Effects of (±)-9α-Hydroxyhexahydrocannabinol on excitability of hippocampal neurons

Cannabinoid-related compounds influence neuronal signalling pathways involved in membrane excitability, synaptic transmission, and neuroplasticity. Several phytocannabinoids and their derivatives modulate neuronal activity through cannabinoid receptor-dependent and receptor-independent mechanisms. However, the neurophysiological effects of hydrogenated cannabinoid derivatives remain poorly characterised. (±)-9α-hydroxyhexahydrocannabinol (9α-OH-HHC) is a hydroxylated hexahydrocannabinol derivative with potential interactions with cannabinoid-sensitive signalling pathways, yet its direct effects on neuronal viability and intrinsic electrophysiological properties have not been systematically investigated. Because hippocampal neurons are critically involved in learning, memory formation, and network excitability, primary hippocampal cultures provide a suitable model for examining cellular responses to novel cannabinoid-related compounds.

The aim of this study was to determine whether 9α-OH-HHC affects neuronal viability and intrinsic electrophysiological properties in primary hippocampal neurons. Based on known modulatory effects of cannabinoids on neuronal excitability, we hypothesised that exposure to this compound could alter membrane properties and action potential firing behaviour.

Primary hippocampal cultures were prepared from postnatal day 0 Wistar rats. Hippocampi were enzymatically dissociated using trypsin and cells were plated on culture plates for viability assessment or on glass coverslips for electrophysiological recordings. Cultures were maintained under standard incubator conditions, and experiments were conducted after 10 days in vitro. Neuronal metabolic activity was assessed using the MTS assay following exposure to 9α-OH-HHC at 100 nanomolar, 1 micromolar, and 10 micromolar concentrations. The compound was dissolved in dimethylsulfoxide and applied either acutely prior to testing or chronically for 48 hours. Electrophysiological recordings were performed using the whole-cell patch clamp technique in current clamp configuration. For electrophysiological experiments, neurons were exposed to 10 micromolar 9α-OH-HHC either acutely or chronically for 48 hours. Resting membrane potential was measured without current injection. Additional parameters included input resistance, rheobase, single action potential amplitude, action potential threshold, and action potential rise time. Neuronal firing during a 300 millisecond depolarising current pulse was analysed with respect to spike count and dynamic changes in action potential threshold and rise time. Statistical analysis was performed using two-way analysis of variance ANOVA followed by Tukey post hoc test.

Exposure to 9α-OH-HHC did not significantly affect neuronal metabolic activity across the tested concentration range. Electrophysiological analysis revealed no statistically significant differences between treated and control neurons in resting membrane potential, input resistance, rheobase, action potential amplitude, action potential threshold, or action potential rise time (two-way ANOVA, p > 0.05). Similarly, analysis of action potential firing during depolarising current stimulation showed no significant differences in spike count or firing dynamics after either acute or chronic exposure (two-way ANOVA, p > 0.05).

In this pilot study, 9α-OH-HHC did not significantly alter metabolic viability or intrinsic membrane excitability of primary hippocampal neurons. The absence of detectable electrophysiological changes may reflect limited interaction of this compound with neuronal ion channels or insufficient engagement of cannabinoid receptor-mediated signalling pathways in this cellular model. Alternatively, functional effects may require synaptic connectivity or network-level processes not fully developed in dissociated neuronal cultures. Further studies examining synaptic transmission and receptor-specific mechanisms may provide additional insight into the neurobiological profile of this compound.

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