Behavior test
The number of entries into (0.80 ± 1.17 times, p < 0.05) and duration in (12.48 ± 13.65s, p < 0.01) open-arms were significantly lower than that of the control group (3.40 ± 1.62 times, 59.74 ± 23.32s), showing stronger anxiety degree. After withdrawal, the anxiety of rats was relieved.
The literature shows that acute administration of 25mg/kg ketamine could rapidly activate the autonomous behavior of rats, which was characterized by excessive exercise, stereotyped behavior, and ataxia20. However, in other studies, it was found that the movement of rats was reduced by low-dose ketamine injection21,22.
In addition, ketamine showed obvious tolerance in the process of long-term use. A patient with severe depression gradually developed the dosage from 50mg/week to 2g/day after 6 months, which also caused drug dependence, loss of consciousness, dissociative stupor, and amnesia23. It also has many data supports that cognitive impairment and memory impairment could be caused by ketamine24,25. For example, the 60 mg/kg/d dose of ketamine administration for a half year could lead to mice spatial recognition memory deficits26.
In the EPM, group K stayed longer in the closed arm and entered the open arm less frequently. Combining the gravity center path with the head path map, it can be found that the three groups of rats have exploratory behavior in the open arm and the border area, but group K could hardly overcome fear and enter the open arm area, and only stay in the closed arm or the outward probe of the central platform, suggesting that their anxiety level was higher. Greer McKendrick et al.reported that a single dose of acute pretreatment with ketamine (10 mg/kg, ip) increased the time spent in the opening arm of mice in the saline group and morphine group, causing an anti-anxiety role27. In general, the frequency of entering the open arm and the open arm duration of Group W were similar to the group S, but the performance of individuals in Group W was significantly different, and there was no significant difference between Group W and Group K. In addition, compared with group S, group W has significantly increased the behavior of extending its head out of the open arm to explore, which was contrary to its performance in new object recognition experiments. This experiment cannot explain this difference.
Addiction pathway
In our study, the influence value of addiction-related pathways enriched by KEGG is relatively small. Most drugs could disturb the dopamine (DA) system of the ventral tegmental area and reconstruct the brain's reward circuitry. It also induces extensive glutamatergic synapse adaptation.Wu et al found that ketamine rapidly enhances glutamate-evoked spinogenesis in the medial prefrontal cortex, it increases evoked cortical spinogenesis through dopamine Drd1 receptor activation that requires dopamine release28.
Other researchers believed that ketamine could activate the mTOR pathway of human mesencephalic dopamine neurons.However, the activation of dopamine neurons caused by ketamine only existed for a few minutes29, which may explain our group did not find the phenomenon of dopamine increase in the previous study of neurotransmitters.
Like dopamine (DA), cannabinoids significantly affect brain functions, which include memory, emotion, and sensory. As a mainly 2-AG receptor, endogenous cannabinoid receptors are expressed widely in PFC.Previous study shows that blockade could alleviate the neuron remodel effect of ketamine30. It also was proved that the use of ketamine could alleviate different drug addictive associated diseases,such as prolong sustain abstinence and relief the withdrawal symptoms. In the morphine-dependent model, ketamine eased naloxone-withdrawal symptoms and eliminated morphine-induced place preference in rats31.
Cysteine and methionine metabolic pathways
Methionine is an essential amino acid, which was the main methyl donor in the body. It generates S-adenosylmethionine through the transmethylation pathway, and the latter participates in almost all methylation reactions in the body32. Much evidence shows that SAA plays a key role in protein structure, metabolism, immunity, and oxidation33. Methionine and cysteine are extremely sensitive to reactive oxygen species, which have an obvious antioxidant effect and reduce oxidative stress damage caused by various factors. Research shows that ketamine can promote the production of ROS and malondialdehyde in the prefrontal cortex, hippocampus, thymocytes, and ureteral epithelial cells, mediate mitochondrial apoptosis pathway and autophagy activation, and produce cytotoxicity34,35. In this experiment, the contents of six different metabolites of cysteine and methionine metabolic pathway in the plasma of the K group were decreased, indicating that the consumption of cysteine and methionine in plasma increased, suggesting that ketamine may cause oxidative stress injury in rats. After one week of withdrawal, the levels of L-methionine, S-adenosylhomocysteine, and L-serine in plasma were still lower than in the control group, indicating that the redox homeostasis imbalance caused by ketamine had not been completely repaired.
Glutamine and glutamate metabolic pathway
The regulation of Glu and Gln metabolism is critical to energy homeostasis and excitatory neural signal transmission. In the cytoplasm, Gln provides nucleotides and hexosamine via amide groups γ- Nitrogen and generates Glu, participates in the synthesis of glutathione (GSH), and maintains the balance of redox reaction.Glu is an excitatory neurotransmitter, it is stored in synaptic vesicles, released into the synaptic space, and absorbed back into the cell by transporters. In addition, Gln can be metabolized into γ-aminobutyricacid (GABA), which is an inhibitory neurotransmitter36, their participation regulates the transmission of neural signals in the nervous system. Evidence shows that the glutamate system is highly correlated with the pathophysiology of depression. Neuro-imaging and autopsy studies found that Glu levels in plasma, cerebrospinal fluid, and brain of patients with depression have increased37.
In addition, the effects of ketamine on the metabolism of Gln and Glu have also been reported. On the one hand, 30 mg/kg ketamine treatment of young rats can increase the levels of Gln and Glu in the prefrontal cortex and nucleus accumbens, and this change was maintained till adulthood, which may be related to ketamine's antagonism against the oxidative stress38. On the other hand, the levels of Gln and Glu in the hippocampus of mice decreased 14h and recovered in 72 h after a single dose of 3mg/kg ketamine administration36. In this experiment, the chronic low-dose ketamine caused a decrease in plasma Gln and Glu content, which was consistent with its antidepressant effect.
Purine metabolic pathway
In the prefrontal cortex, the levels of adenosine, adenine, inosine, hypoxanthine, and guanosine were decreased, and the content of xanthine increased. In vivo,purine mainly exists in the form of purine nucleotides, which can participate in the synthesis of nucleic acids, and also participate in energy metabolism, oxidative stress, neural regulation, and other important metabolic pathways,as a signal molecule39. It has been proved that purine metabolites and their receptors are related to various mental diseases. The imbalance between extracellular ATP and adenosine concentrations is considered to be the cause of central nervous system diseases, including mental disorders40. In the early work of our research group, we also found that repeated administration of 30 mg/kg/d ketamine for 10 days could disturb the purine metabolic pathways in the rat hippocampus and striatum, and the levels of various metabolites also showed significant differences41.
The correlation between purine and depression has also been reported. Studies have shown that the polymorphism of the P2RX7 gene encoding purinergic ion channel is related to the development of refractory depression42, and the inhibition of adenosine A1 and A2A receptors also mediates antidepressant-like effects43. In this experiment, it was found that the chronic low-dose ketamine had an impact on the purine metabolic pathway in the prefrontal cortex of rats, but the specific consequences caused by the changes of these metabolites were not clear, and the changes of specific substances in the pathway should be accurately described by other methods, such as the method of tracer.