The Influence of Polyphenols on Calcium Dynamics in Synaptosomes of Model Rats with Attention Deficit Hyperactivity Disorder of Varying Ages

Abstract

Intrоduсtiоn: ADHD is characterized by disrupted neuronal excitability, often associated with dysregulated calcium (Ca²⁺) signaling. We investigated the effects of the polyphenol ANK-2 on intracellular Ca²⁺ dynamics in synaptosomes isolated from the brains of healthy and ADHD-model rats of different ages. Mаtеriаls аnd Mеthоds: In the studies, rats weighing 150 - 200 g were subjected to an ADHD model by administering 6-OHDA at 2 different doses of 8 and 4 mg/kg orally for 14 days using a special probe. Also, in order to induce the АDHD model in the prenatal period in young rats, it was first established that rats of both sexes were placed together, their condition was determined using a swab, and then, on average 3 - 4 days before birth, CdCl₂ was dissolved (50 ppm) and administered orally to pregnant rats weighing 200 - 250 g using a special probe, as a result of which the ADHD model was manifested in the offspring. Using the calcium-sensitive fluorescent probe Fluo-4AM, we found that ANK-2 significantly reduced cytosolic Ca²⁺ levels in a dose-dependent manner. Results and discussion: In ADHD models, baseline Ca²⁺ content was markedly elevated compared to age-matched controls, consistent with hyperactive synaptic transmission. GABA (100 μM) decreased [Ca²⁺] in by 14 % in healthy rats and 20 % in ADHD rats, suggesting enhanced compensatory GABAergic inhibition in the ADHD condition. Co-incubation with ANK-2 (10 - 100 μM) further suppressed calcium influx, indicating a synergistic enhancement of GABAergic effects. Additionally, ANK-2 potentiated the calcium-lowering effect of levetiracetam (5 μM), a selective N-type Ca²⁺ channel blocker, further reducing cytosolic Ca²⁺ levels. Соnсlusiоns: These findings suggest that ANK-2 acts through multiple mechanisms, including enhancement of GABAergic signaling, direct or indirect inhibition of voltage-gated calcium channels, and modulation of neurotransmitter release. Our results position ANK-2 as a promising candidate for correcting excitatory-inhibitory imbalance in ADHD by targeting calcium-dependent synaptic transmission.

HIGHLIGHTS

• 6-OHDA selectively damages dopaminergic and noradrenergic neurons, mimicking ADHD-like hyperactivity. Provide a description for each abbreviation at the beginning: Dopamine (DA) levels alter voltage-gated calcium channels (VGCC) and intracellular calcium buffering, leading to abnormal calcium influx. 6-OHDA induces dopaminergic neurodegeneration, particularly in the mesocortical and nigrostriatal pathways. This results in compensatory activation of glutamate receptors and VGCC in postsynaptic neurons, resulting in an increase in intracellular Ca²⁺.
• On the other hand, cadmium chloride disrupts calcium channels (e.g., L-type Ca²⁺ channels) and calcium-dependent functions in mitochondria, which aggravates synaptic dysfunction. Cd²⁺ mimics Ca²⁺ and blocks Ca²⁺ ATPases and Na⁺/Ca²⁺ exchangers, disrupting calcium transport. Impaired Ca²⁺ buffering and elevated synaptosomal calcium levels cause oxidative stress and mitochondrial dysfunction.
• The results of behavioral tests on rats with ADHD model during our experiments show that in the control groups the experimental animals were very active and hyperactive in the Open Field test, moved quickly and hardly stood still. At the same time, the model showed that the ADHD groups were very hyperactive, with nervous system processes being activated, while the animals remained motionless.
• The polyphenol ANK-2 significantly reduces intracellular calcium levels in Fluo-4 AM synaptosome suspensions from rat brains of different ages. This may explain that polyphenol ANK-2 enhances the inhibitory effect of GABA (gamma-aminobutyric acid), helping to reduce neuronal excitability, or probably acts as a direct or indirect modulator of presynaptic calcium channels, particularly N-type Ca²⁺ channels, thereby reducing calcium influx.

GRAPHICAL ABSTRACT