Mechanism Of Basal Forebrain Cholinergic Neurons In Regulating The Cognitive Functions In Hypothyroid Mice

Objective: Hypothyroidism is a common endocrine disease in clinic,which is caused by insufficient synthesis,secretion or biological effects of thyroid hormones(TH).The etiology of hypothyroidism is complex,and many of which is primary hypothyroidism.Adult hypothyroidism is accompanied by neuropsychiatric symptoms,muscular and articular lesions,in addition to hypometabolic syndrome,myxedema and other symptoms.Clinical hypothyroidism requires thyroid hormone replacement therapy,but the neuropsychiatric symptoms of some patients are still not completely relieved when the thyroid hormone level is normal.This project intends to establish hypothyroid animal model with transgenic mice to study the changes of cognitive behavior of hypothyroid mice,and treated by thyroxine(LT4),chemogenetics and pharmacological methods were employed to specifically regulate the activity of cholinergic neurons,so as to elucidate the mechanism of basal forebrain cholinergic neurons in hypothyroidism induced cognitive dysfunctions.Method: The specific pathogen free ChAT-Cre transgenic mice(The Cre gene was inserted downstream of choline acetyltransferase,which was regulated by the ChAT gene promoter and specifically expressed in cholinergic neurons)was used in the current study,The body weight of the mice was about 22-25 g(age 8-10 weeks)at the beginning of the experiment,The AAV-DIO-h M3Dq-m Cherry(20 nl / side)was stereotactically injected into the basal forebrain of the ChAT Cre mice.After the operation,the mice were fed in the laboratory environment with temperature of 22 ° C ± 1 ° C,humidity of 55%-65%,light and dark cycle(12 h / 12 h,light on time of 7:00).The hypothyroid model mice were induced by propylthiouracil(PTU)(dissolved in drinking water at the concentration of 1 mg / ml),and PTU solution was prepared and changed once a week.At the same time,the animal bedding chips were changed and body weight were recored.From the fourth week,the LT4 replacement treatment mice were received LT4 10 mg / kg on the basis of PTU solution,and electrophysiological and behavioral experiments were carried out after 6-7 weeks from the beging of the experiment.The electrophysiological characteristics of cholinergic neurons in basal forebrain of normal mice and hypothyroid mice were detected by patch clamp technique;the changes of learning and memory ability of hypothyroid mice after specific activation of cholinergic neurons in basal forebrain were studied by chemogenetics,combined with passive avoidance test or new object recognition test.After all experiments,blood was collected from heart to measure the levels of T3,T4 and TSH to assess the thyroid function of different mice.The C57BL/6j mice were divided into 5 groups: control group,hypothyroidism group,LT4 treatment group,LT4 + donepezil(5 mg / kg)group and LT4 + donepezil(10 mg / kg)group.The hypothyroid model mice were incudec by PTU(1 mg / ml)in drinking water,and which was changed once a week.At the same time,the animal bedding chips were changed and body weight were recored.From the fourth week,LT4 replacement treated mice were received LT4 10 mg / kg on the basis of PTU solution;LT4 combined with donepezil treatment mice were given LT4 10 mg / kg and donepezil 5,10 mg / kg on the basis of PTU for additional 2-3 weeks.Then the passive avoidance test and new object recognition test were used to determine the learning and memory ability of different mice.After the behavioral test,blood samples were collected from the heart after anesthesia of animals.Serum T3,LT4 and TSH levels were measured to evaluate the thyroid function of different mice.To explore the effect and mechanism of cholinergic neurons in basal forebrain on the cognitive ability of hypothyroid mice.Result: For ChAT Cre mice,the body weight of the hypothyroid mice in the first and second weeks was significantly lower than that of the normal control group,tehn there was no significant difference in the body weight of the mice in the third,fourth,fifth and sixth weeks.The serum T3 and T4 levels of hypothyroidism mice were significantly lower than those of the control group and LT4 mice,while the serum TSH level of hypothyroidism mice was significantly higher.The amplitude of action potential of cholinergic neurons decreased,the level of resting membrane potential increased in hypothyroid mice.The expression of h M3 Dq in cholinergic neurons was confirmed by immunofluorescence staining.Clozapine oxide(CNO)can specifically activate the h M3 Dq protein on basal forebrain cholinergic neurons by activation of the Gq signal pathway and excite the cholinergic neurons in basal forebrain.The results of whole cell patch clamp showed that CNO could induce the discharge of cholinergic neurons to increase continuously and excite cholinergic neurons.Passive avoidance test showed that the latency to enter the dark box of hypothyroidism mice was significantly shorter than that of normal control mice,while LT4 replacement therapy could not completely restore it to the level of normal control mice.Specific activation of basal forebrain cholinergic neurons by chemogenetics could significantly increase the latency to enter into the dark box of hypothyroidism mice and LT4 treated mice,and improve their learning and memory ability.For C56BL/6j mice,the body weight of the hypothyroid mice in the first and second weeks was significantly lower than that of the normal control group,and there was no significant difference in the weight of the mice in the third,fourth,fifth and sixth weeks.The serum T3 and T4 levels of hypothyroidism mice were lower than those of the control group,LT4 treated or LT4 combined with donepezil treated mice,while the serum TSH level of hypothyroidism mice was significantly higher.After LT4 combined with different doses of donepezil(5,10 mg / kg),donepezil can shorten the latecy to enter into the dark box of mice in a dose-dependent manner.Similarly,the new object recognition test results also indicated that the new object recognition scores of hypothyroidism or LT4 replacement treatment mice were lower.The donepezil is an ACh E inhibitor in the central nervous system,which has little effect on peripheral tissues.Donepezil can inhibit ACh E activity,slow down the decomposition of acetylcholine(ACh)in the synaptic space,increase ACh content in the synaptic space,improve cholinergic activity,thus improve cognitive function,and effectively improve the learning and memory ability of mice.Conclusion: 1.A decreased electrophysiological characteristics of basal forebrain cholinergic neurons was found in hypothyroid mice,and not all the cholinergic neurons recovery to physiological condition after LT4 replacement therapy,suggesting that basal forebrain cholinergic neurons play an important role in the memory impairemnts in hypothyroid mice.2.Activation of basal forebrain cholinergic neurons by chemogenetics can significantly improve the learning and memory ability of hypothyroid mice.3.Combine the LT4 and donepezil to treat the hypothyroid mice,the cognitive ability of hypothyroid mice can also be effectively improved.