Cannabinoid Type Ⅱ Receptors Protect Rat Hippocampal Neurons Against OAβ-induced Neuronal Hyperexcitation

Alzheimer’s disease（AD）is a common chronic neurodegenerative disease.It is characterized by an increase in the accumulation ofβamyloid peptides（Aβ）and a gradual loss of learning and memory.Patients often have a cognitive impairment,loss of language and visual ability,and changes in mental personality and behavior.In recent years,studies have found a significant increase in the incidence of epilepsy in patients with AD,which may be related to the hyperexcitation of neurons and their circuits due to the increased aggregation of Aβ.Studies have shown that soluble Aβoligomer（o Aβ）plays a crucial role in driving neuronal hyperactivity in patients with AD and is the main form of neurotoxicity of Aβ.Inhibiting the toxic effect of Aβmay be an important therapeutic strategy for protecting hippocampal neurons and reducing damage.Aβ is a polypeptide that is highly susceptible to aggregation.Aβmonomer can aggregate to soluble oligomers and insoluble fibrous,further deposited to form amyloid plaques.The formation of amyloid plaques is a common feature of AD neuropathology.Studies have confirmed that,contrary to Aβplaques,soluble Aβoligomers,especially the oligomeric Aβ_1-42is a key driver of neuronal dysfunction,which can induce neuronal hyperactivity and damage hippocampal neurons.Cannabinoid type 2 receptor（CB₂R）plays the neuroprotective roles in various neurological diseases,including AD.Studies have shown that activating CB₂R can exert an anti-inflammatory effect and enhance the clearance of amyloid proteins to reduce the production of amyloid peptides.Experiments have confirmed that the activation of CB₂R inhibits the firing rate of VTA dopaminergic neurons and reduces the excitability of dopaminergic neurons.However,whether CB₂R activation plays a role in protecting hippocampal neurons against o Aβ-induced neuronal hyperexcitation remains unclear.In this study,Hippocampal primary cultures of SD rats within 24 hours of birth were used to evaluate the o Aβ_1-42-induced hyperexcitation,and the effects of CB₂R agonist JWH133 on the o Aβ_1-42induced neuronal hyperexcitation.We used single-cell patch-clamp technology,Lactate Dehydrogenase（LDH）kit to investigate whether CB₂R can protect hippocampal neurons against the neuronal hyperexcitation and neurotoxicity induced by o Aβ_1-42.The results of the experiment are as follows:1.We first identify neuronal phenotypes of cultured rat hippocampal neurons according to their morphological and electrophysiological features.The results showed that the averaged action potential firing rate evoked by an injection of 90 p A depolarizing current for pyramidal neurons（15.9±0.8 Hz）was slower than that for non-pyramidal neurons（29.8±1.3 Hz）,and the difference was statistically significant（P<0.001）.In following experiments,pyramidal neurons were selected.2.After exposed to 100 n M o Aβ_1-42for 7 days,cultured hippocampal neurons exhibited an increased action potential firing rate after current injection（90 p A）compared to control cells（P<0.01）.The frequency of spontaneous excitatory postsynaptic currents（s EPSCs）was increased（P<0.01）.In addition,LDH release was also significantly increased compared with the control group（P<0.05）.These results suggest that chronic treatment with o Aβ_1-42 results in neuronal hyperexcitation and toxicity in primary hippocampal cultures.3.Single-cell patch-clamp recordings showed that in chronic o Aβ_1-42 treated neurons,when depolarized current stimulation of 60,90,120,or 150 p A was administered to hippocampal neurons,JWH133 significantly shifted the input-output relationship curve to the right compared with o Aβ_1-42 group alone.Statistical comparisons of the slope of AP firing with different injected currents showed that chronic treatment with o Aβ_1-42 induced the hyperexcitation in cultured hippocampal neurons（P<0.01）,and the CB₂R agonist JWH133 protected neurons against the o Aβ_1-42-induced hyperexcitation（P<0.05）,which was eliminated by a selective CB₂R antagonist,AM630（P<0.001）.This suggests that the activation of CB₂R protects hippocampal neurons against hyperexcitation induced by chronic o Aβtreatment.4.Analysis of the action potential parameters showed that when depolarizing stimulation of 90 p A of hippocampal neurons was administered,compared with the o Aβ_1-42 group alone,o Aβ_1-42 and 1μmol/L JWH133 co-treatment prolonged action potential initiation latency（P<0.05）and increased the post-hyperpolarization potential level（P<0.001）.These effects were eliminated by AM630（P<0.05）.5.CB₂R agonist JWH133 reduced the o Aβ_1-42 induced neuronal hyperexcitation in a concentration-dependent manner.There was no significant change in the firing frequency of neurons in the 0.1μmol/L JWH133 group compared with the o Aβ_1-42 group（P>0.05）.However,action potential firing rate in both JWH133 groups of 1μmol/L and 3μmol/L were reduced（P<0.001）.6.Compared with the control group,chronic treatment of o Aβ_1-42 alone for 5 days increased neuronal action potential firing rate（P<0.05）,but thereafter,o Aβ_1-42 and 1μmol/L JWH133 co-treatment for 2 days reduced the established neuronal hyperexcitation caused by o Aβ_1-42 treatment for 5 days（P<0.05）.The above results show that under the toxic effect of chronic o Aβ_1-42,neurons undergo hyperexcitation and apoptosis in primary cultured hippocampal neurons.This can be used as a cellular model for the neurotoxicity of o Aβ_1-42.We use this cell model to evaluate the effects of CB₂R agonist on the o Aβ_1-42-induced hyperexcitation of hippocampal neurons.We find that the CB₂R agonist JWH133 not only prevents the o Aβ_1-42-induced neuronal hyperexcitation,but also eliminates the established hyperexcitation of o Aβ_1-42.The application of the selective CB₂R antagonist AM630 can eliminate the effects of JWH133,suggesting that the neuroprotective effect of JWH133 is mediated by CB₂Rs on primary cultured hippocampal neurons.Our findings provide new insights into the neuroprotective effects mediated by CB₂Rs.