999). 5-lipoxygenase contributes to Alzheimer's disease pathology, prion protein toxicity as well as multiple sclerosis demyelination. Post-mortem brain tissues from subjects with clinical and pathological diagnosis of progressive supranuclear palsy, one of the most common form of tauopathy, have a significant up-regulation of the 5-lipoxygenase (Giannopoulos et al. 2019).

Mental illness including depression and schizophrenia has been associated with elevated levels of both inflammation and stress (Guan et al. 2014; Locachevic et al. 2019; Miller et al. 2018; Goldsmith et al. 2019). The 5-lipoxygenase activating protein which is involved in the leukotriene’s pathway in association with 5-lipoxygenase, has been linked to neuropsychiatry conditions, suggesting a connection between the 5-lipoxygenase and dopamine (Joshi et al. 2013). Previous data from our group with 5-lipoxygenase-deficient mice (5-LO^-/-) revealed that 5-lipoxygenase activity is critical for regulating stress-induced symptoms (Locachevic et al. 2019). Interestingly, inflammation and stress, two vulnerability factors for psychiatric disorders may have a common pathway.

Besides the complex and multiple mechanisms involved in neurodegenerative conditions, Parkinson's disease is neurochemically associated with the loss of nigrostriatal dopaminergic tone. There is evidence of 5-lipoxygenase/leukotrienes involvement in the dopaminergic mediated effects, mainly dependent on the nigrostriatal dopaminergic pathway (Chou et al. 2013; Kang et al. 2013, 2021). Levels of 5-lipoxygenase and leukotrienes were found to be upregulated, accompanied by the degeneration of dopamine neurons in the nigrostriatal pathway of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), challenged mice (Chou et al., 2013, 2014; Kang et al., 2013). Several inflammatory mechanisms involved in this degenerative process, such as astrogliosis end microglial activation, have been demonstrated (Lazzarini et al. 2013; Tristão et al. 2016; Kouli et al. 2020; Ferrari et al. 2021). Other studies demonstrated a protective effect of 5-lipoxygenase inhibitors in dopaminergic neurons (Li et al. 2008; Kang et al. 2013, Wallin et al., 2021). LTB₄ was found to be increased in the striatum and substantia nigra following MPTP injection in mice (Kang et al. 2013).

Mice deficient in 5-lipoxygenase/leukotrienes were found to have lower striatal levels of tyrosine hydroxylase and dopamine than wild-type littermates. The animals are protected against MPTP-induced neurotoxicity as shown by unchanged striatal dopamine levels compared to saline-treated 5-LO^-/- mice, and also had no signs of inflammation indicated by the absence of microglia or astrocyte reactivity (Chou et al. 2013). Similar results were seen in another study where the use of a 5-lipoxygenase inhibitor protected against neuronal cell death in both a human dopaminergic cell line and mouse model (Kang et al. 2021). Kurtuncu and coworkers demonstrated that 5-LO^-/- mice do not present changes in the general spontaneous locomotor behavior but have a profound effect on their behavioral response to cocaine (Kurtuncu et al. 2008). Recently, Barbosa-Silva and co-authors demonstrated that 5-LO^-/- adult mice present motor deficits and increased repetitive behavior observed in the rotarod test and marble burying test (Barbosa-Silva et al. 2022).

Current evidence is pointing towards an undeniable involvement of 5-lipoxygenase/leukotrienes in the pathogenesis of Parkinson’s disease. Investigating the interaction between 5-lipoxygenase/leukotrienes and the dopaminergic system may improve dopamine-related pathologies knowledge.

The availability of mouse models with 5-lipoxygenase gene deletion provides critical tools for understanding the role of the 5-lipoxygenase/leukotrienes system in dopamine-mediating behavioral and molecular responses. Since dopamine signaling is involved in the onset and progression of several diseases in the nervous system, we investigated 5-lipoxygenase/leukotrienes-deficient mice in pharmacologically changing conditions of the dopaminergic system. These investigated conditions also require dopamine function in dopaminergic pathways other than those particularly involved in motor control. The selected experimental strategy includes the behavioral response of 5-LO^-/- adult mice to different mechanisms of action of dopaminergic drugs, besides glial reactivity.

Animals Male 5-lipoxygenase-deficient mice (5-LO^−/−) were age-matched in all procedures with their genetic background, the wild-type 129sv mouse strain. The Alox5^−/− (129-Alox5^tm1Fun/J) and 129sv mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and were bred in the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (Ribeirão Preto, SP, Brazil). C57Bl/6 mice, obtained from the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, were used as controls to validate our methodology used in the 129sv mice. Mice were kept in 12 h light/dark cycles. All procedures were performed following the ethical standards established by the National Council for Control of Animal Experimentation (CONCEA) and were approved by the Ethics Committee on Animal Use (CEUA) of the Campus of Ribeirão Preto.

Drugs Amphetamine and MK801 (Sigma Chemical Co.; USA) were dissolved in sterile isotonic saline and administered intraperitoneally (10 ml/kg). Reserpine (Sigma Chemical Co.; USA) was dissolved in distillate water and a 0.5% solution of glacial acetic acid, then administered subcutaneously (1 ml/kg). Apomorphine (Sigma Chemical Co.; USA) was dissolved in distillate water and was administered subcutaneously (1 ml/kg).

Experimental Design

Experiment 1 For evaluation of psychotic-like effects of dopaminergic drugs independent cohorts of 5-LO^−/− and wild-type mice were acutely treated with both intraperitoneal amphetamine (10 mg/kg) or subcutaneous apomorphine (0.5 mg/kg) (Issy et al. 2009, 2020) or with their respective controls (saline or distillated water). An additional group of 5-LO^−/− mice received an intraperitoneal injection of glutamatergic antagonist MK801 (0.5 mg/kg; Issy et al. 2018). Mice were subjected to the PPI test 15 min after pharmacological treatment (n = 7–8 per treatment).

Experiment 2 For evaluation of dopamine mediated hyperlocomotion 5-LO^−/− and wild-type mice were acutely treated with subcutaneous distillated water (1 ml/kg) or apomorphine (0.5, 1, or 10 mg/kg; Issy et al. 2020) injection, and 30 min later mice were submitted to the locomotor activity analyses in the actimeter apparatus (n = 5–7 per treatment).

Experiment 3 To evaluate the effects of dopamine-induced oral involuntary movements, 5-LO⁻/⁻ and wild-type mice received two subcutaneous injections of reserpine (1 mg/kg) with an interval of 48 hours and were subjected to the evaluation of oral involuntary movements five days after the first reserpine injection (Issy et al. 2018). An additional experimental group was analyzed twirty-five days after the first reserpine injection. Locomotor activity was analyzed immediately after the evaluation of oral involuntary movements. Brains of both 5-LO^−/− and wild-type mice were processed for immunohistochemical analyses of striatal GFAP and Iba-1-positive cells (n = 4–6 per group).

Prepulse inhibition reaction The test was conducted simultaneously in two identical startle response systems (Med Associates, Inc., USA), as previously reported (Issy et al., 2018). A continuous acoustic signal provided a background white noise level of 65 dB. The pulse (pulse alone, P) was a burst of white noise of 105dB with a rise/decay of 5 ms and duration of 20 ms. Prepulses (PP) were used at intensities of 80, 85 or 90 dB, with a frequency of 7 kHz, and duration of 10 ms. The PPI test consisted of 64 trials pseudorandomly divided into eight different categories presented with an inter-stimulus interval of 30 s. These trials were further categorized into four groups: (1) pulse alone, (2) prepulse alone, (3) prepulse plus pulse, and (4) no stimulus. The level of PPI was determined by expressing the prepulse plus pulse startle amplitude as a percentage of the pulse-alone startle amplitude using the formula: %PPI = [100 × (startle mean of pulse alone trials – startle mean of prepulse-pulse trials)/startle mean to pulse alone trials]. Platform calibration was performed by adjusting the gain on the load cell amplifier to 150 arbitrary units (AU) at a standard weight of 40 g. The load cell had ranging from − 2047 to + 2047 AU.

Locomotor activity An actimeter was used to assess the locomotor activity exhibited by the animals in a novel cage over a 5 min period. The measurements obtained included total horizontal and vertical exploratory activity, as well as stereotyped behavior. All movement without specific displacement was considered stereotyped-like behavior. The apparatus used (Actimeter, Cibertec, Madrid, Spain) consists of an individual novel cage (30 cm × 14 cm × 12 cm) attached to a continuous recording system that detects the animal’s displacement using an infrared photocell system.

Orofacial involuntary movements The parameters of orofacial involuntary movements were assessed by manually counting continuous single mouth openings in a vertical plane, not directed towards a physical material, tongue protrusion and/or mandibular tremor during a 10 min period. A blind observer conducted the analysis of these three parameters, 24 h after the second reserpine injection. The analysis of orofacial movements was conducted in a glass cylinder (19 cm diameter and 22 cm height) with mirrors positioned on the floor and behind the apparatus to enable observation of orofacial involuntary movements when mice were not facing the observer.

Immunohistochemical analysis

Tissue preparation Mice were deeply anesthetized with a lethal dose of ketamine (100 mg/kg) and xylazine (14 mg/kg; ip) combination and transcardially perfused with phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA) followed by cryoprotection with 30% sucrose in 0.1M phosphate buffer (PB) over 48 hours. The brains were frozen at -40 ºC on dry ice and isopentano and 30-µm-thick serial coronal sections were obtained using a cryostat microtome.

Immunohistochemistry Free-floating sections underwent antigen recovery (heating sections for 30 min in a water bath at 90 ◦C in a 0.1 M citrate buffer solution; pH 6.0). After rinsed in the washing buffer (0.1 M PBS + 0.15% Triton-X; pH 7.4) sections were incubated for 30 min with 2% hydrogen peroxide (diluted in PBS + Triton) to remove endogenous peroxidase activity. Nonspecific binding sites were blocked in a solution containing 5% BSA, washing buffer and 5% normal serum (from secondary antibody species) for 1 h. Next, sections were incubated with the primary antibody for GFAP (glial fibrillary acid protein; rabbit anti-GFAP; 1:1000, Sigma-Aldrich, St. Louis) or Iba-1 (rabbit anti-Iba-1; 1:400; Waco laboratory Chemicals). Following primary antibody incubation for 48 hours, sections were successively washed (washing buffer) and incubated in a secondary antibody solution (PBS) for 90 min (goat anti-rabbit; 1:400). Sections were then incubated with the avidin-biotin immunoperoxidase method for 2 hours (Vectastain ABC kit, Vector Lab, Burlingame, CA, U.S.A.), and the immunoreactivity was revealed by the addition of chromogen diaminobenzidine (Sigma).

Quantitative evaluation Striatal GFAP-positive cells were quantified at 20X magnification. The striatum (Bregma 0.38 mm) was unilaterally assessed in the dorsolateral and dorsomedial quadrants. The regions were located based on the coordinates of the Franklin and Paxinos (1997) mouse brain atlas. All analyses were performed on the right hemisphere of the brain by an experimentally blind investigator. Images were digitized with a video camera (Leica DFC420), captured real magnification in grayscale and evaluated with ImageJ (http://rsb.info.nih.gov). Integrated optical density values (product of area and mean gray value) of corresponding sectors in each section were averaged. For this procedure, a mean gray value of the stain was expressed in arbitrary gray scale units where the scale ranges from 0 to 255 (0 representing the darkest, most intense labeling) to form one density measurement performed in the sections. After this, integrated optical density was calculated by multiplying the selected area with the mean gray value. Sampled areas consisted of regions of interest measuring 0.1 mm². The average gray value from an unstained area was subtracted from each section to correct for background immunoreactivity.

Quantitative assessment of microglia morphology A quantitative analysis of microglia morphology was conducted, considering both activated and resting microglia labeled by Iba-1. The striatum (Bregma 0.38 mm) was unilaterally assessed in the dorsolateral and dorsomedial quadrants. Cells were categorized as resting or reactive microglia based on established morphological criteria (Diz-Chaves et al., 2012; Lopez-Rodriguez et al., 2015): cells with few cellular processes (2 or fewer), or showing 3–5 short branches, were classified as resting microglia, while cells with numerous (> 5) longer processes, large soma with retracted and thicker processes, or cells exhibiting an amoeboid cell body, numerous short processes, and intense Iba-1 immunostaining, were classified as reactive microglia. Images at 40 × objective were used for this analysis. The data is presented in the form of the percentage of reactive cells relative to the total number of cells positively labeled for Iba-1 (considered as 100%).

Data analysis One- or two-way analysis of variance (ANOVA), followed by post-hoc Tukey test was applied for multiple comparisons as indicated in the results section. In the two-way analysis, the factors analyzed were prepulse intensities and condition (genotype plus treatment) for the PPI test, and genotype (plus time) and treatment for immunohistochemical analyses of GFAP and Iba-1. Data were expressed as mean ± standard error of mean (SEM). Values of p < 0.05 were considered statistically significant.

Amphetamine effect in the PPI test

Our data revealed a significant difference in amphetamine-induced effects in the PPI test in 5-LO^−/− mice compared to wild-type mice (Fig. 1A). Amphetamine induced a slight PPI disruption in the 5-LO^−/− mice of prepulse intensity at the intensity of 85dB, when compared to 5-LO^−/− mice receiving vehicle. The two-way repeated measures analysis showed a main effect of prepulse intensity (80, 85 or 90 dB; F_2,12 = 5.939, p = 0.016) and condition (wild-type or 5-LO^−/− mice x saline or amphetamine; F_3,18 = 6.323, p = 0.004), and an interaction (F_6,63 = 2.761, p = 0.026) between prepulse intensities x condition (genotype plus treatment). The basal startle reactivity was not significantly different between 5-LO^−/− and wild-type mice. Amphetamine treatment reduced startle amplitude only compared to wild-type mice with saline (F_3,24 = 4.031, p = 0.018; Table 1).

Apomorphine effect in the PPI test

Our data did not reveal any difference in PPI response in either wild-type or 5-LO⁻/⁻ mice acutely treated with apomorphine 0.5 mg/kg (Fig. 1B). The basal startle reactivity was not significantly different between 5-LO^−/− and wild-type mice (data not shown).

MK801 effect in the PPI test

Our data did not reveal any difference in MK801-induced effects in the PPI test in 5-LO^−/− mice when compared to wild-type mice (Fig. 1C). The two-way repeated measures analysis revealed a main effect of condition (wild-type or 5-LO^−/− mice x saline or MK801; F_3,63 = 28.63, p = 0.001), but not a prepulse intensity effect (80, 85 or 90 dB; F_2,21 = 0.03653, p = 0.964), or an interaction (F_6,63 = 0.542, p = 0.773) between prepulse intensities x condition (genotype plus treatment). The basal startle reactivity was not significantly different between 5-LO^−/− and wild-type mice (data not shown).

Table 1

Startle reflex amplitude in arbitrary units.
Treatment	WT Saline	5-LO^−/− Saline	WT amphetamine	5-LO^−/− Amphetamine
Startle reflex	984 ± 149	621 ± 68	648 ± 98	*516 ± 106

Data presented as mean ± SEM. *5-LO^−/− amphetamine vs. wild-type saline. *p < 0.05; n = 7 per group. One-way ANOVA followed by Tukey post hoc test.

Apomorphine reduced motor activity and stereotyped-like behavior in the 5-LO^−/− mice

The basal horizontal and vertical activity, and stereotyped-like behavior was found higher in the 5-LO^−/− mice when compared to wild-type mice when both received vehicle (horizontal: F_7,47 = 10.87, p ˂ 0.0001; vertical: F_7,44 = 22.95, p ˂ 0.000; stereotyped-like behavior: F_7,48 = 12.27, p ˂ 0.0001; one-way ANOVA, Fig. 2). Apomorphine treatment induced a significant decrease in the 5-LO^−/− mice horizontal and vertical activity, and stereotyped-like behavior at all used doses (0.5, 1 and 10 mg/kg) when compared to 5-LO^−/− mice with received vehicle. Under apomorphine dose of 0.5 mg/kg the horizontal activity, and stereotyped-like behavior of 5-LO^−/− mice was significantly higher when compared to wild-type mice receiving a comparable apomorphine dose (Fig. 2). Under all apomorphine doses the vertical activity of 5-LO^−/− mice decreased to similar level of wild-type mice (Fig. 2).

Reserpine effects in the 5-LO^−/− mice

Two days after the second reserpine injection, mice were evaluated regarding the presence of orofacial involuntary movements. Reserpine treatment induced a significant increase in the frequency of orofacial involuntary movements in 5-LO^−/− mice when compared to 5-LO^−/− mice that received vehicle (F_3,15 = 13.67, p = 0.001; one-way ANOVA, Fig. 3A). The basal horizontal and vertical locomotor activity and stereotyped-like behavior were found increased in the 5-LO^−/− mice when compared to wild-type mice when both received vehicle (Fig. 3B-D). The 5-LO^−/− mice presented a significant decrease in locomotor activity (horizontal and vertical) and in stereotyped-like behavior after reserpine treatment when compared to 5-LO^−/− mice that received vehicle (horizontal: F_3,16 = 31.34, p ˂ 0.0001; vertical: F_3,16 = 27.06, p ˂ 0.0001; stereotyped-like behavior: F_3,16 = 23.59, p ˂ 0.0001; one-way ANOVA, Fig. 3). The wild-type mice presented no alteration in locomotor activity and in stereotyped-like behavior after reserpine treatment when compared to wild-type mice that received vehicle.

An additional experimental group was analyzed 35 days after reserpine treatment. In this condition, the orofacial involuntary movements in 5-LO^−/− mice were similar to 5-LO^−/− mice receiving vehicle. All motor activity measurements were significant higher in 5-LO^−/− mice (Table 2), similar to basal conditions measurement (Fig. 3).

Table 2

Motor activity analyses thirty-five days after the first injection of reserpine.
Genotype vs Treatment	WT Vehicle	5-LO^−/− Vehicle	WT Reserpine	5-LO^−/− Reserpine	Statistical analyses
Orofacial involuntary movements	13 ± 2	*3 ± 1	11 ± 2	4 ± 2	F_3,18=6.111, p = 0.0047
Horizontal activity	337 ± 150	*1354 ± 125	355 ± 94	^#1658 ± 173	F_3,17=30.23, p˂0.0001
Vertical activity	2 ± 1	*42 ± 6	1 ± 1	^#41 ± 7	F_3,17=52.34, p˂0.0001
Stereotyped-like behavior	396 ± 92	*778 ± 59	384 ± 58	^#1190 ± 140	F_3,17=22.03, p˂0.0001

Data presented as mean ± SEM. *5-LO^−/− vehicle vs. WT vehicle. ^#5-LO^−/− reserpine vs. WT reserpine. *^#p < 0.05; n = 4–6 per group. One-way ANOVA followed by Tukey post hoc test.

Striatal astrocyte (GFAP) expression

The immunohistochemistry analysis was conducted five or thirty-five days after reserpine treatment. The total levels of GFAP were evaluated at dorsal and ventral striatum portions in the lateral end medial quadrants.

Reserpine treatment induced a significant increase in GFAP immunoreactivity in wild-type mice but not in the 5-LO^−/− genotype, five days after reserpine first injection in the dorsomedial (DM) and dorsolateral (DL) striatum (Tukey test, p < 0.05; Fig. 4). At thirty-five days, wild-type mice had lesser GFAP immunoreactivity than at five days (Tukey test, p < 0.05; Fig. 4).

The two-way ANOVA revealed a main effect of genotype in the DL (F_3,32 = 49.52, p ˂ 0.0001) and DM (F_3,32 = 10.05, p ˂ 0.0001) striatum. Also, it was found a main effect of treatment in the DL (F_1,32 = 4.823, p = 0.0354) and DM (F_1,32 = 13.22, p = 0.0010) striatum, and an interaction between genotype and treatment in the DL (F_3,32 = 9.246, p = 0.0001) and DM (F_3,32 = 9.158, p = 0.0002) striatum. The GFAP immunoreactivity in the DL striatum is represented at Fig. 4.

Striatal microglia (Iba 1) expression and reactivity

The total levels of Iba 1 expressing cells were found similar in wild-type mice and 5-LO^−/− genotype in dorsal striatum (Table 3). Reserpine treatment did not induce any modification in the microglia immunoreactivity either in wild-type or 5-LO^−/− mice in dorsal striatum (Table 3; Two-way ANOVA) or ventral striatum (data not shown).

The percentage of reactive microglia was found significantly increased in WT mice after five days of reserpine first injection in both DM and DL striatal quadrants (Fig. 5). The images clearly show cells with amoeboid cell body, numerous short processes, and intense Iba-1 immunostaining after reserpine treatment, suggesting a microglia reactive state (Fig. 5). The two-way ANOVA revealed a main effect of genotype [DM F_3,32 = 66.44, p˂0.0001 and DL F_3,32 = 40.20, p˂0.0001], treatment [DM F_1,32 = 49.51, p˂0.0001 and DL F_1,32 = 31.29, p˂0.0001], and an interaction between genotype and treatment [DM F_3,32 = 64.36, p˂0.0001 and DL F_3,32 = 38.26, p ˂ 0.0001]. In the 5-LO^−/− genotype mice reserpine treatment did not increase reactive microglia percentage (Fig. 5).

Table 3

Striatal microglia (Iba 1) expression.
Genotype vs Treatment	WT Vehicle	5-LO^−/− Vehicle	WT Reserpine	5-LO^−/− Reserpine
5 days after reserpine first injection
DM	429.2 ± 30	468 ± 22	483.4 ± 30	431.6 ± 29
Dl	461 ± 6	438.4 ± 15	441 ± 14	426.2 ± 4
35 days after reserpine first injection
DM	427.8 ± 23	449.8 ± 15	465.8 ± 12	411.4 ± 39
Dl	433.4 ± 19	434 ± 9	451 ± 13	366.6 ± 52

Data presented as mean ± SEM. n = 5 per group. One-way ANOVA followed by Tukey post hoc test.

In this study, we conducted pharmacological manipulations of the dopaminergic system in the 5-LO^−/− genotype. Overall, our data revealed modified behaviors in 5-LO^−/− mice, suggesting a basal altered condition of the dopaminergic system in this genotype. Corroborating this hypothesis, we found distinct behavioral responses of 5-LO^−/− mice to drugs with dopaminergic-related mechanisms compared to wild-type mice. The characterization of leukotriene-deficient mice has revealed numerous phenotypic findings in these mice (Funk and Chen, 2000).

The basal percentage of PPI response was found similar between wild-type and 5-LO^−/− mice. However, the disruptive effect of amphetamine (10 mg/kg) in the PPI test, confirmed in all prepulse intensities in the wild-type mice was not observed in 5-LO^−/− mice. Nevertheless, apomorphine at the selected dose (0.5 mg/kg) did not induce PPI disruption in both 5-LO^−/− or wild-type. The disruptive effects of the glutamatergic antagonist MK801 (0.5 mg/kg) were comparable in wild-type and 5-LO^−/− mice. These results suggest a selective amphetamine-induced effect in the 5-LO^−/− mice. PPI, representing sensorimotor integration response, examines the efficiency of central inhibitory mechanisms necessary for fundamental neurobiological processes (Swerdlow et al. 2017). Our results suggest that in the absence of 5-lipoxygenase, there is no modification of forebrain structures' basal function required for the integrated response of normal PPI. However, we found decreased responsiveness of 5-LO^−/− mice to amphetamine-mediated responses in the PPI which might be a functional indication of the non-integrity of dopaminergic neurons.

On the other hand, a significantly increased basal motor activity was revealed in 5-LO^−/− mice, measured through horizontal and vertical exploratory activity and stereotyped-like behavior compared to wild-type mice. Apomorphine acts as a potent, and broad-spectrum dopamine agonist activating D1- and D2-like receptors (Jenner and Katzenschlager, 2016).

The well-known apomorphine-induced increased motor activity and stereotyped-like behavior were not found in wild-type or 5-LO^−/− mice, with doses of 0.5, 1.0 or 10 mg/kg. In contrast, the elevated basal motor activity of 5-LO deficient mice was attenuated by apomorphine. Similar effects were found in reserpine treated rodents in which apomorphine reverses motor deficits (Kaur et al. 1994). The influence of deficient 5-LO genotype in the regulation of striatal dopaminergic tone, as suggested before, should warrant our findings (Severson et al. 1981).

In agreement with these findings, reserpine treatment (1 mg/kg) induced a significant increase in the frequency of orofacial involuntary movements in the 5-LO^−/− but did not affect wild-type mice. Oral involuntary movements can be induced by long-term use of some drugs acting on the dopaminergic system as antipsychotics and levodopa (Bergman and Soares-Weiser, 2018; Calabresi et al. 2000). Reserpine, a monoamine-depleting agent, that irreversibly blocks the vesicular monoamine transporter 2 (VMAT2), reduces the sequestration of monoamines into synaptic vesicles, mostly notably dopamine, and promotes oral involuntary movements suitable for investigating this condition (Cunha et al. 2016; Issy et al. 2018; Reinheimer et al. 2020). The increased sensitivity to oral involuntary movements found in 5-LO^−/− mice suggests a pre-existing condition that affects reserpine effects. Data from an experimental group in which reserpine effects were analyzed 35 days after the first reserpine injection showed no long-lasting behavior change in the 5-LO^−/− mice. These results confirmed the persistent no-drug-induced alteration condition in the 5-LO^−/− mice corroborating its distinctive phenotype.

Dopaminergic tone and nigrostriatal vulnerability were evaluated in 5-lipoxygenase/leukotrienes-deficient mice. The authors found a modest basal reduction in the striatal dopamine level that was concomitant with a decline in the striatal tyrosine hydroxylase of 5-lipoxygenase/leukotrienes-deficient mice (Chou et al. 2013). Additionally, in the absence of 5-lipoxygenase, dopamine depletion and astrogliosis induced by the intoxicant MPTP were significantly lower compared to the wild-type mice (Chou et al. 2013).

Here we found higher motor activity of 5-LO^−/− mice compared to its counterparts. Recently, Barbosa-Silva and cols. described impairment in the baseline motor performance of 5-LO^−/− mice in the rotarod test and increased repetitive behavior, suggesting that these altered behavioral responses could occur by altered regulation of synapse pruning and neural connectivity in this genotype (Barbosa-Silva et al. 2022). Evidence suggests that the interactions between neural and immune cells during development play a role in synapse formation (pruning, and plasticity) mechanisms that may persist in both physiological and pathological conditions (Phillis et al. 2006; Chu and Praticò, 2009; Bilbo and Schwarz, 2009; Chavan et al. 2017; Barbosa-Silva et al. 2022). Altered behavioral responses might be a functional indication changes in the connectivity of dopaminergic neurons in 5-LO^−/− mice.

The involvement of arachidonic acid metabolites, including leukotrienes in neurodegenerative diseases has been demonstrated (Kursun et al. 2022). As mentioned before, the formation of LTB₄ is catalyzed by 5-lipoxygenase encoded by the Alox5 gene. LTB₄ is important for the regulation of the hypothalamus/hypophsis axis and consequently corticosterone levels (Locachevic et al. 2019). The altered phenotype of Alox5^−/− mice includes reduced sucrose preference, suggesting a depressive-like behavior, and reduced reward sensitivity (Locachevic et al. 2019). Given that dopamine is considered an essential element in the brain reward system (Di Chiara and Bassareo, 2007; Arias-Carrión et al. 2010), 5-lipoxygenase-leukotrienes-deficient mice might show an altered dopaminergic function. Our data corroborates this hypothesis since 5-LO^−/− mice demonstrated decreased responsiveness to amphetamine-mediated responses.

In addition, LTB₄ is recognized as a TRPV1 ligand (Vigna et al. 2011). Endogenous agonist ligands of TRPV1 include the endocannabinoids, lipoxygenase products such as LTB₄, 2-arachidonic glyceryl ether, and N-arachidonoyl-dopamine (Vigna et al. 2011). The association of LTB₄ mediating TRPV1-induced effects was already demonstrated in inflammatory conditions (Vigna et al. 2011). Recently, a distinct TRPV1 positive ventral tegmental area neuron subpopulation was identified as a critical modulatory component in responsiveness to amphetamine (Serra et al., 2021). These neurons co-express both dopamine and glutamate markers (Viereckel et al. 2016; Serra et al. 2021). Dopamine neurons from the ventral tegmental area, where the TRPV1 gene is highly expressed, send extensive forebrain projections, primarily to the nucleus accumbens and cerebral cortex. An interesting mesoaccumbal dopamine projection is strongly associated with reward processing, motivation, and behavioral reinforcement (Viereckel et al. 2016; Arias-Carrión et al. 2010; Serra et al. 2021). 5-LO^−/− mice is deficient in several leukotrienes and also the LTB₄ conversion pathway. Further investigation is needed to reveal if the decreased LTB₄ source has a role in the altered dopaminergic response of the 5-LO^−/− genotype.

Finally, reserpine treatment induced a significant increase in both astrocyte expression (GFAP-ir) and in the percentage of reactive microglia (Iba1-ir) in wild-type mice in the dorsal striatum, but these effects were not observed in the 5-LO^−/− genotype. These events were time-dependent, and no longer changes in microglia profile occurred. Considering that LTB₄ is one of the main products of 5-lipoxygenase activation and it is released and acts in the microglia, it is possible to propose a direct correlation between lesser sensitivity microglia and the absence of 5-lipoxygenase/LTB₄. According, decreased astrogliosis in the 5-lipoxygenase deficient mice was previously reported (Chou et al. 2013). These findings give sufficient reason to hypothesize that the 5-lipoxygenase/LTB₄ pathway might be a key regulator of glial cell activation (Strempfl et al. 2022).

We cannot exclude the possibility that the deregulated basal inflammatory profile of 5-lipoxygenase deficient mice could influence the results found here. This genotype presents at basal condition increased levels of splenic IL-1β and IL-17 (Locachevic et al. 2019). In fact, 5-LO^−/− mice develop exacerbated inflammatory responses that includes increased stimulated PGE2 production induced by exogenous toxin or stress (Zoccal et al., 2016; Locachevic et al. 2019). On the other hand, it was described that human inhibitors of 5-lypoxygenase suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages and prostaglandin export (Rossi et al. 2010; Kahnt et al. 2013; 2022).

In conclusion, we found changes in the behavioral responses modulated by the dopaminergic system in 5-lipoxygenase-deficient mice related to the motor and sensorimotor functions. We also found a distinctive glial reactive profile in this genotype. Whether there is a correlation between these response profiles is still unknow. Together, these findings suggest that 5-lypoxygenase contributes to physiologic striatal dopamine homeostasis but, when overactivated, leads to neuroinflammation and contributes to neurodegeneration.

Acknowledgments

We would like to thank Célia Ap. da Silva for her helpful technical assistance.

Author Contribution

ACI, JFP, GCN performed experiments and analyzed the data. ACI; EDB wrote the paper. ACI; JFP; LHF; GCN and E.D.B. designed the experiments, supervised the project, and contributed to the writing the paper. All authors discussed the results and commented on the manuscript at all stages. All authors participated in the review of the manuscript.

Financial Support

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and from Conselho Nacional de Pesquisa (CNPq). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.

Ethics Statement

This study was carried out in accordance with the recommendations of National Council for Control of Animal Experimentation (CONCEA).

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Differential dopamine-mediated effects in the 5-lipoxygenase deficient mice

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Abstract

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Introduction

Material and Methods

Experimental Design

Immunohistochemical analysis

Results

Amphetamine effect in the PPI test

Apomorphine effect in the PPI test

MK801 effect in the PPI test

Apomorphine reduced motor activity and stereotyped-like behavior in the 5-LO^−/− mice

Reserpine effects in the 5-LO^−/− mice

Striatal astrocyte (GFAP) expression

Striatal microglia (Iba 1) expression and reactivity

Discussion

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References

Additional Declarations

Status:

Journal Publication

Version 1

Differential dopamine-mediated effects in the 5-lipoxygenase deficient mice

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Material and Methods

Experimental Design

Immunohistochemical analysis

Results

Amphetamine effect in the PPI test

Apomorphine effect in the PPI test

MK801 effect in the PPI test

Apomorphine reduced motor activity and stereotyped-like behavior in the 5-LO−/− mice

Reserpine effects in the 5-LO−/− mice

Striatal astrocyte (GFAP) expression

Striatal microglia (Iba 1) expression and reactivity

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1

Apomorphine reduced motor activity and stereotyped-like behavior in the 5-LO^−/− mice

Reserpine effects in the 5-LO^−/− mice