Apparent absence of hypothalamic cholinergic neurons in the common ostrich and emu: Implications for global brain states during sleep.

We examined the presence/absence and parcellation of cholinergic neurons in the hypothalami of five birds: a Congo grey parrot (Psittacus erithacus), a Timneh grey parrot (P. timneh), a pied crow (Corvus albus), a common ostrich (Struthio camelus), and an emu (Dromaius novaehollandiae). Using immunohistochemistry to an antibody raised against the enzyme choline acetyltransferase, hypothalamic cholinergic neurons were observed in six distinct clusters in the medial, lateral, and ventral hypothalamus in the parrots and crow, similar to prior observations made in the pigeon. The expression of cholinergic nuclei was most prominent in the Congo grey parrot, both in the medial and lateral hypothalamus. In contrast, no evidence of cholinergic neurons in the hypothalami of either the ostrich or emu was found. It is known that the expression of sleep states in the ostrich is unusual and resembles that observed in the monotremes that also lack hypothalamic cholinergic neurons. It has been proposed that the cholinergic system acts globally to produce and maintain brain states, such as those of arousal and rapid-eye-movement sleep. The hiatus in the cholinergic system of the ostrich, due to the lack of hypothalamic cholinergic neurons, may explain, in part, the unusual expression of sleep states in this species. These comparative anatomical and sleep studies provide supportive evidence for global cholinergic actions and may provide an important framework for our understanding of one broad function of the cholinergic system and possible dysfunctions associated with global cholinergic neural activity.

PIK3CA regulates development of diabetes retinopathy through the PI3K/Akt/mTOR pathway.

OBJECTIVE: To explore their association with the development of diabetes retinopathy (DR), single nucleotide polymorphism (SNP) mutations were screened out by high-throughput sequencing and validated in patients diagnosed with DR. To understand the role of PIK3CA in the pathogenesis of DR and explore the relationship between PIK3CA,phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR),and DR, the effect of PIK3CA.rs17849079 mutation was investigated in a DR cell model. METHODS: Twelve patients diagnosed with DR at the Qinghai Provincial People's Hospital from September 2020 to June 2021 were randomly selected as the case group, while 12 healthy subjects of similar age and gender who underwent physical examination in Qinghai Provincial People's Hospital physical examination center during the same period were randomly selected as the control group. Blood samples (2 mL) were collected from both groups using EDTA anticoagulant blood collection vessels and frozen at -20°C for future analysis. SNP mutations were detected by high-throughput sequencing, and the shortlisted candidates were subjected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The detected SNP candidates were verified by expanding the sample size (first validation: 56 patients in the case group and 58 controls; second validation: 157 patients in the case group and 96 controls). A lentivirus vector carrying mutated or wild-type PIK3CA.rs17849079 was constructed. ARPE-19 cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS) to establish a DR cell model. PIRES2-PIK3CA-MT and PIRES2-PIK3CA-WT vectors were transfected into DR model cells, which were categorized into control, mannitol, model, empty vector, PIK3CA wild-type, and PIK3CA mutant-type groups. Cell activity was detected by the cell counting kit (CCK)-8 assay, and cellular apoptosis was evaluated by flow cytometry. Glucose concentration and levels of cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1ß were detected using enzyme-linked immunosorbent assay kits. The expression of PIK3CA, AKT1, mTOR, and VEGF genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR), while the expression of PI3K, p-PI3K, AKT1, p-AKT1, mTOR, p-mTOR, and VEGF proteins was detected by western blotting. RESULTS: The mutated SNPs were mainly enriched in the PI3K/AKT pathway, calcium ion pathway, and glutamatergic synaptic and cholinergic synaptic signaling pathways. Seven SNPs, including PRKCE.rs1533476, DNAH11.rs10485983, ERAP1.rs149481, KLHL1.rs1318761, APOBEC3C.rs1969643, FYN.rs11963612, and KCTD1.rs7240205, were not related to the development of DR. PIK3CA.rs17849079 was prone to C/T mutation. The risk of DR increased with the presence of the C allele and decreased in the presence of the T allele. High glucose induced the expression of PIK3CA and VEGF mRNAs as well as the expression of PI3K, p-PI3K, p-AKT1, p-mTOR, and VEGF proteins in ARPE-19 cells, which led to secretion of inflammatory factors TNF-αand IL-1, cell apoptosis, and inhibition of cell proliferation. The PIK3CA.rs17849079 C allele accelerated the progression of DR. These biological effects were inhibited when the C allele of PIK3CA.rs17849079 was mutated to T allele. CONCLUSION: The mutated SNP sites in patients with DR were mainly enriched in PI3K/AKT, calcium ion, and glutamatergic synaptic and cholinergic synaptic signaling pathways. The rs17849079 allele of PIK3CA is prone to C/T mutation where the C allele increases the risk of DR. High glucose activates the expression of PIK3CA and promotes the phosphorylation of PI3K, which leads to the phosphorylation of AKT and mTOR. These effects consequently increase VEGF expression and accelerate the development of DR. The C to T allele mutation in PIK3CA.rs17849079 can play a protective role and reduce the risk of DR.

Sinomenine regulates the cholinergic anti-inflammatory pathway to inhibit TLR4/NF-κB pathway and protect the homeostasis in brain and gut in scopolamine-induced Alzheimer's disease mice.

Sinomenine (SIN), an alkaloid extracted from the Chinese herbal medicine Sinomenium acutum, has great potential in anti-inflammatory, immune regulation, analgesic and sedative, and is already a clinical drug for the treatment of rheumatoid arthritis in China. Our previous studies show SIN inhibits inflammation by regulating ɑ7nAChR, a key receptor of cholinergic anti-inflammatory pathway (CAP), which plays an important role in regulating peripheral and central nervous system inflammation. Growing evidence supports the cholinergic dysregulation and inflammatory responses play the key role in the pathogenesis of AD. The intervention effects of SIN on AD by regulating CAP and homeostasis in brain and gut were analyzed for the first time in the present study using scopolamine-induced AD model mice. Behavioral tests were used to assess the cognitive performance. The neurons loss, cholinergic function, inflammation responses, biological barrier function in the mouse brain and intestinal tissues were evaluated through a variety of techniques, and the gut microbiota was detected using 16SrRNA sequencing. The results showed that SIN significantly inhibited the cognitive decline, dysregulation of cholinergic system, peripheral and central inflammation, biological barrier damage as well as intestinal flora disturbance caused by SCOP in mice. More importantly, SIN effectively regulated CAP to suppress the activation of TLR4/NF-κB and protect the homeostasis in brain and gut to alleviate cognitive impairment.

Morus Alba Fruit Extract and its Fractions Ameliorate Streptozotocin Induced Cognitive Deficit in Mice via Modulating Oxidative and Cholinergic Systems.

Increased oxidative stress and acetylcholinesterase (AChE) activity are key pathological characters contributing to the memory disorders. Thus, drugs targeting both oxidative stress and AChE are being explored for the management of cognitive dysfunction. Morus alba fruits (commonly consumed for its high nutritious value) are known to have antioxidant and AChE inhibitory effects. However, the role of Morus alba fruits in the management of memory disorders has not reported yet. This investigation was conducted to assess the antioxidant and AChE inhibitory potential of Morus alba fruit extracts in-vitro and to identify the components responsible for such effects. Further, the obtained bioactive component was studied for possible memory improvement effects against streptozotocin (STZ) induced dementia. To isolate the bioactive component in-vitro DPPH and AChE assays guided fractionation was performed. Memory functions in mice were determined using Morris Water Maze test while brain biochemical parameters were measured to understand the mechanism of action. In-vitro assays revealed strong AChE and DPPH inhibitory potential of methanol extract (ME), therefore, it was further fractionated. Among various fractions obtained, ethyl-acetate fraction (EAF) was found to possess marked AChE and DPPH inhibitory activities. On subsequent fractionation of EAF, bioactivity of obtained sub-fractions was found to be inferior to EAF. Further, both ME and EAF improved STZ (intracerebroventricular) induced cognitive dysfunction in animals by restoring endogenous antioxidant status (superoxide dismutase and reduced glutathione) and reducing thiobarbituric acid reactive species and nitric oxide levels along with brain AChE and myeloperoxidase activity. TLC densitometric studies showed appreciable levels of phenolic acids and quercetin in both EAF and ME. It can be concluded that Morus alba fruit extract has the ability to modulate cholinergic and oxidative system due to presence of phenolic and flavonoid compounds and hence, could aid in the management of memory disorders.

LPC20K modified from krill oil ameliorates the scopolamine-induced cognitive impairment.

Alzheimer's disease (AD) is characterized by cognitive impairment. It is common in the elderly. Etiologically, dysfunction of cholinergic neurotransmitter system is prominent in AD. However, disease modifying drug for AD is still unavailable. We hypothesized that krill oil and modified krill oil containing 20 % lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA, LPC20K) could play a crucial role in AD by improving cognitive functions measured by several behavioral tests. We found that LPC20K could ameliorate short-term, long-term, spatial, and object recognition memory under cholinergic hypofunction states. To find the underlying mechanism involved in the effect of LPC20K on cognitive function, we investigated changes of signaling molecules using Western blotting. Expression levels of protein kinase C zeta (PKCζ) and postsynaptic density protein 95 (PSD-95), and phosphorylation levels of extracellular signal-regulated kinase (ERK), Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), and cAMP response element-binding protein (CREB) were significantly increased in LPC20K-administered group compared to those in the memory impairment group. Moreover, the expression levels of BDNF were temporally increased especially 6 or 9 h after administration of LPC20K compared with the control group. These results suggest that LPC20K could ameliorate memory impairment caused by hypocholinergic state by enhancing the expression levels of PKCζ and PSD-95, and phosphorylation levels of ERK, CaMKⅡ and CREB and increasing BDNF expression levels. Therefore, LPC20K could be used as a dietary supplement against cognitive impairment observed in diseases such as AD with a hypocholinergic state.

[Research progress in mechanism of puerarin in treating vascular dementia].

Vascular dementia(VD) is a condition of cognitive impairment due to acute and chronic cerebral hypoperfusion. The available therapies for VD mainly focus on mitigating cerebral ischemia, improving cognitive function, and controlling mental behavior. Achievements have been made in the basic and clinical research on the treatment of VD with traditional Chinese medicine(TCM) active components, including Ginkgo leaf extract, puerarin, epimedium, tanshinone, and ginsenoside. Most of these components have anti-inflammatory, anti-apoptotic, anti-oxidant, and neuroprotective effects, and puerarin demonstrates excellent performance in mitigating cholinergic nervous system disorders and improving synaptic plasticity. Puerarin, ginkgetin, and epimedium are all flavonoids, while tanshinone is a diterpenoid. Puerariae Lobatae Radix, pungent in nature, can induce clear Yang to reach the cerebral orifices and has the wind medicine functions of ascending, dispersing, moving, and scurrying. Puerariae Lobatae Radix entering collaterals will dredge blood vessels to promote blood flow, and that entering the sweat pore will open the mind, which is in line with the TCM pathogenesis characteristics of VD. This study reviews the progress in the mechanism of puerarin, the main active component of Puerariae Lobatae Radix, in treating VD. Puerarin can ameliorate cholinergic nervous system disorders, reduce excitotoxicity, anti-inflammation, inhibit apoptosis, alleviate oxidative stress injury, enhance synaptic plasticity, up-regulate neuroprotective factor expression, promote cerebral circulation metabolism, and mitigate Aß injury. The pathways of action include activating nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE), vascular endothelial growth factor(VEGF), extracellular regulated protein kinases(ERK), phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt), Janus-activating kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3), AMP-activated protein kinase(AMPK), as well as inhibiting the tumor necrosis factor α(TNF-α), transient receptor potential melastatin 2(TRPM2)/N-methyl-D-aspartate receptor(NMDAR), p38 mitogen-activated protein kinase(p38 MAPK), Toll-like receptor 4(TLR4)/nuclear factor-kappaB(NF-κB), early growth response 1(Egr-1), and matrix metalloproteinase 9(MMP-9). By reviewing the papers about the treatment of VD by puerarin published by CNKI, Wanfang, VIP, PubMed, and Web of Science in the last 10 years, this study aims to support the treatment and drug development for VD.

Omega-3 fatty acids prevent nicotine withdrawal-induced impairment of learning and memory via affecting oxidative status, inflammatory response, cholinergic activity, BDNF and amyloid-B in rat hippocampal tissues.

In the present study, the main objective was to reveal whether treatment by Omega-3 fatty acids could prevent the adverse effects of adolescent nicotine withdrawal on spatial and avoidance memory in male rats. For this purpose, Morris water maze and passive avoidance tests were performed on male Wistar rats and the hippocampal levels of oxidative stress markers, inflammatory indices, brain-derived neurotrophic factor, nitrite, amyloid-B and acetylcholinesterase (AChE) were measured. Moreover, density of dark neurons were assessed in CA1 and CA3 regions. Results showed that adolescent nicotine exposure followed by a period of drug cessation exacerbates the behavioral indices of learning and memory through affecting a variety of biochemical markers within the hippocampal tissues. These changes lead to elevation of oxidative and inflammatory markers, reduction of neurotrophic capacity and increased AChE activity in hippocampal tissues. In addition, it was observed that co-administration of nicotine with Omega-3 fatty acids significantly prevents nicotine withdrawal-induced adverse effects through restoration of the mentioned biochemical disturbances. Therefore, we suggest administration of Omega-3 fatty acids as a safe, inexpensive and effective therapeutic strategy for prevention of memory dysfunctions associated with nicotine abstinence during adolescence.

Oral Administration of Egg- and Soy-Derived Lysophosphatidylcholine Mitigated Acetylcholine Depletion in the Brain of Scopolamine-Treated Rats.

Enzyme-modified lecithin that contains lysophosphatidylcholine (LPC) is generally recognized as safe. However, its potential as a functional ingredient has been less investigated than other choline (Ch)-containing compounds, such as glycerophosphocholine (GPC). Reports on the possibility of LPC functioning as a cholinergic precursor in vivo and on its kinetics are limited to docosahexaenoic acid-bound LPC. Herein, three experiments were performed to investigate these processes in scopolamine (SCO)-treated rats. First, an egg-derived LPC reagent was orally administered to rats, and brain acetylcholine (ACh), Ch, plasma Ch, and LPC were measured. Second, soy- and rapeseed-derived enzyme-modified lecithins and GPC were administered for comparison. Third, soy-derived enzyme-modified lecithins with different fat contents were administered for comparison. The LPC reagent mitigated SCO-induced ACh depletion at 500 mg/kg body weight and increased plasma Ch, but not LPC, concentrations. Additionally, soy-derived LPC-containing food additive counteracted brain ACh depletion similarly to GPC. Interestingly, plasma Ch and linoleoyl-LPC levels were higher when soy-derived LPC with a higher fat content was administered, whereas the plasma levels of palmitoyl-LPC decreased and those of total LPC remained constant. In conclusion, egg- and soy-derived LPC species function as cholinergic precursors in vivo, and future studies should explore this potential.

A Study on the Effects of Muscarinic and Serotonergic Regulation by Bojanggunbi-tang on the Pacemaker Potential of the Interstitial Cells of Cajal in the Murine Small Intestine.

In traditional Korean medicine, the 16-herb concoction Bojanggunbi-tang (BGT) is used to treat various gastrointestinal (GI) diseases. In this study, we investigated the regulatory mechanism underlying the influence of BGT on the interstitial cells of Cajal (ICCs), pacemaker cells in the GI tract. Within 12 h of culturing ICCs in the small intestines of mice, the pacemaker potential of ICCs was recorded through an electrophysiological method. An increase in the BGT concentration induced depolarization and decreased firing frequency. This reaction was suppressed by cholinergic receptor muscarinic 3 (CHRM3) antagonists, as well as 5-hydroxytryptamine receptor (5HTR) 3 and 4 antagonists. Nonselective cation channel inhibitors, such as thapsigargin and flufenamic acid, along with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors, also suppressed the BGT reaction. Guanylate cyclase and protein kinase G (PKG) antagonists inhibited BGT, but adenylate cyclase and protein kinase A antagonists had no effect. In conclusion, we demonstrated that BGT acts through CHRM3, 5HTR3, and 5HTR4 to regulate intracellular Ca2+ concentrations and the PKC, MAPK, guanylate cycle, and PKG signaling pathways.

Potential value of neuroimmunotherapy for COVID-19: efficacies and mechanisms of vagus nerve stimulation, electroacupuncture, and cholinergic drugs.

COVID-19 is an inflammatory disease with multiple organs involved, mainly respiratory symptoms. Although the majority of patients with COVID-19 present with a mild to moderate self-limited course of illness, about 5-10% of patients with inflammatory disorders in severe COVID-19 have life-threatening progression. With the exception of a few drugs that have shown outstanding anti-COVID-19 effects, the efficacy of most drugs remains controversial. An increasing number of animal and clinical studies have shown that neuromodulation has a significant effect on reducing inflammatory markers of COVID-19, thus exerting an effective neuroimmunotherapeutic value. Currently, the main neuroimmunomodulatory measures effective against COVID-19 include vagus nerve stimulation, electroacupuncture, and cholinergic drugs. In this review, we will summarize the research progress of potential value of this neuroimmunotherapy measures for COVID-19 and elaborate its efficacies and mechanisms, in order to provide reliable evidence for clinical intervention.

Multi-target bioactive compound screening from the infructescence of Platycarya strobilacea Sieb. et Zucc. by affinity chromatography using immobilized ß2 -adrenoceptor and muscarinic-3 acetylcholine receptor as the stationary phase.

As a main source for the recognition and identification of lead compounds, traditional Chinese medicine plays a pivotal role in preventing diseases for years. However, screening bioactive compounds from traditional Chinese medicine remains challenging because of the complexity of the systems and the occurrence of the synergic effect of the compounds. The infructescence of Platycarya strobilacea Sieb. et Zucc is prescribed for allergic rhinitis treatment with unknown bioactive compounds and unclear mechanisms. Herein, we immobilized the ß2 -adrenoceptor and muscarine-3 acetylcholine receptor onto the silica gel surface to prepare the stationary phase in a covalent bond through one step. The feasibility of the columns was investigated by the chromatographic method. Ellagic acid and catechin were identified as the bioactive compounds targeting the receptors. The binding constants of ellagic acid were calculated to be (1.56 ± 0.23)×107  M-1 for muscarine-3 acetylcholine receptor and (2.93 ± 0.15)×107  M-1 for ß2 -adrenoceptor by frontal analysis. While catechin can bind with muscarine-3 acetylcholine receptor with an affinity of (3.21 ± 0.05)×105  M-1 . Hydrogen bonds and van der Waals' force were the main driving forces for the two compounds with the receptors. The established method provides an alternative for multi-target bioactive compound screening in complex matrices.

Cognitive Enhancer Donepezil Attenuates Heroin-Seeking Behavior Induced by Cues in Rats.

PURPOSE: Opioid use disorder is a significant global problem. Chronic heroin use is associated with impairment of cognitive function and conscious control ability. The cholinergic system can be disrupted following heroin administration, indicating that activation of the cholinergic system may prevent chronic heroin misuse. Donepezil as an inhibitor of cholinesterase has been reported to clinically improve cognition and attention. In this study, the inhibition of heroin self-administration and heroin-seeking behaviours by donepezil were evaluated in rats. METHODS: Rats were trained to self-administer heroin every four hours for 14 consecutive days under a fixed ratio 1 (FR1) reinforcement schedule, then underwent withdrawal for two weeks. A progressive ratio schedule was then used to evaluate the relative motivational value of heroin reinforcement. After withdrawal, a conditioned cue was introduced for the reinstatement of heroin-seeking behaviour. Donepezil (0.3-3 mg/kg, i.p.) was used during both the FR1 heroin self-administration and progressive ratio schedules. Immunohistochemistry was used to investigate the mechanism of action of donepezil in the rat brain. RESULTS: Pre-treatment with high dose donepezil (3 mg/kg) but not low doses (0.3-1 mg/kg) significantly inhibited heroin self-administration under the FR1 schedule. Donepezil decreased motivation values under the progressive ratio schedule in a dose-dependent manner. All doses of donepezil (1-3 mg/kg) decreased the reinstatement of heroin seeking induced by cues. Correlation analysis indicated that the inhibition of donepezil on heroin-seeking behaviour was positively correlated with an increased expression of dopamine receptor 1 (D1R) and dopamine receptor 2 (D2R) in the nucleus accumbens (NAc) and increased expression of choline acetyltransferase (ChAT) in the ventral tegmental area (VTA). CONCLUSIONS: The present study demonstrated that donepezil could inhibit heroin intake and heroin-seeking behaviour. Further, donepezil could regulate dopamine receptors in the NAc via an increase of acetylcholine. These results suggested that donepezil could be developed as a potential approach for the treatment of heroin misuse.

Essential oil of Sicilian Prangos ferulacea (L.) Lindl. and its major component, ß-ocimen, affect contractility in rat small and large intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Prangos ferulacea (L.) Lindl is an Apiaceae plant, widely used in traditional medicine. Recently, chemical composition and biological activities of its essential oil (Prangroil) have been reported, but there are no studies on possible effects on intestinal contractility. AIMS OF THE STUDY: We investigated the effects of essential oil Sicilian Prangoil on the contractility of rat small (duodenum) and large (colon) intestine and the related action mechanism. MATERIALS AND METHODS: Responses to Prangoil and to its major component ß-ocimen in intestinal segments were assessed in vitro as changes in isometric tension. RESULTS: Prangoil, induced in duodenum, depending upon doses, contraction and/or muscular relaxation. Instead, in colon Prangoil only reduced the phasic contractions and induced muscular relaxation. ß-ocimen, in both segments, produced only reduction of the spontaneous contractions without affecting basal tone. Prangoil contractile effects were abolished by ω-conotoxin, neural N-type Ca2+ channels blocker, atropine, muscarinic receptor antagonist, neostigmine, acetylcholinesterase (AChE) inhibitor, suggesting that Prangoil-induced contraction would be the result of an increase in neuronal cholinergic activity. Prangoil and ß-ocimen inhibitory effects were unaffected by ω-conotoxin, L-NAME, blocker of the NO synthase, ODQ, soluble guanylate cyclase inhibitor, excluding involvement of neurotransmitter release or NO synthesis in the inhibitory effects. Potassium channel blocker did not affect Prangoil or ß-ocimen inhibitory responses. Prangoil or ß-ocimen inhibited the Ca2+ and high-KCl solution -induced contractions and the Carbachol-induced contractions in calcium free solution. CONCLUSION: Prangoil affects the contractility of small and large intestine in rat, with regional differences, via potentiation of neural cholinergic activity, blockade of L-type voltage-gated calcium channel and reduction of Ca2+ release from the intracellular store. The Prangroil main components, ß-ocimen, contributes to the inhibitory effects.

Radical Caging Strategy for Cholinergic Optopharmacology.

Photo-caged methodologies have been indispensable for elucidating the functional mechanisms of pharmacologically active molecules at the cellular level. A photo-triggered removable unit enables control of the photo-induced expression of pharmacologically active molecular function, resulting in a rapid increase in the concentration of the bioactive compound near the target cell. However, caging the target bioactive compound generally requires specific heteroatom-based functional groups, limiting the types of molecular structures that can be caged. We have developed an unprecedented methodology for caging/uncaging on carbon atoms using a unit with a photo-cleavable carbon-boron bond. The caging/uncaging process requires installation of the CH2-B group on the nitrogen atom that formally assembles an N-methyl group protected with a photoremovable unit. N-Methylation proceeds by photoirradiation via carbon-centered radical generation. Using this radical caging strategy to cage previously uncageable bioactive molecules, we have photocaged molecules with no general labeling sites, including acetylcholine, an endogenous neurotransmitter. Caged acetylcholine provides an unconventional tool for optopharmacology to clarify neuronal mechanisms on the basis of photo-regulating acetylcholine localization. We demonstrated the utility of this probe by monitoring uncaging in HEK cells expressing a biosensor to detect ACh on the cell surface, as well as Ca2+ imaging in Drosophila brain cells (ex vivo).

Contextual fear expression engages a complex set of interactions between ventromedial prefrontal cortex cholinergic, glutamatergic, nitrergic and cannabinergic signaling.

Rats re-exposed to an environment previously associated with the onset of shocks evoke a set of conditioned defensive responses in preparation to an eventual flight or fight reaction. Ventromedial prefrontal cortex (vmPFC) is mutually important for controlling the behavioral/physiological consequences of stress exposure and the one's ability to satisfactorily undergo spatial navigation. While cholinergic, cannabinergic and glutamatergic/nitrergic neurotransmissions within the vmPFC are shown as important for modulating both behavioral and autonomic defensive responses, there is a gap on how these systems would interact to ultimately coordinate such conditioned reactions. Then, males Wistar rats had guide cannulas bilaterally implanted to allow drugs to be administered in vmPFC 10 min before their re-exposure to the conditioning chamber where three shocks were delivered at the intensity of 0.85 mA for 2 s two days ago. A femoral catheter was implanted for cardiovascular recordings the day before fear retrieval test. It was found that the increment of freezing behavior and autonomic responses induced by vmPFC infusion of neostigmine (acetylcholinesterase inhibitor) were prevented by prior infusion of a transient receptor potential vanilloid type 1 (TRPV1) antagonist, N-methyl-d-aspartate receptor antagonist, neuronal nitric oxide synthase inhibitor, nitric oxide scavenger and soluble guanylate cyclase inhibitor. A type 3 muscarinic receptor antagonist was unable to prevent the boosting in conditioned responses triggered by a TRPV1 agonist and a cannabinoid receptors type 1 antagonist. Altogether, our results suggest that expression of contextual conditioned responses involves a complex set of signaling steps comprising different but complementary neurotransmitter pathways.

Polygoni Multiflori Radix Praeparata and Acori Tatarinowii Rhizoma ameliorate scopolamine-induced cognitive impairment by regulating the cholinergic and synaptic associated proteins.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Polygoni Multiflori Radix Praeparata (PMRP) and Acori Tatarinowii Rhizoma (ATR) is often used in traditional Chinese medicine to prevent and treat Alzheimer's disease (AD). However, it is not clear whether the effects and mechanisms of the decoction prepared by traditional decocting method (PA) is different from that prepared by modern decocting method (P + A). AIM OF THE STUDY: The present study aimed to investigate the differences in the protective effects of PA and P + A on scopolamine induced cognitive impairment, and to explore its potential mechanism. MATERIALS AND METHODS: To assess the protective effect of PA and P + A on cognitive dysfunction, the mice were orally administrated with PA (1.56, 6.24 g kg-1•day-1) and P + A (1.56, 6.24 g kg-1•day-1) for 26 days before co-treatment with scopolamine (4 mg kg-1•day-1, i.p.). The learning and memory abilities of mice were examined by Morris water maze test, and the expressions of proteins related to cholinergic system and synaptic function were detected by the methods of ELISA, real-time PCR and Western blotting. Then, molecular docking technique was used to verify the effect of active compounds in plasma after PA administration on Acetylcholinesterase (AChE) protein. Finally, the Ellman method was used to evaluate the effects of different concentrations of PA, P + A (1 µg/mL-100 mg/mL) and the compounds (1-100 µM) on AChE activity in vitro. RESULTS: On one hand, in the scopolamine-induced cognitive impairment mouse model, both of PA and P + A could improve the cognitive impairment, while the effect of PA on cognitive amelioration was better than that of P + A. Moreover, PA regulated the cholinergic and synaptic functions by enhancing the concentration of acetylcholine (ACh), the mRNA levels of CHT1, Syn, GAP-43 and PSD-95, and the related proteins (CHT1, VACHT, Syn, GAP-43 and PSD-95), and significantly inhibiting the expression of AChE protein. Meanwhile, P + A only up-regulated the mRNA levels of GAP-43 and PSD-95, increased the expressions of CHT1, VACHT, Syn, GAP-43 and PSD-95 proteins, and inhibited the expression of AChE protein. On the other hand, the in vitro study showed that some compounds including emodin-8-o-ß-d-Glucopyranoside, THSG and α-Asarone inhibited AChE protein activity with the IC50 values 3.65 µM, 5.42 µM and 9.43 µM, respectively. CONCLUSIONS: These findings demonstrate that both of PA and P + A can ameliorate the cognitive deficits by enhancing cholinergic and synaptic related proteins, while PA has the stronger improvement effect on the cholinergic function, which may be attributed to the compounds including THSG, emodin, emodin-8-O-ß-D-glucopyranoside and α-asarone. The present study indicated that PA has more therapeutic potential in the treatment of neurodegenerative diseases such as AD. The results provide the experimental basis for the clinical use of PA.

Effects of Marginal Zn Excess and Thiamine Deficiency on Microglial N9 Cell Metabolism and Their Interactions with Septal SN56 Cholinergic Cells.

Mild thiamine deficiency aggravates Zn accumulation in cholinergic neurons. It leads to the augmentation of Zn toxicity by its interaction with the enzymes of energy metabolism. Within this study, we tested the effect of Zn on microglial cells cultivated in a thiamine-deficient medium, containing 0.003 mmol/L of thiamine vs. 0.009 mmol/L in a control medium. In such conditions, a subtoxic 0.10 mmol/L Zn concentration caused non-significant alterations in the survival and energy metabolism of N9 microglial cells. Both activities of the tricarboxylic acid cycle and the acetyl-CoA level were not decreased in these culture conditions. Amprolium augmented thiamine pyrophosphate deficits in N9 cells. This led to an increase in the intracellular accumulation of free Zn and partially aggravated its toxicity. There was differential sensitivity of neuronal and glial cells to thiamine-deficiency-Zn-evoked toxicity. The co-culture of neuronal SN56 with microglial N9 cells reduced the thiamine-deficiency-Zn-evoked inhibition of acetyl-CoA metabolism and restored the viability of the former. The differential sensitivity of SN56 and N9 cells to borderline thiamine deficiency combined with marginal Zn excess may result from the strong inhibition of pyruvate dehydrogenase in neuronal cells and no inhibition of this enzyme in the glial ones. Therefore, ThDP supplementation can make any brain cell more resistant to Zn excess.

Treatment of cholinergic-induced status epilepticus with polytherapy targeting GABA and glutamate receptors.

Despite new antiseizure medications, the development of cholinergic-induced refractory status epilepticus (RSE) continues to be a therapeutic challenge as pharmacoresistance to benzodiazepines and other antiseizure medications quickly develops. Studies conducted by Epilepsia. 2005;46:142 demonstrated that the initiation and maintenance of cholinergic-induced RSE are associated with trafficking and inactivation of gamma-aminobutyric acid A receptors (GABAA R) thought to contribute to the development of benzodiazepine pharmacoresistance. In addition, Dr. Wasterlain's laboratory reported that increased N-methyl-d-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) contribute to enhanced glutamatergic excitation (Neurobiol Dis. 2013;54:225; Epilepsia. 2013;54:78). Thus, Dr. Wasterlain postulated that targeting both maladaptive responses of reduced inhibition and increased excitation that is associated with cholinergic-induced RSE should improve therapeutic outcome. We currently review studies in several animal models of cholinergic-induced RSE that demonstrate that benzodiazepine monotherapy has reduced efficacy when treatment is delayed and that polytherapy with drugs that include a benzodiazepine (eg midazolam and diazepam) to counter loss of inhibition, concurrent with an NMDA antagonist (eg ketamine) to reduce excitation provide improved efficacy. Improved efficacy with polytherapy against cholinergic-induced seizure is demonstrated by reduction in (1) seizure severity, (2) epileptogenesis, and (3) neurodegeneration compared with monotherapy. Animal models reviewed include pilocarpine-induced seizure in rats, organophosphorus nerve agent (OPNA)-induced seizure in rats, and OPNA-induced seizure in two mouse models: (1) carboxylesterase knockout (Es1-/- ) mice which, similarly to humans, lack plasma carboxylesterase and (2) human acetylcholinesterase knock-in carboxylesterase knockout (KIKO) mice. We also review studies showing that supplementing midazolam and ketamine with a third antiseizure medication (valproate or phenobarbital) that targets a nonbenzodiazepine site rapidly terminates RSE and provides further protection against cholinergic-induced SE. Finally, we review studies on the benefits of simultaneous compared with sequential drug treatments and the clinical implications that lead us to predict improved efficacy of early combination drug therapies. The data generated from seminal rodent studies of efficacious treatment of cholinergic-induced RSE conducted under Dr. Wasterlain's guidance suggest that future clinical trials should treat the inadequate inhibition and temper the excess excitation that characterize RSE and that early combination therapies may provide improved outcome over benzodiazepine monotherapy.

Effect of diet supplemented with African Star Apple Fruit Pulp on purinergic, cholinergic and monoaminergic enzymes, TNF-α expression and redox imbalance in the brain of hypertensive rats.

OBJECTIVE: This study examined whether diet supplemented with African star apple fruit pulp (FP) can mitigate the effect of high blood pressure on brain neurochemicals, histopathology and expression of genes linked with neuroinflammation. METHODS: Rats were administered with cyclosporine (25 mg/kg.bw) to induce hypertension and were fed with or without FP supplemented diet. Purinergic (Nucleoside triphosphate diphosphohydrolases [NTPdase] and adenosine deaminase [ADA]) cholinergic (acetylcholinesterase [AChE]) and monoaminergic (monoamine oxidase-B) enzymes were assessed in treated and untreated hypertensive rats' brains. Oxidative stress biomarkers (catalase, glutathione-S-transferase, thiols, reactive oxygen species [ROS] and malondialdehyde [MDA]), as well as AChE, tumour necrosis factor and receptor (TNF-α and TNF-α-R) expression, were also determined. RESULTS: FP supplemented diet significantly reduced NTPdase and ADA activities and increased Na+/K+-ATPase activities in hypertensive rats' brains compared to the untreated group. Furthermore, FP reduced acetylcholinesterase and monoamine oxidase-B activities compared to the hypertensive group. Redox imbalance was observed in hypertensive rats with inhibition of antioxidant enzymes and high levels of ROS and MDA. However, FP supplemented diet improved antioxidant enzymes, reduced ROS and MDA production in the brain of hypertensive rats. High blood pressure also triggered upregulation of AChE, TNF-α and TNF-α-R while feeding with FP supplemented diet downregulated the genes. CONCLUSION: This study demonstrates the neuroprotective role of FP supplemented diet against alterations in neurochemicals associated with Alzheimer's disease, oxidative stress-induced neuronal damage and expression of genes linked with neuroinflammation. Moreover, studies on animal behaviour and human subjects are required to confirm these beneficial effects.

Differential Regulation of Prelimbic and Thalamic Transmission to the Basolateral Amygdala by Acetylcholine Receptors.

The amygdalar anterior basolateral nucleus (BLa) plays a vital role in emotional behaviors. This region receives dense cholinergic projections from basal forebrain which are critical in regulating neuronal activity in BLa. Cholinergic signaling in BLa has also been shown to modulate afferent glutamatergic inputs to this region. However, these studies, which have used cholinergic agonists or prolonged optogenetic stimulation of cholinergic fibers, may not reflect the effect of physiological acetylcholine release in the BLa. To better understand these effects of acetylcholine, we have used electrophysiology and optogenetics in male and female mouse brain slices to examine cholinergic regulation of afferent BLa input from cortex and midline thalamic nuclei. Phasic ACh release evoked by single pulse stimulation of cholinergic terminals had a biphasic effect on transmission at cortical input, producing rapid nicotinic receptor-mediated facilitation followed by slower mAChR-mediated depression. In contrast, at this same input, sustained ACh elevation through application of the cholinesterase inhibitor physostigmine suppressed glutamatergic transmission through mAChRs only. This suppression was not observed at midline thalamic nuclei inputs to BLa. In agreement with this pathway specificity, the mAChR agonist, muscarine more potently suppressed transmission at inputs from prelimbic cortex than thalamus. Muscarinic inhibition at prelimbic cortex input required presynaptic M4 mAChRs, while at thalamic input it depended on M3 mAChR-mediated stimulation of retrograde endocannabinoid signaling. Muscarinic inhibition at both pathways was frequency-dependent, allowing only high-frequency activity to pass. These findings demonstrate complex cholinergic regulation of afferent input to BLa that is pathway-specific and frequency-dependent.SIGNIFICANCE STATEMENT Cholinergic modulation of the basolateral amygdala regulates formation of emotional memories, but the underlying mechanisms are not well understood. Here, we show, using mouse brain slices, that ACh differentially regulates afferent transmission to the BLa from cortex and midline thalamic nuclei. Fast, phasic ACh release from a single optical stimulation biphasically regulates glutamatergic transmission at cortical inputs through nicotinic and muscarinic receptors, suggesting that cholinergic neuromodulation can serve precise, computational roles in the BLa. In contrast, sustained ACh elevation regulates cortical input through muscarinic receptors only. This muscarinic regulation is pathway-specific with cortical input inhibited more strongly than midline thalamic nuclei input. Specific targeting of these cholinergic receptors may thus provide a therapeutic strategy to bias amygdalar processing and regulate emotional memory.