Impaired Glutamatergic and GABAergic Transmission by Amitraz in Primary Hippocampal Cells
Keywords: Amitraz, Primary hippocampal cells, Glutamate, GABA, Glutaminase, GAD 65
Abstract
Amitraz is a formamidine pesticide reported as a neurotoxic compound that induces convulsions among other effects. Excitatory and inhibitory neurotransmission is mediated mainly by glutamate and GABA, respectively, so their alteration could be responsible for induction of seizures. Amitraz’s α₂ adrenergic agonist action, which has been suggested as likely responsible for this effect, could alter these neurotransmitter systems and lead to seizure induction. Moreover, other mechanisms such as histamine H₁ receptor inhibition could be involved. To confirm if amitraz disrupts glutamatergic/GABAergic transmission by these mechanisms, we evaluated, in primary hippocampal neurons, the effect of amitraz (0.01 μM to 100 μM) with or without the α₂ adrenergic antagonist idazoxan (1 μM) and/or the H₁ receptor agonist n-methylhistaprodifen (30 μM) co-treatment on 4-aminobutyrate aminotransferase, glutamate decarboxylase 65 (GAD 65), succinate-semialdehyde dehydrogenase and glutaminase gene expression and on glutamate and GABA levels after 24 h treatment. We observed that amitraz disrupts glutaminase and GAD 65 gene expression, altering glutamatergic and GABAergic transmission. These effects were mediated partially by H₁ and α₂ receptors, suggesting that other mechanisms could be involved. These data could help explain the mechanism by which amitraz induces seizures and provide a therapeutic strategy to protect against this effect in case of poisoning.
1. Introduction
Amitraz (1,5 di-(2,4-dimethylphenyl)-3-methyl-1,3,5-triaza-penta-1,4-diene) is a formamidine pesticide used worldwide on animals and crops to control pests. Many cases of amitraz poisoning have been reported, which is a cause of concern for health authorities.
Amitraz is a potent neurotoxic compound that induces sedation, loss of righting reflex, motor incoordination, coma, and seizures. It exerts neurotoxic effects through several mechanisms: monoamine oxidase (MAO) inhibition, H₁ receptor inhibition, and α₂ receptor agonism. Amitraz is also an endocrine disruptor affecting hormone levels such as estradiol and testosterone.
Previous reports indicate that amitraz can induce seizures in the hippocampus of rats, potentially via its α₂ adrenergic agonist properties. Inhibition of histamine H₁ receptors has also been implicated in seizure induction. Alternatively, seizures could be induced through dysfunctions of glutamatergic and GABAergic systems.
Based on these findings, we hypothesized that amitraz could alter glutamatergic and GABAergic transmission through inhibition of α₂ and H₁ receptors. To test this, we studied primary hippocampal neurons treated with amitraz (0.01 μM to 100 μM) with or without the α₂ receptor antagonist idazoxan (1 μM), and the H₁-agonist n-methylhistaprodifen (30 μM).
2. Methods
2.1. Chemicals
Amitraz (98%), idazoxan, n-methylhistaprodifen, GABA, glutamate, poly-L-lysine, and DMSO were from Sigma. Anti-MAP2 and anti-GFAP antibodies were from Millipore. All other chemicals were of analytical grade.
2.2. Primary Hippocampal Neuron Culture
All procedures followed EU and Spanish regulations for animal use. Hippocampi from embryonic day 17–18 Wistar rats were dissected and cultured as previously described. Viability was >95% by trypan blue exclusion. Cultures were 92% neurons (MAP2+) and 8% astrocytes (GFAP+).
On day 7 in vitro, cells were treated with amitraz (0.01–100 μM) with or without idazoxan (1 μM) and/or n-methylhistaprodifen (30 nM) for 24 h. A vehicle group with 0.1% DMSO was used as control.
2.3. Measurement of Cell Viability (MTT Assay)
After 24 h treatment, cell viability was assessed by MTT reduction. Absorbance was read at 562 nm and expressed as % of control (0.1% DMSO).
2.4. Amino Acid Determination
Glutamate and GABA levels were measured in culture supernatant by HPLC after perchlorate precipitation and o-phthalaldehyde derivatization. Results were normalized to total protein (BCA assay) and expressed as % of untreated control.
2.5. Real-Time PCR Analysis
Total RNA was extracted (Trizol), quantified, and reverse-transcribed. qPCR was performed for glutaminase, GABAT, GAD 65, SSADH, and ACTB (internal control). Relative gene expression was calculated using the ΔΔCt method.
2.6. Statistical Analysis
Data are mean ± SD of at least three replicates. One-way ANOVA with Tukey post-hoc test was used. p ≤ 0.05 was considered significant.
3. Results
3.1. Effect of Amitraz on Cell Viability
MTT assay showed reduced cell viability only at the highest amitraz concentration (100 μM) compared to control. Idazoxan, n-methylhistaprodifen, or their combination did not affect viability alone, but co-treatment with amitraz reduced cell death compared to amitraz alone at 100 μM.
3.2. Amino Acid Determination
After 24 h amitraz exposure, GABA levels increased at 0.1–1 μM but decreased at ≥10 μM. Glutamate showed the opposite pattern. Idazoxan or n-methylhistaprodifen partially reversed these changes; combined co-treatment did not fully restore levels. Changes were not due to cell loss, as normalization by protein content confirmed altered synthesis.
3.3. Real-Time PCR Analysis
Amitraz upregulated GAD 65 expression at 0.1–1 μM and downregulated it at ≥10 μM. Glutaminase was significantly upregulated at ≥10 μM. Idazoxan or n-methylhistaprodifen partially reversed GAD 65 changes; n-methylhistaprodifen reversed glutaminase upregulation. Combined co-treatment had a greater effect on GAD 65 but did not fully restore expression.
4. Discussion
Amitraz disrupts glutamate and GABA levels in hippocampal neurons, likely by altering their synthesis through changes in glutaminase and GAD 65 gene expression. These effects are partially mediated by α₂ adrenergic activation and H₁ receptor inhibition, but other mechanisms may contribute. The reduction in GABA and increase in glutamate at higher amitraz concentrations could underlie the compound’s pro-convulsant effects.
α₂ adrenoceptor agonists can be anticonvulsant at low doses and pro-convulsant at high doses, modulating GABA and glutamate release. H₁ receptor inhibition is associated with seizure induction and altered GABA/glutamate synthesis. Amitraz’s effects on these receptors may thus explain its neurotoxicity and seizure-inducing potential. The concentrations used in vitro are relevant to those producing toxicity in vivo.
Further studies are needed to clarify the involvement of JHU395 neurotransmitter uptake/metabolism and to fully assess the risk of amitraz and related compounds.