Activation of medial septum cholinergic neurons restores cognitive function in temporal lobe epilepsy

Cognitive impairment is the most common complication in patients with temporal lobe epilepsy with hippocampal scle rosis.There is no effective treatment for cognitive impairment.Medial septum cholinergic neurons have been reported to be a potential target for controlling epileptic seizures in tempo ral lobe epile psy.However,their role in the cognitive impairment of temporal lobe epilepsy remains unclear.In this study,we found that patients with temporal lobe epile psy with hippocampal sclerosis had a low memory quotient and severe impairment in verbal memory,but had no impairment in nonverbal memory.The cognitive impairment was slightly correlated with reduced medial septum volume and medial septum-hippocampus tra cts measured by diffusion tensor imaging.In a mouse model of chronic temporal lobe epilepsy induced by kainic acid,the number of medial septum choline rgic neurons was reduced and acetylcholine release was reduced in the hippocampus.Furthermore,selective apoptosis of medial septum cholinergic neurons mimicked the cognitive deficits in epileptic mice,and activation of medial septum cholinergic neurons enhanced hippocampal acetylcholine release and restored cognitive function in both kainic acid-and kindling-induced epile psy models.These res ults suggest that activation of medial septum cholinergic neurons reduces cognitive deficits in temporal lobe epilepsy by increasing acetylcholine release via projections to the hippocampus.

Emerging non-invasive therapeutic approaches targeting hypocholinergic neural systems in Parkinson's disease

Cholinergic system associated DOPA-refractory motor and cognitive symptoms-a need for novel therapeutic approaches:Accumulating evidence points to significant motor and non-motor morbidities associated with hypocholinergic deficits in central and peripheral neural systems in Parkinson’s disease(PD)(Bohnen et al.,2018,2022).This so-called“malignant”hypocholinergic disease phenotype is associated with DOPA-refractory dementia and mobility disturbances,such as falls and freezing of gait,and augur novel therapeutic approaches targeting cholinergic systems in PD.Cholinergic pharmacotherapy in PD has been an interest for a long time.However,the development of cholinergic augmentation pharmacotherapy has been hampered by limited clinical efficacy,the presumption that changes in cholinergic activity are homogeneous in the central nervous system(CNS)and peripheral nervous system,tolerance or safety of cholinesterase inhibitor drugs,low CNS penetrance,high rate of peripheral autonomic side-effects and clinical contra-indications.The development of nicotinic or muscarinic receptor modulating drugs appears more promising but is still in the development stage.Given the current unmet need for managing DOPA-refractory cognitive and mobility impairments associated with hypocholinergic neural systems,there is a need for novel and complementary therapeutic and more personalized approaches.

Anticonvulsant Effects of Chrysanthellum americanum L. (Vatke) Aqueous Extract in Mice Pilocarpine Model of Epilepsy and Associated Memory Impairment: Role of Antioxidant Defense System and Cholinergic Transmission

Chrysanthellum americanum (L.) Vatke is a medicinal plant used by the traditional healers to treat epilepsy and associated memory impairment. This work aims at evaluating the anticonvulsant effects of Chrysanthellum americanum aqueous extract in mice pilocarpine model of epilepsy and associated memory loss. Mice were administered orally Chrysanthellum americanum aqueous extract (27.69, 69.22, 138.45, 276.9 mg/kg, prepared from the whole part) for test groups, intraperitoneally 300 mg/kg sodium valproate for the positive control group or orally 10 mL/kg distilled water for the negative control group, respectively, during a period of seven consecutive days. On the first day, temporal lobe epilepsy was induced by intraperitoneal injection of 360 mg/kg pilocarpine one hour after the administration of different treatment to mice, and the occurrence of status epilepticus was evaluated. On the second day, the anticonvulsant property was measured after the intraperitoneal injection of a sub-convulsive dose of picrotoxin (1 mg/kg). On the seventh day, the anti-amnesic properties of the extract were evaluated in the epileptic mice using the T-maze and open field paradigms. The results show that Chrysanthellum americanum extract significantly (p Chrysanthellum americanum (276.9 mg/kg) likewise sodium valproate (300 mg/kg) significantly (p Chrysanthellum americanum aqueous extract has anticonvulsant effects against pilocarpine induced-epileptic seizures and memory impairment. These properties could be mediated by the amelioration of antioxidant defense system and cholinergic neurotransmission in epileptic mice, which could partly justify the use of Chrysanthellum americanum in the traditional medicine for the treatment of epilepsy.

Effects of repetitive transcranial magnetic stimulation on cognitive function and cholinergic activity in the rat hippocampus after vascular dementia

Repetitive transcranial magnetic stimulation （rTMS） is a non-invasive treatment that can enhance the recovery of neurological function after stroke. Whether it can similarly promote the recovery of cognitive function after vascular dementia remains unknown, In this study, a rat model for vascular dementia was established by the two-vessel occlusion method. Two days after injury, 30 pulses of rTMS were ad- ministered to each cerebral hemisphere at a frequency of 0.5 Hz and a magnetic field intensity of 1,33 T. The Morris water maze test was used to evaluate learning and memory function. The Karnovsky-Roots method was performed to determine the density of cholinergic neurons in the hippocampal CA1 region. Immunohistochemical staining was used to determine the number of brain-derived neurotroph- ic factor （BDNF）-immunoreactive cells in the hippocampal CA1 region, rTMS treatment for 30 days significantly improved learning and memory function, increased acetylcholinesterase and choline acetyltransferase activity, increased the density of cholinergic neurons, and increased the number of BDNF-immunoreactive cells. These results indicate that rTMS can ameliorate learning and memory deficiencies in rats with vascular dementia, The mechanism through which this occurs might be related to the promotion of BDNF expression and subsequent restoration of cholinergic system activity in hippocampal CA 1 region.

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Impairment of the nerve growth factor pathway driving amyloid accumulation in cholinergic neurons the incipit of the Alzheimer's disease story?

The current idea behind brain pathology is that disease is initiated by mild disturbances of common physiological processes. Overtime, the disruption of the neuronal homeostasis will determine irreversible degeneration and neuronal apoptosis. This could be also true in the case of nerve growth factor （NGF） al- terations in sporadic Alzheimer＇s disease （AD）, an age-related pathology characterized by cholinergic loss, amyloid plaques and neurofibrillary tangles. In fact, the pathway activated by NGF, a key neurotrophin for the metabolism of basal forebrain cholinergic neurons （BFCN）, is one of the first homeostatic systems affected in prodromal AD. NGF signaling dysfunctions have been thought for decades to occur in AD late stages, as a mere consequence of amyloid-driven disruption of the retrograde axonal transport of neuro- trophins to BFCN. Nowadays, a wealth of knowledge is potentially opening a new scenario： NGF signaling impairment occurs at the onset of AD and correlates better than amyloid load with cognitive decline. The recent acceleration in the characterization of anatomical, functional and molecular profiles of early AD is aimed at maximizing the efficacy of existing treatments and setting novel therapies. Accordingly, the elucidation of the molecular events underlying APP metabolism regulation by the NGF pathway in the sep- to-hippocampal system is crucial for the identification of new target molecules to slow and eventually halt mild cognitive impairment （MCI） and its progression toward AD.

Wnt3a expression during the differentiation of adipose-derived stem cells into cholinergic neurons

The present study analyzed changes in Wnt3a expression during differentiation of adipose-derived stern cells into cholinergic neurons. Immunocytochemistry and immunofluorescence revealed significantly increased nestin, neuron-specific enolase, microtubule-associated protein 2, and choline acetyltransferase expression in adipose-derived stem cells isolated from Sprague-Dawley rats and cultured in vitro in neural-induced medium. These expressions increased with prolonged induction time. Real-time reverse transcription-PCR and western blot assay results demonstrated significantly increased choline acetyltransferase and Wnt3a protein and mRNA expressions, respectively, in adipose-derived stem cells following induction. Choline acetyltransferase expression positively correlated with Wnt3a protein and mRNA expressions. These results demonstrated that neural-induced medium induced differentiation of adipose-derived stem cells into cholinergic neuronal-like cells, with subsequent increased Wnt3a expression.

Age-dependent loss of cholinergic neurons in learning and memory-related brain regions and impaired learning in SAMP8 mice with trigeminal nerve damage

The tooth belongs to the trigeminal sensory pathway. Dental damage has been associated with impairments in the central nervous system that may be mediated by injury to the trigeminal nerve. In the present study, we investigated the effects of damage to the inferior alveolar nerve, an important peripheral nerve in the trigeminal sensory pathway, on learning and memory be-haviors and structural changes in related brain regions, in a mouse model of Alzheimer’s disease. Inferior alveolar nerve transection or sham surgery was performed in middle-aged （4-month-old） or elderly （7-month-old） senescence-accelerated mouse prone 8 （SAMP8） mice. When the middle-aged mice reached 8 months （middle-aged group 1） or 11 months （middle-aged group 2）, and the elderly group reached 11 months, step-down passive avoidance and Y-maze tests of learn-ing and memory were performed, and the cholinergic system was examined in the hippocampus （Nissl staining and acetylcholinesterase histochemistry） and basal forebrain （choline acetyltrans-ferase immunohistochemistry）. In the elderly group, animals that underwent nerve transection had fewer pyramidal neurons in the hippocampal CA1 and CA3 regions, fewer cholinergic ifbers in the CA1 and dentate gyrus, and fewer cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band, compared with sham-operated animals, as well as showing impairments in learning and memory. Conversely, no signiifcant differences in histology or be-havior were observed between middle-aged group 1 or group 2 transected mice and age-matched sham-operated mice. The present ifndings suggest that trigeminal nerve damage in old age, but not middle age, can induce degeneration of the septal-hippocampal cholinergic system and loss of hippocampal pyramidal neurons, and ultimately impair learning ability. Our results highlight the importance of active treatment of trigeminal nerve damage in elderly patients and those with Alzheimer’s disease, and indicate that tooth extraction should be avoided in these populations.

Disturbance of cholinergic Grb2-associated-binding protein 1 signaling participate in the pathological process of cognitive dysfunction

OBJECTIVE A causal relationshiphas been postulated between cholinergic dysfunction and the progression of cognitive decline in neurodegenerative disorders. However,the cause of the cognitive dysfunction remains unclear. METHODS Gab1^(loxP/loxP) were bred with ChAT-Cre mice to generate ChAT-Cre; Gab1^(f/f) mice. Excitability of cholinergic neurons wererecorded using whole-cel patch clump. A series of behavioral analyses were used to address the changes of cognitive function in ChAT-Cre; Gab1^(f/f) mice. Neurochemical changes on brain of conditional knockout mice were evaluated by using immunohistochemistry and Western blotting analysis. RESULTS Grb2-associated-binding protein 1(Gab1) is adocking/scaffolding molecule known to play an important role in cell growth and survival. Here,wereport that Gab1 is decreased in cholinergic neurons in a mousemodel of AD. We found that selective downregulation of Gab1 in the septum impaired learning and memory and hippocampal long-term potentiation,whereas overexpression of Gab1 in the same area rescued the cognitive deficitsseen in ChAT-Cre; Gab1^(f/f) and APP^(swe)/PS1 mice.^(18)F-FDGmicroP ET imaging data indicated that Gab1 treatment had no effect on metabolic activity of glucose in APPswe/PS1 mice. Moreover,we identify abnormal function of SKchannelscontributes to increased firing in cholinergic neuronsof ChAT-Cre; Gab1^(f/f) mice. CONCLUSION Gab1 signaling may serve as a potential treatment target for neurological disorders involving dysfunction of central cholinergic neurons.

Berberine Relieves Insulin Resistance via the Cholinergic Anti-inflammatory Pathway in HepG2 Cells

Berberine（BBR） is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus（T2DM） in China. The development of T2 DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in Hep G2 cells. Cellular glucose uptake, quantified by the 2-[N-（7-Nitrobenz-2-oxa-1,3-diazol-4-yl）-amino]-2-deoxy-D-glucose（2-NBDG）, was inhibited by 21% after Hep G2 cells were incubated with insulin（10-6 mol/L） for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor（α7n ACh R） protein was reduced without the change of acetylcholinesterase（ACh E） activity. The level of interleukin-6（IL-6） in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β（IKKβ） Ser181/IKKβ and the expression of nuclear factor-kappa B（NF-κB） p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7n ACh R protein and inhibited ACh E activity. These changes were also accompanied with the decrease of the ratio of p IKKβ Ser181/IKKβ, NF-κB p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in Hep G2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of ACh E activity.

The synthetic thyroid hormone, levothyroxine, protects cholinergic neurons in the hippocampus of naturally aged mice

The thyroid hormones, triiodothyronine and thyroxine, play important roles in cognitive func- tion during the mammalian lifespan. However, thyroid hormones have not yet been used as a therapeutic agent for normal age-related cognitive deficits. In this study, CD-1 mice （aged 24 months） were intraperitoneally injected with levothyroxine （L-T4; 1.6 gg/kg per day） for 3 consecutive months. Our findings revealed a significant improvement in hippocampal cyto- skeletal rearrangement of actin and an increase in serum hormone levels of L-T4-treated aged mice. Furthermore, the survival rate of these mice was dramatically increased from 60% to 93.3%. The Morris water maze task indicated that L-T4 restored impaired spatial memory in aged mice. Furthermore, level of choline acetyltransferase, acetylcholine, and superoxide dismutase were in- creased in these mice, thus suggesting that a possible mechanism by which L-T4 reversed cognitive impairment was caused by increased activity of these markers. Overall, supplement of low-dosage L-T4 may be a potential therapeutic strategy for normal age-related cognitive deficits.

Involvement of M3 Cholinergic Receptor Signal Transduction Pathway in Regulation of the Expression of Chemokine MOB-1, MCP-1 Genes in Pancreatic Acinar Cells

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10 -3 mol/L, 10 -4 mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10 -3 mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10 -3 mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10 -5 mol/L atropine) or NF-κB inhibitor (10 -2 mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1genes in pancreatic acinar cells in vitro through the activation of NF-κB.

Nicotinic cholinergic receptors in esophagus:Early alteration during carcinogenesis and prognostic value

AIM: To compare expression of nicotinic cholinergic receptors(CHRNs) in healthy and squamous cell carcinoma-affected esophagus and determine the prognostic value.METHODS: We performed RT-q PCR to measure the expression of CHRNs in 44 esophageal samples from healthy individuals and in matched normal surrounding mucosa, and in tumors from 28 patientsdiagnosed with esophageal squamous cell carcinoma(ESCC). Next, we performed correlation analysis for the detected expression of these receptors with the habits and clinico-pathological characteristics of all study participants. In order to investigate the possible correlations between the expression of the different CHRN subunits in both healthy esophagus and tissues from ESCC patients, correlation matrices were generated. Subsequently, we evaluated whether the detected alterations in expression of the various CHRNs could precede histopathological modifications during the esophageal carcinogenic processes by using receiver operating characteristic curve analysis. Finally, we evaluated the impact of CHRNA5 and CHRNA7 expression on overall survival by using multivariate analysis.RESULTS: CHRNA3, CHRNA5, CHRNA7 and CHRNB4, but not CHRNA1, CHRNA4, CHRNA9 or CHRNA10, were found to be expressed in normal(healthy) esophageal mucosa. In ESCC, CHRNA5 and CHRNA7 were overexpressed as compared with patient-matched surrounding non-tumor mucosa(ESCC-adjacent mucosa; P < 0.0001 and P = 0.0091, respectively). Positive correlations were observed between CHRNA3 and CHRNB4 expression in all samples analyzed. Additionally, CHRNB4 was found to be differentially expressed in the healthy esophagus and the normalappearing ESCC-adjacent mucosa, allowing for distinguishment between these tissues with a sensitivity of 75.86% and a specificity of 78.95%(P = 0.0002). Finally, CHRNA5 expression was identified as an independent prognostic factor in ESCC; patients with high CHRNA5 expression showed an increased overall survival, in comparison with those with low expression. The corresponding age- and tumor stage-adjusted hazard ratio was 0.2684(95%CI: 0.075-0.97, P = 0.0448).CONCLUSION: Expression of CHRNs is homogeneous along healthy esophagus and deregulated in ESCC, suggesting a pathogenic role for these receptors in ESCC development and progression.

MicroRNA-124 mediates the cholinergic anti-inflammatory action through inhibiting the production of pro-inflammatory cytokines

The vagus nerve can control inflammatory response through a ＇ cholinergic anti-inflammatory pathway＇, which is mediated by the α7-nicotinic acetylcholine receptor （α7nAChR） on macrophages. However, the intracel- lular mechanisms that link α7nAChR activation and pro-inflammatory cytokine production remain not well under- stood. In this study, we found that miR-124 is upregulated by cholinergic agonists in LPS-exposed cells and mice. Utilizing miR-124 mimic and siRNA knockdown, we demonstrated that miR-124 is a critical mediator for the cho- linergic anti-inflammatory action. Furthermore, our data indicated that miR-124 modulates LPS-induced cytokine production by targeting signal transducer and activator of transcription 3 （STAT3） to decrease IL-6 production and TNF-α converting enzyme （TACE） to reduce TNF-ot release. These results also indicate that miR-124 is a potential therapeutic target for the treatment of inflammatory diseases.

Mangiferin,a natural xanthone,accelerates gastrointestinal transit in mice involving cholinergic mechanism

AIM:To investigate the effects of mangiferin on gastrointestinal transit(GIT) in normal and constipated mice,together with the possible mechanism.METHODS:Intragastrically-administered charcoal mealwas used to measure GIT in overnight starved Swiss mice.In the first experiments,mangiferin(3 mg/kg,10 mg/kg,30 mg/kg,and 100 mg/kg,po) or tegaserod(1 mg/kg,ip) were administered 30 min before the charcoal meal to study their effects on normal transit.In the second series,mangiferin(30 mg/kg) was tested on delayed GIT induced by several different pharmacological agonists(morphine,clonidine,capsaicin) or antagonists(ondansetron,verapamil,and atropine) whereas in the third series,mangiferin(30 mg/kg,100 mg/kg and 300 mg/kg) or tegaserod(1 mg/kg) were tested on 6 h fecal pellets outputted by freely fed mice.The ratio of wet to dry weight was calculated and used as a marker of fecal water content.RESULTS:Mangiferin administered orally significantly(P < 0.05) accelerated GIT at 30 mg/kg and 100 mg/kg(89% and 93%,respectively),similarly to 5-hydroxytryptamine4(5-HT4) agonist tegaserod(81%) when compared to vehicle-treated control(63%).Co-administered mangiferin(30 mg/kg) totally reversed the inhibitory effect of opioid agonist morphine,5-HT3-receptor antagonist ondansetron and transient receptor potential vanilloid-1 receptor agonist capsaicin on GIT,but only to a partial extent with the GIT-delay induced by 2-adrenoceptor agonist clonidine,and calcium antagonist verapamil.However,co-administered atropine completely blocked the stimulant effect of mangiferin on GIT,suggesting the involvement of muscarinic acetylcholine receptor activation.Although mangiferin significantly enhanced the 6 h fecal output at higher doses(245.5 ± 10.43 mg vs 161.9 ± 10.82 mg and 227.1 ± 20.11 mg vs 161.9 ± 10.82 mg of vehicle-treated control,at 30 and 100 mg/kg,P < 0.05,respectively),the effect of tegaserod was more potent(297.4 ± 7.42 mg vs 161.9 ± 10.82 mg of vehicle-treated control,P < 0.05).Unlike tegaserod,which showed an enhanced water content in fecal pellets(59.20% ± 1.09% vs 51.44% ± 1.19% of control,P < 0.05),mangiferin evidenced no such effect,indi-cating that it has only a motor and not a secretomotor effect.CONCLUSION:Our data indicate the prokinetic action of mangiferin.It can stimulate the normal GIT and also overcome the drug-induced transit delay,via a cholinergic physiological mechanism.

Impact of Nonlethal Levels of Metacid-50 and Carbaryl on Thyroid Function and Cholinergic System of Channa punctatus

With regard to brain acetylcholinesterase and acetylcholine and serum triiodothyronine(T_3) and thyroxine(T_4)profiles,a biphasic response pattern was elicited in Channa punctatus chroni- cally exposed to nonlethal doses of locally used pesticides,namely,Metacid-50 and Carbaryl. Data revealed that these xenobiotics caused significant inhibition of brain acetylcholinesterase activity and a decrease in thyroxine level accompanied by a concurrent increase in acetylcholine accumulation and T_3 level.It is surmised that Metacid-50 and Carbaryl influence both neural and hormonal functions.1989 Academic Press.Inc.

Cholinergic input from the pedunculopontine nucleus to the cerebellum: implications for deep brain stimulation in Parkinson's disease

Deep brain stimulation（DBS）is a well established electrophysiological treatment initially applied to treat medication-refractory motor symptoms in Parkinson＇s disease（PD）,and is now being explored for several neurological and psychiatric disorders.The specific physiological mechanisms underlying the effectiveness of DBS are not fully understood.

Innervation of Cholinergic Vestibular Efferent System in Vestibular Periphery of Rats

The innervation of cholinergic efferent fibers in the vestibular endorgans of the rat was investigated using a modified preembedding immunostaining technique of immunoelectron microscopy. A monoclonal antibody to choline acetyltransferase (ChAT) was used as a marker of cholinergic fibers. It was found that there were four types of cholinergic innervation in the vestibular endorgans of the rat: (1) cholinergic nerve endings formed axo-dendritic synapses with afferent chalice surrounding the type I sensory hair cells; (2) cholinergic nerve endings formed axo-somatic synapses with type Ⅱ hair cells; (3) cholinergic fibers synapse with afferent nerve fibers and (4) a synaptic contact developed between cholinergic nerve endings. The results demonstrated that a multiform innervation of the cholinergic efferents exists in the rat vestibular periphery.

Effect of growth hormone-releasing peptide on cardiac cholinergic nerve fiber density distribution in a rat model of heart failure

BACKGROUND： Changes in the cardiac autonomic nerve are considered to be important factors in the mechanisms of heart failure. It is possible to reduce or slow down nerve degeneration and necrosis, provided that patients take effective neuroprotectants during the early stages of heart failure. Moreover, it is possible to relieve the pathological process and reduce the risk of death. OBJECTIVE： To study the effect of growth hormone releasing peptide （GHRP） on cardiac cholinergic nerve fiber density distribution in a rat model of heart failure, and verify whether GHRP can ameliorate denervation. DESIGN, TIME AND SETTING： A randomized controlled study was performed at the Key Laboratory of Anatomy, Harbin Medical University, between June and October 2009. MATERIALS： Fifty adult, healthy, female, Wistar rats, weighing （200± 20） g, were randomly divided into GHRP （n = 30）, model （n = 10）, and sham operation （n = 10） groups. GHRP-2 was made in Shanghai, China （batch No. z071212-03）. METHODS： Acute myocardial infarction was established by ligating the left anterior descending coronary artery in the GHRP and model groups. Five weeks later, myocardial function was detected using color ultrasound electrocardiograph a successful marker of chronic heart failure models Ejection fraction 〈 60% was considered to be However, the left anterior descending coronary artery was not ligated in the sham operation group. The GHRP group was injected with 100 μ g/kg GHRP-2, and the other two groups were injected with the same volume of physiological saline, once per day. MAIN OUTCOME MEASURES： After 4 weeks, pathological changes in cardiac cholinergic nerve fibers were detected under optic microscopy following hematoxylin/eosin staining. In addition, density distribution was measured using a multi-function color pathological image system. RESULTS： In the sham operation group, myocardial cells were regular, uniformly stained, and no inflammatory cells were present. In the model group, myocardial cells were unevenly stained, exhibited nuclear atrophy, degeneration, dissolution, or disappearance. In the GHRP group, myocardial damage was less than in the model group; cardiac muscle fibers exhibited slight degeneration. The myocardium in the sham operation group was serried, spreading the cholinergic innervations along the cardiac fiber. In the model group, there was a decreased number of cholinergic nerve fibers decreased, which also became shorter and smaller, compared with the sham operation group （P 〈 0.01）. In the GHRP group, cholinergic positive nerve fibers were significantly increased compared with the model group （P 〈 0.01）, but still less than the sham surgery group （P 〈 0.05）. CONCLUSION： GHRP delayed denervation and reduced nerve reconstitution following heart failure in rats.

Bone morphogenetic protein 4 induces cholinergic differentiation of brain-derived neural stem cells in vivo and in vitro

In vitro studies have demonstrated that many factors of bone morphogenetic proteins （BMPs） induce cholinergic differentiation of neural stem cells. However, BMP retains the potential to induce increased numbers of cholinergic neurons in central nervous system regions that are rich in cholinergic cells, which is an important determinant of BMP. Therefore, BMP-4 was added to neural stem cell culture medium or the adult rat hippocampal dentate gyrus. Results demonstrated that BMP-4 induced cholinergic differentiation of neural stem cells in vitro and increased the number of cholinergic neurons in the adult rat hippocampal dentate gyrus.