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Abiotic stress that plants face may impact their growth and limit their productivity. In response to abiotic stress, several endogenous survival mechanisms get activated, including the synthesis of quaternary amines in plants. Acetylcholine (ACh), a well-known quaternary amine, and its components associated with cholinergic signaling are known to contribute to a variety of physiological functions. However, their role under abiotic stress is not well documented. Even after several studies, there is a lack of a comprehensive understanding of how cholinergic components mitigate abiotic stress in plants. Acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE) belongs to the GDSL lipase/acylhydrolase protein family and has been found in several plant species. Several studies have demonstrated that GDSL members are involved in growth, development, and abiotic stress. This review summarizes all the possible mitigating effects of the ACh-AChE system on abiotic stress tolerance and will try to highlight all the progress made so far in this field. [ABSTRACT FROM AUTHOR]

Gonadal steroids exert different effects on the central nervous system (CNS), such as preserving neuronal function and promoting neuronal survival. Estradiol, progesterone, and testosterone reduce neuronal loss in the CNS in animal models of neurodegeneration. However, hormone replacement therapy has been associated with higher rates of endometrial, prostate, and breast cancer. Tibolone (TIB), the metabolites of which show estrogenic and progestogenic effects, is an alternative to reduce this risk. However, the impact of TIB on memory and learning, as well as on choline acetyltransferase (ChAT) and tryptophan hydroxylase (TPH) levels in the hippocampus of aging males, is unknown. We administered TIB to aged C57BL/6J male mice at different doses (0.01 or 1.0 mg/kg per day for 12 weeks) and evaluated its effects on memory and learning and the content of ChAT and TPH. We assessed memory and learning with object recognition and elevated T-maze tasks. Additionally, we determined ChAT and TPH protein levels in the hippocampus by Western blotting. TIB administration increased the percentage of time spent on the novel object in the object recognition task. In addition, the latency of leaving the enclosed arm increased in both TIB groups, suggesting an improvement in fear-based learning. We also observed decreased ChAT content in the group treated with the 0.01 mg/kg TIB dose. In the case of TPH, no changes were observed with either TIB dose. These results show that long-term TIB administration improves memory without affecting locomotor activity and modulates cholinergic but not serotonergic systems in the hippocampus of aged male mice. [ABSTRACT FROM AUTHOR]

Abiotic stress that plants face may impact their growth and limit their productivity. In response to abiotic stress, several endogenous survival mechanisms get activated, including the synthesis of quaternary amines in plants. Acetylcholine (ACh), a well-known quaternary amine, and its components associated with cholinergic signaling are known to contribute to a variety of physiological functions. However, their role under abiotic stress is not well documented. Even after several studies, there is a lack of a comprehensive understanding of how cholinergic components mitigate abiotic stress in plants. Acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE) belongs to the GDSL lipase/acylhydrolase protein family and has been found in several plant species. Several studies have demonstrated that GDSL members are involved in growth, development, and abiotic stress. This review summarizes all the possible mitigating effects of the ACh-AChE system on abiotic stress tolerance and will try to highlight all the progress made so far in this field.

Acetylcholine plays an important role as a neurotransmitter in the central and peripheral nervous systems. Its biosynthesis is catalyzed by choline acetyltransferase (ChAT). In mice, the ChAT gene has three 5′-noncoding (R, N, and M) and 14 coding exons, from which seven mRNA isoforms (M, N1, N2, R1, R2, R3, and R4) are transcribed. They differ in the 5′-noncoding ends and encode the same protein (common ChAT). An additional isoform that lacks coding exons from 5 to 8 encodes a smaller protein (peripheral ChAT). This research aimed to design and check the specificity of primers targeting R3, N1, N2, M, peripheral ChAT, and common ChAT isoforms. The optimal PCR conditions and specificity of the primers were tested in a series of PCR reactions. Specially designed DNA fragments or plasmids containing isoform-specific nucleotide sequences were used as templates. The appropriate annealing temperature, which yields sufficient specificity for each tested primer pair, was as follows: R3 – 60 °C; N1 – 63 °C; N2 – 65 °C; M – 65 °C; peripheral ChAT – 65 °C and common ChAT – 63 °C.

INTRODUCTION: The central and peripheral nervous systems provide cholinergic innervation in the colon. The ability to assess their neuroanatomical distinctions is still a challenge. The pig is regarded as a relevant translational model due to the close similarity of its enteric nervous system (ENS) with that of human. Opioid-induced constipation is one of the most common side effects of opioid therapy. METHODS: We developed an approach to differentiate the central and peripheral cholinergic innervation of the pig colon using double immunolabeling with a novel mouse anti-human peripheral type of choline acetyltransferase (hpChAT) antibody combined with a rabbit anti-common type of ChAT (cChAT) antibody, a reliable marker of cholinergic neurons in the central nervous system. We examined their spatial configurations in 3D images of the ENS generated from CLARITY-cleared colonic segments. The density was quantitated computationally using Imaris 9.7. We assessed changes in the distal colon induced by daily oral treatment for 4  weeks with the μ opioid receptor agonist, loperamide (0.4 or 3  mg/kg). RESULTS: The double labeling showed strong cChAT immunoreactive (ir) fibers in the cervical vagus nerve and neuronal somata and fibers in the ventral horn of the sacral (S2) cord while hpChAT immunoreactivity was visualized only in the ENS but not in the vagus or sacral neural structures indicating the selectivity of these two antibodies. In the colonic myenteric plexus, dense hpChAT-ir neurons and fibers and varicose cChAT-ir fibers surrounding hpChAT-ir neurons were simultaneously visualized in 3D. The density of cChAT-ir varicose fibers in the outer submucosal plexus of both males and females were higher in the transverse and distal colon than in the proximal colon and in the myenteric plexus compared to the outer submucosal plexus and there was no cChAT innervation in the inner submucosal plexus. The density of hpChAT in the ENS showed no segmental or plexus differences in both sexes. Loperamide at the highest dose significantly decreased the density hpChAT-ir fibers + somata in the myenteric plexus of the distal colon. DISCUSSION: These data showed the distinct density of central cholinergic innervation between myenteric and submucosal plexuses among colonic segments and the localization of cChAT-ir fibers around peripheral hpChAT neurons in 3D. The reduction of cholinergic myenteric innervation by chronic opiate treatment points to target altered prokinetic cholinergic pathway to counteract opiate constipation.

Rbfox1 is a multifunctional RNA-binding protein that regulates alternative splicing, transcription, mRNA stability, and translation. Rbfox1 is an important regulator of gene networks involved in neurogenesis and neuronal function. Disruption of Rbfox function has been associated with several neurodevelopmental and neuropsychiatric disorders. We have shown earlier that Rbfox1 is expressed in retinal ganglion and amacrine cells (ACs) and that its down-regulation in adult mouse retinas leads to deficiency of depth perception. In the present study, we used several markers of ACs, including gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), neuropeptide Y (NPY), glycine transporter (GlyT1), and vesicular glutamate transporter 3 (VGlut3) to identify types of ACs that express Rbfox1. Expression of Rbfox1 was observed predominantly in GABAergic ACs located in the inner nuclear layer (INL) and ganglion cell layer (GCL). All GABAergic/cholinergic starburst ACs and virtually all NPY-positive GABAergic ACs were also Rbfox1-positive. Among glycinergic ACs, a sparse population of Rbfox1/VGlut3-positive cells was identified, indicating that Rbfox1 is expressed in a very small population of glycinergic ACs. These data contribute to our understanding about molecular differences between various types of amacrine cells and the cell-specific gene networks regulated by Rbfox1.

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene (CHAT) result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for Chat (Chat-/-) die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing loxP sites flanking Chat exons 4 and 5 with mice that expressed Cre-ERT2. Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of Chat, autonomic failure, weakness, and death. However, a proportion of Chatflox/flox-Cre-ERT2 mice receiving at birth an intracerebroventricular injection of 2 × 1013 vg/kg adeno-associated virus type 9 (AAV9) carrying human CHAT (AAV9-CHAT) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9-CHAT by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of Chatflox/flox-Cre-ERT2 mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human CHAT RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CHAT-CMS. [ABSTRACT FROM AUTHOR]

Gonadal steroids exert different effects on the central nervous system (CNS), such as preserving neuronal function and promoting neuronal survival. Estradiol, progesterone, and testosterone reduce neuronal loss in the CNS in animal models of neurodegeneration. However, hormone replacement therapy has been associated with higher rates of endometrial, prostate, and breast cancer. Tibolone (TIB), the metabolites of which show estrogenic and progestogenic effects, is an alternative to reduce this risk. However, the impact of TIB on memory and learning, as well as on choline acetyltransferase (ChAT) and tryptophan hydroxylase (TPH) levels in the hippocampus of aging males, is unknown. We administered TIB to aged C57BL/6J male mice at different doses (0.01 or 1.0 mg/kg per day for 12 weeks) and evaluated its effects on memory and learning and the content of ChAT and TPH. We assessed memory and learning with object recognition and elevated T-maze tasks. Additionally, we determined ChAT and TPH protein levels in the hippocampus by Western blotting. TIB administration increased the percentage of time spent on the novel object in the object recognition task. In addition, the latency of leaving the enclosed arm increased in both TIB groups, suggesting an improvement in fear-based learning. We also observed decreased ChAT content in the group treated with the 0.01 mg/kg TIB dose. In the case of TPH, no changes were observed with either TIB dose. These results show that long-term TIB administration improves memory without affecting locomotor activity and modulates cholinergic but not serotonergic systems in the hippocampus of aged male mice.

Major features of aging might be progressive decreases in cognitive function and physical activity, in addition to withered appearance. Previously, we reported that the intracerebroventricular injection of human neural stem cells (NSCs named F3) encoded the choline acetyltransferase gene (F3.ChAT). The cells secreted acetylcholine and growth factors (GFs) and neurotrophic factors (NFs), thereby improving learning and memory function as well as the physical activity of aged animals. In this study, F344 rats (10 months old) were intravenously transplanted with F3 or F3.ChAT NSCs (1 × 106 cells) once a month to the 21st month of age. Their physical activity and cognitive function were investigated, and brain acetylcholine (ACh) and cholinergic and dopaminergic system markers were analyzed. Neuroprotective and neuroregenerative activities of stem cells were also confirmed by analyzing oxidative damages, neuronal skeletal protein, angiogenesis, brain and muscle weights, and proliferating host stem cells. Stem cells markedly improved both cognitive and physical functions, in parallel with the elevation in ACh levels in cerebrospinal fluid and muscles, in which F3.ChAT cells were more effective than F3 parental cells. Stem cell transplantation downregulated CCL11 and recovered GFs and NFs in the brain, leading to restoration of microtubule-associated protein 2 as well as functional markers of cholinergic and dopaminergic systems, along with neovascularization. Stem cells also restored muscular GFs and NFs, resulting in increased angiogenesis and muscle mass. In addition, stem cells enhanced antioxidative capacity, attenuating oxidative damage to the brain and muscles. The results indicate that NSCs encoding ChAT improve cognitive function and physical activity of aging animals by protecting and recovering functions of multiple organs, including cholinergic and dopaminergic systems, as well as muscles from oxidative injuries through secretion of ACh and GFs/NFs, increased antioxidant elements, and enhanced blood flow. [ABSTRACT FROM AUTHOR]

Introduction: The central and peripheral nervous systems provide cholinergic innervation in the colon. The ability to assess their neuroanatomical distinctions is still a challenge. The pig is regarded as a relevant translational model due to the close similarity of its enteric nervous system (ENS) with that of human. Opioid-induced constipation is one of the most common side effects of opioid therapy. Methods: We developed an approach to differentiate the central and peripheral cholinergic innervation of the pig colon using double immunolabeling with a novel mouse anti-human peripheral type of choline acetyltransferase (hpChAT) antibody combined with a rabbit anti-common type of ChAT (cChAT) antibody, a reliable marker of cholinergic neurons in the central nervous system. We examined their spatial configurations in 3D images of the ENS generated from CLARITY-cleared colonic segments. The density was quantitated computationally using Imaris 9.7. We assessed changes in the distal colon induced by daily oral treatment for 4 weeks with the µ opioid receptor agonist, loperamide (0.4 or 3 mg/kg). Results: The double labeling showed strong cChAT immunoreactive (ir) fibers in the cervical vagus nerve and neuronal somata and fibers in the ventral horn of the sacral (S2) cord while hpChAT immunoreactivity was visualized only in the ENS but not in the vagus or sacral neural structures indicating the selectivity of these two antibodies. In the colonic myenteric plexus, dense hpChAT-ir neurons and fibers and varicose cChAT-ir fibers surrounding hpChAT-ir neurons were simultaneously visualized in 3D. The density of cChAT-ir varicose fibers in the outer submucosal plexus of both males and females were higher in the transverse and distal colon than in the proximal colon and in the myenteric plexus compared to the outer submucosal plexus and there was no cChAT innervation in the inner submucosal plexus. The density of hpChAT in the ENS showed no segmental or plexus differences in both sexes. Loperamide at the highest dose significantly decreased the density hpChAT-ir fibers + somata in the myenteric plexus of the distal colon. Discussion: These data showed the distinct density of central cholinergic innervation between myenteric and submucosal plexuses among colonic segments and the localization of cChAT-ir fibers around peripheral hpChAT neurons in 3D. The reduction of cholinergic myenteric innervation by chronic opiate treatment points to target altered prokinetic cholinergic pathway to counteract opiate constipation. [ABSTRACT FROM AUTHOR]

The enteric nervous system is the largest and most complex division of the peripheral nervous system. Located in the wall of the gastrointestinal tract, it is a regulatory and coordination unit of the nervous system. Neurochemical, pharmacological, and functional studies describe three main classes of neurons in the enteric nervous system– primary afferent, interneurons and motor neurons. These are grouped in ganglia, which are connected and form plexuses. Acetylcholine is a major neurotransmitter that plays a pivotal role in the enteric nervous system and several non-neuronal structures. Internal cholinergic neurons and vagus terminals in the enteric nervous system use acetylcholine as the main excitatory neurotransmitter, regulating motility and mucosal function in the digestive system. The enzyme choline acetyltransferase (ChAT) that catalyzes the synthesis of acetylcholine represents the most specific cholinergic marker. Recent markers used to visualize cholinergic structures are the splicing variants of ChAT mRNA that are transcribed from the ChAT gene. Different alternatively spliced ChAT mRNA variants are transcribed in many animal species, including humans. In the mouse, seven variants in the 5’-noncoding region and two variants that differ in their coding region are described. Morphological, genetic and molecular analysis of ChAT and its splicing variants, as the most reliable and frequently used marker for cholinergic structures, would contribute to a better understanding of the physiological and pathological states of cholinergic neurons in the enteric nervous system.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. In AD patients, amyloid-β (Aβ) peptide-mediated degeneration of the cholinergic system utilizing acetylcholine (ACh) for memory acquisition is observed. Since AD therapy using acetylcholinesterase (AChE) inhibitors are only palliative for memory deficits without reversing disease progress, there is a need for effective therapies, and cell-based therapeutic approaches should fulfil this requirement. We established F3.ChAT human neural stem cells (NSCs) encoding the choline acetyltransferase (ChAT) gene, an ACh-synthesizing enzyme, HMO6.NEP human microglial cells encoding the neprilysin (NEP) gene, an Aβ-degrading enzyme, and HMO6.SRA cells encoding the scavenger receptor A (SRA) gene, an Aβ-uptaking receptor. For the efficacy evaluation of the cells, first, we established an appropriate animal model based on Aβ accumulation and cognitive dysfunction. Among various AD models, intracerebroventricular (ICV) injection of ethylcholine mustard azirinium ion (AF64A) induced the most severe Aβ accumulation and memory dysfunction. Established NSCs and HMO6 cells were transplanted ICV to mice showing memory loss induced by AF64A challenge, and brain Aβ accumulation, ACh concentration and cognitive function were analyzed. All the transplanted F3.ChAT, HMO6.NEP and HMO6.SRA cells were found to survive up to 4 weeks in the mouse brain and expressed their functional genes. Combinational treatment with the NSCs (F3.ChAT) and microglial cells encoding each functional gene (HMO6.NEP or HMO6.SRA) synergistically restored the learning and memory function of AF64A-challenged mice by eliminating Aβ deposits and recovering ACh level. The cells also attenuated inflammatory astrocytic (glial fibrillary acidic protein) response by reducing Aβ accumulation. Taken together, it is expected that NSCs and microglial cells over-expressing ChAT, NEP or SRA genes could be strategies for replacement cell therapy of AD. [ABSTRACT FROM AUTHOR]

The cholinergic efferent network from the medial septal nucleus to the hippocampus is crucial for learning and memory. This study aimed to clarify whether hippocampal cholinergic neurostimulating peptide (HCNP) has a rescue function in the cholinergic dysfunction of HCNP precursor protein (HCNP-pp) conditional knockout (cKO). Chemically synthesized HCNP or a vehicle were continuously administered into the cerebral ventricle of HCNP-pp cKO mice and littermate floxed (control) mice for two weeks via osmotic pumps. We immunohistochemically measured the cholinergic axon volume in the stratum oriens and functionally evaluated the local field potential in the CA1. Furthermore, choline acetyltransferase (ChAT) and nerve growth factor (NGF) receptor (TrkA and p75NTR) abundances were quantified in wild-type (WT) mice administered HCNP or the vehicle. As a result, HCNP administration morphologically increased the cholinergic axonal volume and electrophysiological theta power in HCNP-pp cKO and control mice. Following the administration of HCNP to WT mice, TrkA and p75NTR levels also decreased significantly. These data suggest that extrinsic HCNP may compensate for the reduced cholinergic axonal volume and theta power in HCNP-pp cKO mice. HCNP may function complementarily to NGF in the cholinergic network in vivo. HCNP may represent a therapeutic candidate for neurological diseases with cholinergic dysfunction, e.g., Alzheimer's disease and Lewy body dementia. [ABSTRACT FROM AUTHOR]

The hypothalamic neurohormone kisspeptin-10 (KP-10) was inherently implicated in cholinergic pathologies when aberrant fluctuations of expression patterns and receptor densities were discerned in neurodegenerative micromilieus. That said, despite variable degrees of functional redundancy, KP-10, which is biologically governed by its cognate G-protein-coupled receptor, GPR54, attenuated the progressive demise of α-synuclein (α-syn)-rich cholinergic-like neurons. Under explicitly modeled environments, in silico algorithms further rationalized the surface complementarities between KP-10 and α-syn when KP-10 was unambiguously accommodated in the C-terminal binding pockets of α-syn. Indeed, the neuroprotective relevance of KP-10's binding mechanisms can be insinuated in the amelioration of α-syn-mediated neurotoxicity; yet it is obscure whether these extenuative circumstances are contingent upon prior GPR54 activation. Herein, choline acetyltransferase (ChAT)-positive SH-SY5Y neurons were engineered ad hoc to transiently overexpress human wild-type or E46K mutant α-syn while the mitigation of α-syn-induced neuronal death was ascertained via flow cytometric and immunocytochemical quantification. Recapitulating the specificity observed on cell viability, exogenously administered KP-10 (0.1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated apoptosis and mitochondrial depolarization in cholinergic differentiated neurons. In particular, co-administrations with a GPR54 antagonist, kisspeptin-234 (KP-234), failed to abrogate the robust neuroprotection elicited by KP-10, thereby signifying a GPR54 dispensable mechanism of action. Consistent with these observations, KP-10 treatment further diminished α-syn and ChAT immunoreactivity in neurons overexpressing wild-type and E46K mutant α-syn. Overall, these findings lend additional credence to the previous notion that KP-10's binding zone may harness efficacious moieties of neuroprotective intent. [ABSTRACT FROM AUTHOR]

Abstract Background An active lifestyle is associated with improved cognitive functions in aged people and may prevent or slow down the progression of various neurodegenerative diseases including Alzheimer’s disease (AD). To investigate these protective effects, male APPNL-G-F mice were exposed to long-term voluntary exercise. Methods Three-month-old AD mice were housed in a cage supplemented with a running wheel for 9 months for long-term exercise. At the age of 12 months, behavioral tests were completed for all groups. After completing behavioral testing, their brains were assessed for amyloid pathology, microgliosis, and cholinergic cells. Results The results showed that APPNL-G-F mice allowed to voluntarily exercise showed an improvement in cognitive functions. Furthermore, long-term exercise also improved anxiety in APPNL-G-F mice as assessed by measuring thigmotaxis in the Morris water task. We also found reductions in amyloid load and microgliosis, and a preservation of cholinergic cells in the brain of APPNL-G-F mice allowed to exercise in their home cages. These profound reductions in brain pathology associated with AD are likely responsible for the observed improvement of learning and memory functions following extensive and regular exercise. Conclusion These findings suggest the potential of physical exercise to mitigate the cognitive deficits in AD.

Aster koraiensis Nakai (AK) leaf reportedly ameliorates health problems, such as diabetes. However, the effects of AK on cognitive dysfunction or memory impairment remain unclear. This study investigated whether AK leaf extract could attenuate cognitive impairment. We found that AK extract reduced the production of nitric oxide (NO), tumour necrosis factor (TNF)-α, phosphorylated-tau (p-tau), and the expression of inflammatory proteins in lipopolysaccharide- or amyloid-β-treated cells. AK extract exhibited inhibitory activity of control specific binding on N-methyl-D-aspartate (NMDA) receptors. Scopolamine-induced AD models were used chronically in rats and acutely in mice. Relative to negative controls (NC), hippocampal choline acetyltransferase (ChAT) and B-cell lymphoma 2 (Bcl2) activity was increased in rats chronically treated with scopolamine and fed an AK extract-containing diet. In the Y-maze test, spontaneous alterations were increased in the AK extract-fed groups compared to NC. Rats administered AK extract showed increased escape latency in the passive avoidance test. In the hippocampus of rats fed a high-AK extract diet (AKH), the expression of neuroactive ligand–receptor interaction-related genes, including Npy2r, Htr2c, and Rxfp1, was significantly altered. In the Morris water maze assay of mice acutely treated with scopolamine, the swimming times in the target quadrant of AK extract-treated groups increased significantly to the levels of the Donepezil and normal groups. We used Tg6799 Aβ-overexpressing 5XFAD transgenic mice to investigate Aβ accumulation in animals. In the AD model using 5XFAD, the administration of AK extract decreased amyloid-β (Aβ) accumulation and increased the number of NeuN antibody-reactive cells in the subiculum relative to the control group. In conclusion, AK extract ameliorated memory dysfunction by modulating ChAT activity and Bcl2-related anti-apoptotic pathways, affecting the expression of neuroactive ligand–receptor interaction-related genes and inhibiting Aβ accumulation. Therefore, AK extract could be a functional material improving cognition and memory. [ABSTRACT FROM AUTHOR]

Background: A historically definitive marker for cholinergic neurons is choline acetyltransferase (ChAT), a synthesizing enzyme for acetylcholine, (ACh), which can be found in high concentrations in cholinergic neurons, both in the central and peripheral nervous systems. ChAT, is produced in the body of the neuron, transported to the nerve terminal (where its concentration is highest), and catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding ACh. The creation of bacterial artificial chromosome (BAC) transgenic mice that express promoter-specific fluorescent reporter proteins (green fluorescent protein—[GFP]) provided an enormous advantage for neuroscience. Both in vivo and in vitro experimental methods benefited from the transgenic visualization of cholinergic neurons. Mice were created by adding a BAC clone into the ChAT locus, in which enhanced GFP (eGFP) is inserted into exon 3 at the ChAT initiation codon, robustly and supposedly selectively expressing eGFP in all cholinergic neurons and fibers in the central and peripheral nervous systems as well as in non-neuronal cells. Methods: This project systematically compared the exact distribution of the ChAT-eGFP expressing neurons in the brain with the expression of ChAT by immunohistochemistry using mapping and also made comparisons with in situ hybridization (ISH). Results: We qualitatively described the distribution of ChAT-eGFP neurons in the mouse brain by comparing it with the distribution of immunoreactive neurons and ISH data, paying special attention to areas where the expression did not overlap, such as the cortex, striatum, thalamus and hypothalamus. We found a complete overlap between the transgenic expression of eGFP and the immunohistochemical staining in the areas of the cholinergic basal forebrain. However, in the cortex and hippocampus, we found small neurons that were only labeled with the antibody and not expressed eGFP or vice versa. Most importantly, we found no transgenic expression of eGFP in the lateral dorsal, ventral and dorsomedial tegmental nuclei cholinergic cells. Conclusion: While the majority of the forebrain ChAT expression was aligned in the transgenic animals with immunohistochemistry, other areas of interest, such as the brainstem should be considered before choosing this particular transgenic mouse line. [ABSTRACT FROM AUTHOR]

Tumor cells have evolved to express immunosuppressive molecules allowing their evasion from the host's immune system. These molecules include programmed death ligands 1 and 2 (PD-L1 and PD-L2). Cancer cells can also produce acetylcholine (ACh), which plays a role in tumor development. Moreover, tumor innervation can stimulate vascularization leading to tumor growth and metastasis. The effects of atropine and muscarinic receptor 3 (M3R) blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), on cancer growth and spread were evaluated in vitro using murine colon cancer cell line, CT-26, and in vivo in an orthotopic mouse model of colorectal cancer. In the in vitro model, atropine and 4-DAMP significantly inhibited CT-26 cell proliferation in a dose dependent manner and induced apoptosis. Atropine attenuated immunosuppressive markers and M3R via inhibition of EGFR/AKT/ERK signaling pathways. However, 4-DAMP showed no effect on the expression of PD-L1, PD-L2, and choline acetyltransferase (ChAT) on CT-26 cells but attenuated M3R by suppressing the phosphorylation of AKT and ERK. Blocking of M3R in vivo decreased tumor growth and expression of immunosuppressive, cholinergic, and angiogenic markers through inhibition of AKT and ERK, leading to an improved immune response against cancer. The expression of immunosuppressive and cholinergic markers may hold potential in determining prognosis and treatment regimens for colorectal cancer patients. This study's results demonstrate that blocking M3R has pronounced antitumor effects via several mechanisms, including inhibition of immunosuppressive molecules, enhancement of antitumor immune response, and suppression of tumor angiogenesis via suppression of the AKT/ERK signaling pathway. These findings suggest a crosstalk between the cholinergic and immune systems during cancer development. In addition, the cholinergic system influences cancer evasion from the host's immunity. [ABSTRACT FROM AUTHOR]

Major features of aging might be progressive decreases in cognitive function and physical activity, in addition to withered appearance. Previously, we reported that the intracerebroventricular injection of human neural stem cells (NSCs named F3) encoded the choline acetyltransferase gene (F3.ChAT). The cells secreted acetylcholine and growth factors (GFs) and neurotrophic factors (NFs), thereby improving learning and memory function as well as the physical activity of aged animals. In this study, F344 rats (10 months old) were intravenously transplanted with F3 or F3.ChAT NSCs (1 × 106 cells) once a month to the 21st month of age. Their physical activity and cognitive function were investigated, and brain acetylcholine (ACh) and cholinergic and dopaminergic system markers were analyzed. Neuroprotective and neuroregenerative activities of stem cells were also confirmed by analyzing oxidative damages, neuronal skeletal protein, angiogenesis, brain and muscle weights, and proliferating host stem cells. Stem cells markedly improved both cognitive and physical functions, in parallel with the elevation in ACh levels in cerebrospinal fluid and muscles, in which F3.ChAT cells were more effective than F3 parental cells. Stem cell transplantation downregulated CCL11 and recovered GFs and NFs in the brain, leading to restoration of microtubule-associated protein 2 as well as functional markers of cholinergic and dopaminergic systems, along with neovascularization. Stem cells also restored muscular GFs and NFs, resulting in increased angiogenesis and muscle mass. In addition, stem cells enhanced antioxidative capacity, attenuating oxidative damage to the brain and muscles. The results indicate that NSCs encoding ChAT improve cognitive function and physical activity of aging animals by protecting and recovering functions of multiple organs, including cholinergic and dopaminergic systems, as well as muscles from oxidative injuries through secretion of ACh and GFs/NFs, increased antioxidant elements, and enhanced blood flow.

The recent development of transgenic rodent lines expressing cre recombinase in a cell-specific manner, along with advances in engineered viral vectors, has permitted in-depth investigations into circuit function. However, emerging evidence has begun to suggest that genetic modifications may introduce unexpected caveats. In the current studies, we sought to extensively characterize male and female mice from both the ChAT(BAC)-Cre mouse line, created with the bacterial artificial chromosome (BAC) method, and ChAT(IRES)-Cre mouse line, generated with the internal ribosome entry site (IRES) method. ChAT(BAC)-Cre transgenic and wild-type mice did not differ in general locomotor behavior, anxiety measures, drug-induced cataplexy, nicotine-mediated hypolocomotion, or operant food training. However, ChAT(BAC)-Cre transgenic mice did exhibit significant deficits in intravenous nicotine self-administration, which paralleled an increase in vesicular acetylcholine transporter and choline acetyltransferase (ChAT) hippocampal expression. For the ChAT(IRES)-Cre line, transgenic mice exhibited deficits in baseline locomotor, nicotine-mediated hypolocomotion, and operant food training compared with wild-type and hemizygous littermates. No differences among ChAT(IRES)-Cre wild-type, hemizygous, and transgenic littermates were found in anxiety measures, drug-induced cataplexy, and nicotine self-administration. Given that increased cre expression was present in the ChAT(IRES)-Cre transgenic mice, as well as a decrease in ChAT expression in the hippocampus, altered neuronal function may underlie behavioral phenotypes. In contrast, ChAT(IRES)-Cre hemizygous mice were more similar to wild-type mice in both protein expression and the majority of behavioral assessments. As such, interpretation of data derived from ChAT-Cre rodents must consider potential limitations dependent on the line and/or genotype used in research investigations.SIGNIFICANCE STATEMENT Altered baseline and/or nicotine-mediated behavioral profiles were discovered in transgenic mice from the ChAT(BAC)-Cre and ChAT(IRES)-Cre lines. Given that these cre-expressing mice have become increasingly used by the scientific community, either independently with chemicogenetic and optogenetic viral vectors or crossed with other transgenic lines, the current studies highlight important considerations for the interpretation of data from previous and future experimental investigations. Moreover, the current findings detail the behavioral effects of either increased or decreased baseline cholinergic signaling mechanisms on locomotor, anxiety, learning/memory, and intravenous nicotine self-administration behaviors.

Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1-60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.

Abstract Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker l-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension.

Background: An active lifestyle is associated with improved cognitive functions in aged people and may prevent or slow down the progression of various neurodegenerative diseases including Alzheimer's disease (AD). To investigate these protective effects, male APPNL-G-F mice were exposed to long-term voluntary exercise. Methods: Three-month-old AD mice were housed in a cage supplemented with a running wheel for 9 months for long-term exercise. At the age of 12 months, behavioral tests were completed for all groups. After completing behavioral testing, their brains were assessed for amyloid pathology, microgliosis, and cholinergic cells. Results: The results showed that APPNL-G-F mice allowed to voluntarily exercise showed an improvement in cognitive functions. Furthermore, long-term exercise also improved anxiety in APPNL-G-F mice as assessed by measuring thigmotaxis in the Morris water task. We also found reductions in amyloid load and microgliosis, and a preservation of cholinergic cells in the brain of APPNL-G-F mice allowed to exercise in their home cages. These profound reductions in brain pathology associated with AD are likely responsible for the observed improvement of learning and memory functions following extensive and regular exercise. Conclusion: These findings suggest the potential of physical exercise to mitigate the cognitive deficits in AD. [ABSTRACT FROM AUTHOR]

Aim: A monoterpene and bioactive component of the plant Rhodiola rosea (R. rosea), rosiridin has beneficial effects on the human central nervous system and enhances brain function. The goal of this scientific study was to determine if rosiridin might shield rats from neurocognitive problems induced by scopolamine. Methods: To track the potential toxicities in rats, the acute toxicity in rats was clarified. Rosiridin at a dose of 10 mg/kg was tested in rats for 14 days. At the conclusion of the investigation, behavioral parameters that were used to identify the rats' cognitive and motor abilities were evaluated. Several biochemical parameters were estimated using the prepared homogenate, including acetylcholine esterase (AChE), choline acetyltransferase (ChAT), radical scavengers produced by the body (Catalase-CAT, superoxide dismutase-SOD, and reduced glutathione-GSH), indicators of oxidative and nitrative burnout, pro-inflammatory (Interleukins- IL-1β, IL-6, interferon gamma IFN-ꝩ, and tumor necrosis factor-TNF-α), and cell apoptosis caspases 3 and 9. Results and Conclusion: A significant behavioral parameter restoration was seen in the rosiridin-treated group, including reduction in latency time during acquisition and retention trial in the Morris water maze test, and percentage of spontaneous alterations in the y-maze test, when compared to the disease control group that received scopolamine; rosiridin also altered the oxidative stress and neuroinflammatory markers, as well as restoring Ach and ChAT activities and normalizing GSH, SOD, MDA, TNF-α, nitrate, IL-1β, IL-6, IFN-ꝩ, caspases 3 and 9 levels. The results imply that rosiridin limits the effect of scopolamine on rat cognitive function. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1) are Tolllike receptor (TLR4) agonists that activate proinflammatory neuroimmune signaling linked to loss of basal forebrain cholinergic neurons (BFCNs) and cognitive deficits. Loss of choline acetyltransferase immunoreactive (ChAT + IR) BFCNs is generally interpreted as cell death, but recent in vivo studies find anti-inflammatory interventions restore adolescent ethanol exposure-induced persistent loss of adult ChAT + IR neurons and cognitive deficits, suggesting proinflammatory signaling-induced reversible gene repression of ChAT in BFCNs. Using an ex vivo Wistar rat basal forebrain slice culture (FSC) model to investigate TLR4 involvement in repression of the BFCN phenotype, we report that direct TLR4 activation with LPS decreases expression of multiple BFCN markers in the absence of observable neuronal loss or cell death. Inhibition of HMGB1 blunts while inhibition of TLR4 blocks the LPS-induced loss of ChAT + IR neurons. TLR4 activation induces the transcriptional repressor RE1-silencing transcription factor (REST) and the methyltransferase G9a while increasing repressive histone 3 lysine 9 dimethylation and REST occupancy at cholinergic gene promoters. G9a inhibitors both prevent and reverse the LPS-induced loss of ChAT + IR whereas siRNA inhibition of REST blocks the LPS-induced loss of ChAT + IR BFCNs. These data suggest in vivo HMGB1-TLR4 signaling in BFCNs leads to a reversible loss of the cholinergic neuron phenotype through epigenetic gene repressive mechanisms. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1) are Toll-like receptor (TLR4) agonists that activate proinflammatory neuroimmune signaling linked to loss of basal forebrain cholinergic neurons (BFCNs) and cognitive deficits. Loss of choline acetyltransferase immunoreactive (ChAT + IR) BFCNs is generally interpreted as cell death, but recent in vivo studies find anti-inflammatory interventions restore adolescent ethanol exposure-induced persistent loss of adult ChAT + IR neurons and cognitive deficits, suggesting proinflammatory signaling-induced reversible gene repression of ChAT in BFCNs. Using an ex vivo Wistar rat basal forebrain slice culture (FSC) model to investigate TLR4 involvement in repression of the BFCN phenotype, we report that direct TLR4 activation with LPS decreases expression of multiple BFCN markers in the absence of observable neuronal loss or cell death. Inhibition of HMGB1 blunts while inhibition of TLR4 blocks the LPS-induced loss of ChAT + IR neurons. TLR4 activation induces the transcriptional repressor RE1-silencing transcription factor (REST) and the methyltransferase G9a while increasing repressive histone 3 lysine 9 dimethylation and REST occupancy at cholinergic gene promoters. G9a inhibitors both prevent and reverse the LPS-induced loss of ChAT + IR whereas siRNA inhibition of REST blocks the LPS-induced loss of ChAT + IR BFCNs. These data suggest in vivo HMGB1-TLR4 signaling in BFCNs leads to a reversible loss of the cholinergic neuron phenotype through epigenetic gene repressive mechanisms.

Pinostrobin, a key bioactive compound found in the medicinal plant Boesenbergia rotunda (L.), has been noted for its beneficial biological properties including antioxidant, anti-inflammation, anti-cancer and anti-amnesia activities. In view of this, the present study purposed to evaluate the neuroprotective potential of pinostrobin in reversing scopolamine-induced cognitive impairment involving oxidative stress and cholinergic function in rats. A total of 30 male Wistar rats were randomly divided into five groups (n=6): Group 1 received vehicle as a control, group 2 received vehicle + scopolamine (3 mg/kg, i.p.), group 3 received pinostrobin (20 mg/kg, p.o.) + scopolamine, group 4 received pinostrobin (40 mg/kg, p.o.) + scopolamine and group 5 received donepezil (5 mg/kg, p.o.) + scopolamine. Treatments were administered orally to the rats for 14 days. During the final 7 days of treatment, a daily injection of scopolamine was administered. Scopolamine impaired learning and memory performance, as measured by the novel object recognition test and the Y-maze test. Additionally, oxidative stress marker levels, acetylcholinesterase (AChE) activity, choline acetyltransferase (ChAT) and glutamate receptor 1 (GluR1) expression were determined. Consequently, the findings demonstrated that the administration of pinostrobin (20 and 40 mg/kg) markedly improved cognitive function as indicated by an increase in recognition index and by spontaneous alternation behaviour. Pinostrobin also modulated the levels of oxidative stress by causing a decrease in malondialdehyde levels accompanied by increases in superoxide dismutase and glutathione activities. Similarly, pinostrobin markedly enhanced cholinergic function by decreasing AChE activity and promoting ChAT immunoreactivity in the hippocampus. Additionally, the reduction in GluR1 expression due to scopolamine was diminished by treatment with pinostrobin. The findings indicated that pinostrobin exhibited a significant restoration of scopolamine-induced memory impairment by regulating oxidative stress and cholinergic system function. Thus, pinostrobin could serve as a potential therapeutic agent for the management of neurodegenerative diseases such as Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Background: Neurodegenerative diseases, such as diabetic retinopathy (DR) and glaucoma, induce retinal neuron loss. Acetylcholine-containing cholinergic neurons, known as starburst amacrine cells (SACs), play critical roles in the generation of precise neuronal activity in the retina and are located in the inner nuclear layer (INL, conventional) and ganglion cell layer (GCL, displaced). Methods: This study investigated the loss of and morphological changes in SACs in the retinas of streptozotocin (STZ)-induced diabetic and insulin-deficient C57BL/6-Tg(pH1-siRNAinsulin/CMV-hIDE)/Korl (IDCK) mice. SACs were immunocytochemically localized with anti-choline acetyltransferase (ChAT) antibody, and ChAT-labeled cells in the INL and GCL in the control and experimental groups were counted along the central vertical meridian in the whole-mounted retina using conventional fluorescent or confocal microscopes. Results: ChAT-immunoreactive (IR) neurons in STZ-induced diabetic mouse retina decreased by 8.34% at 4–6 weeks and by 14.89% at 42 weeks compared with the control group. Localized ChAT-IR neuron counts in the retinas of 20-week-old IDCK mice were 16.80% lower than those of age-matched control mice. Cell body deformation and aggregation were detected in the retinas of mice with DR. Single-cell injection experiments revealed the loss and deformation of dendritic branches in ChAT-IR neurons in DR. All ChAT-IR neurons expressed the calcium-binding protein calretinin, whereas no ChAT-IR neuron colocalized with calbindin-D28K or parvalbumin. Conclusions: Our results revealed that the neurodegenerative effects of the loss and deformation of ChAT-IR neurons can provide a reference for future study of this disease.

In recent years, people in the United States and other countries have been using smart drugs, called nootropic or cognitive enhancers, to improve concentration and memory learning skills. However, these drugs were originally prescribed for attention-deficit hyperactivity disorder and dementia, and their efficacy in healthy people has not yet been established. We focused on acetylcholine in the hippocampus, which is responsible for memory learning, and elucidate the long-term effects of smart drugs on the neural circuits. Smart drugs were administered orally in normal young mice for seven weeks. The hippocampus was sectioned and compared histologically by hematoxylin and eosin (HE) staining, immunohistochemistry for acetylcholine, and immunoelectron microscopy. There were no significant changes in acetylcholinesterase staining. However, in HE, we found perivascular edema, and choline acetyltransferase staining showed increased staining throughout the hippocampus and new signal induction in the perivascular area in the CA3, especially in the aniracetam and α-glyceryl phosphoryl choline group. Additionally, new muscarinic acetylcholine receptor signals were observed in the CA1 due to smart drug intake, suggesting that vasodilation might cause neuronal activation by increasing the influx of nutrients and oxygen. Moreover, these results suggest a possible new mechanism of acetylcholine-mediated neural circuit activation by smart drug intake. [ABSTRACT FROM AUTHOR]

The native function of amyloid-β (Aβ) peptides is still unexplored. However, several recent reports suggest a prominent role of Aβ peptides in acetylcholine homeostasis. To clarify this role of Aβ, we have reported that Aβ peptides at physiological concentrations can directly enhance the catalytic efficiency of the key cholinergic enzyme, choline acetyltransferase (ChAT), via an allosteric interaction. In the current study, we further aimed to elucidate the underlying ChAT-Aβ interaction mechanism using in silico molecular docking and dynamics analysis. Docking analysis suggested two most probable binding clusters on ChAT for Aβ40 and three for Aβ42. Most importantly, the docking results were challenged with molecular dynamic studies of 100 ns long simulation in triplicates (100 ns × 3 = 300 ns) and were analyzed for RMSD, RMSF, RoG, H-bond number and distance, SASA, and secondary structure assessment performed together with principal component analysis and the free-energy landscape diagram, which indicated that the ChAT-Aβ complex system was stable throughout the simulation time period with no abrupt motion during the evolution of the simulation across the triplicates, which also validated the robustness of the simulation study. Finally, the free-energy landscape analysis confirmed the docking results and demonstrated that the ChAT-Aβ complexes were energetically stable despite the unstructured nature of C- and N-terminals in Aβ peptides. Overall, this study supports the reported in vitro findings that Aβ peptides, particularly Aβ42, act as endogenous ChAT-Potentiating-Ligand (CPL), and thereby supports the hypothesis that one of the native biological functions of Aβ peptides is the regulation of acetylcholine homeostasis. [ABSTRACT FROM AUTHOR]

The neuropathological substrate of dementia with Lewy bodies (DLB) is defined by the inextricable cross-seeding accretion of amyloid-β (Aβ) and α-synuclein (α-syn)-laden deposits in cholinergic neurons. The recent revelation that neuropeptide kisspeptin-10 (KP-10) is able to mitigate Aβ toxicity via an extracellular binding mechanism may provide a new horizon for innovative drug design endeavors. Considering the sequence similarities between α-syn's non-amyloid-β component (NAC) and Aβ's C-terminus, we hypothesized that KP-10 would enhance cholinergic neuronal resistance against α-syn's deleterious consequences through preferential binding. Here, human cholinergic SH-SY5Y cells were transiently transformed to upsurge the mRNA expression of α-syn while α-syn-mediated cholinergic toxicity was quantified utilizing a standardized viability-based assay. Remarkably, the E46K mutant α-syn displayed elevated α-syn mRNA levels, which subsequently induced more cellular toxicity compared with the wild-type α-syn in choline acetyltransferase (ChAT)-positive cholinergic neurons. Treatment with a high concentration of KP-10 (10 µM) further decreased cholinergic cell viability, while low concentrations of KP-10 (0.01–1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated toxicity. Correlating with the in vitro observations are approximations from in silico algorithms, which inferred that KP-10 binds favorably to the C-terminal residues of wild-type and E46K mutant α-syn with CDOCKER energy scores of −118.049 kcal/mol and −114.869 kcal/mol, respectively. Over the course of 50 ns simulation time, explicit-solvent molecular dynamics conjointly revealed that the docked complexes were relatively stable despite small-scale fluctuations upon assembly. Taken together, our findings insinuate that KP-10 may serve as a novel therapeutic scaffold with far-reaching implications for the conceptualization of α-syn-based treatments. [ABSTRACT FROM AUTHOR]

Background: Basal forebrain cholinergic neurons are dependent on nerve growth factor (NGF) for growth and survival and these cells are among the first to degenerate in Alzheimer's disease (AD). Targeted delivery of NGF has been suggested as a potential therapy for AD. This hypothesis was tested in a clinical trial with encapsulated cell biodelivery of NGF (NGF-ECB) in AD patients. Three of six patients showed improved biomarkers for cognition by the end of the study. Here, we report on the effects of targeted delivery of NGF on human resting EEG. Materials and methods: NGF-ECB implants were implanted bilaterally in the basal forebrain of six AD patients for 12 months. EEG recordings and quantitative analysis were performed at baseline, 3 and 12 months of NGF delivery, and analyzed for correlation with changes in Mini-mental state examination (MMSE) and levels of the cholinergic marker choline acetyltransferase (ChAT) in cerebrospinal fluid (CSF). Results: We found significant correlations between the topographic variance of EEG spectral power at the three study points (baseline, 3 and 12 months) and changes in MMSE and CSF ChAT. This possible effect of NGF was identified in a narrow window of alpha frequency 10–11.5 Hz, where a stabilization in MMSE score during treatment was related to an increase in EEG alpha power. A similar relation was observed between the alpha power and ChAT. More theta power at 6.5 Hz was on the contrary associated with a decrease in CSF ChAT during the trial period. Conclusion: In this exploratory study, there was a positive correlative pattern between physiological high-frequency alpha activity and stabilization in MMSE and increase in CSF ChAT in AD patients receiving targeted delivery of NGF to the cholinergic basal forebrain. [ABSTRACT FROM AUTHOR]

BackgroundBasal forebrain cholinergic neurons are dependent on nerve growth factor (NGF) for growth and survival and these cells are among the first to degenerate in Alzheimer’s disease (AD). Targeted delivery of NGF has been suggested as a potential therapy for AD. This hypothesis was tested in a clinical trial with encapsulated cell biodelivery of NGF (NGF-ECB) in AD patients. Three of six patients showed improved biomarkers for cognition by the end of the study. Here, we report on the effects of targeted delivery of NGF on human resting EEG.Materials and methodsNGF-ECB implants were implanted bilaterally in the basal forebrain of six AD patients for 12 months. EEG recordings and quantitative analysis were performed at baseline, 3 and 12 months of NGF delivery, and analyzed for correlation with changes in Mini-mental state examination (MMSE) and levels of the cholinergic marker choline acetyltransferase (ChAT) in cerebrospinal fluid (CSF).ResultsWe found significant correlations between the topographic variance of EEG spectral power at the three study points (baseline, 3 and 12 months) and changes in MMSE and CSF ChAT. This possible effect of NGF was identified in a narrow window of alpha frequency 10–11.5 Hz, where a stabilization in MMSE score during treatment was related to an increase in EEG alpha power. A similar relation was observed between the alpha power and ChAT. More theta power at 6.5 Hz was on the contrary associated with a decrease in CSF ChAT during the trial period.ConclusionIn this exploratory study, there was a positive correlative pattern between physiological high-frequency alpha activity and stabilization in MMSE and increase in CSF ChAT in AD patients receiving targeted delivery of NGF to the cholinergic basal forebrain.

T lymphocytes are increasingly implicated in pain signaling. A subset of T lymphocytes, termed TChAT, express the rate-limiting enzyme for acetylcholine (ACh) production, choline acetyltransferase (ChAT), and mediate numerous physiological functions. Given that cholinergic signaling has long been known to modulate pain processing and is the basis for several analgesics used clinically, we asked whether TChAT could be the intersection between T lymphocyte and cholinergic mediation of pain signaling. In this study, we used a mouse gene knockout strategy to ablate ChAT specifically from T lymphocytes and examined the development and expression of mechanical and thermal hypersensitivity in a spared nerve injury (SNI) mouse model of neuropathic pain. We found that mice with ChAT knockout in T cells (floxed Chat plus CD4-Cre recombinase) did not differ from control mice with intact ChAT (floxed Chat, but no Cre recombinase) in their expression of mechanical sensitivity before or after injury. Similarly, thermal sensitivity was unaffected after injury, with control mice expressing similar patterns of thermal preference to mice whose T cells do not express ChAT. Our experiments demonstrate that cholinergic signaling initiated by T lymphocytes neither dampens nor exacerbates the expression of mechanical or thermal sensitivity in neuropathic mice. Thus, while both cholinergic signaling and T lymphocytes have established roles in modulating pain phenotypes, it is not cholinergic signaling initiated by T lymphocytes that drive this. Our findings will help to narrow in on which aspects of T-cell modulation may prove useful as therapies. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker l-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension. [ABSTRACT FROM AUTHOR]

Accumulated data have evidenced that brain cholinergic circuits play a crucial role in learning and memory; however, our knowledge about the participation of neocortical and hippocampal cholinergic systems in spatial learning needs to be refined. The aim of this study was to evaluate the association of the activity of membrane-bound and soluble choline acetyltransferase (ChAT) in the synaptosomal sub-fractions of the neocortex and hippocampus with performance of the spatial navigation task in the Morris water maze at different temporal stages of memory trace formation. To identify distinct stages of memory formation, rats were trained using a 5-day protocol with four trials per day. The mean escape latency for each trial was collected, and the entire dataset was subjected to principal component analysis. Based on the Morris water maze protocol, there were three relatively distinct stages of memory formation: days 1–2, day 3, and days 4–5. The remotely stored memory trace tested in repeated and reversal learning beginning on day 19 (14 days after the end of initial learning) was associated at the individual level mainly with performance during the second trial on day 21 (the third day or repeated or reversal learning). The ChAT activity data suggest the participation of cortical cholinergic projections mainly in the first stage of spatial learning (automatic sensory processing) and the involvement of hippocampal interneurons in the second stage (error-corrected learning). Cholinergic cortical interneurons participated mainly in the stage of asymptotic performance (days 4–5). It is advisable to evaluate other signalling pathways at the identified stages of memory formation. [ABSTRACT FROM AUTHOR]

Accumulated data have evidenced that brain cholinergic circuits play a crucial role in learning and memory; however, our knowledge about the participation of neocortical and hippocampal cholinergic systems in spatial learning needs to be refined. The aim of this study was to evaluate the association of the activity of membrane-bound and soluble choline acetyltransferase (ChAT) in the synaptosomal sub-fractions of the neocortex and hippocampus with performance of the spatial navigation task in the Morris water maze at different temporal stages of memory trace formation. To identify distinct stages of memory formation, rats were trained using a 5-day protocol with four trials per day. The mean escape latency for each trial was collected, and the entire dataset was subjected to principal component analysis. Based on the Morris water maze protocol, there were three relatively distinct stages of memory formation: days 1–2, day 3, and days 4–5. The remotely stored memory trace tested in repeated and reversal learning beginning on day 19 (14 days after the end of initial learning) was associated at the individual level mainly with performance during the second trial on day 21 (the third day or repeated or reversal learning). The ChAT activity data suggest the participation of cortical cholinergic projections mainly in the first stage of spatial learning (automatic sensory processing) and the involvement of hippocampal interneurons in the second stage (error-corrected learning). Cholinergic cortical interneurons participated mainly in the stage of asymptotic performance (days 4–5). It is advisable to evaluate other signalling pathways at the identified stages of memory formation.

Growth arrest-specific 5 (GAS5) is an anti-oncogene that has been extensively studied in tumors. However, research on GAS5 in the context of nervous system disease is rare at present. This study aimed to investigate the role of the long non-coding RNA GAS5 in rat pheochromocytoma cells (PC12 cells). GAS5-overexpressing lentivirus was transfected into PC12 cells, and expression levels of GAS5 and C-myc were detected by real-time PCR. Ratios of cells in S phase were detected by 5-ethynyl-2′-deoxyuridine. Immunohistochemical staining was used to detect the immunoreactivity of neuron microtubule markers Tuj1, doublecortin, and microtubule-associated protein 2. Apoptosis was detected by flow cytometry, while expression of acetylcholine in cells was detected by western blot assay. We found that GAS5 can promote PC12 cells to differentiate into Tuj1-positive neuron-like cells with longer processes. In addition, cell proliferation and cell cycle were significantly suppressed by GAS5, whereas it had no effect on apoptosis of PC12 cells. Our results indicate that GAS5 could increase the expression of choline acetyltransferase and acetylcholine release. Thus, we speculate that GAS5 is beneficial to the recovery of neurons and the cholinergic nervous system.

Aim: A monoterpene and bioactive component of the plant Rhodiola rosea (R. rosea), rosiridin has beneficial effects on the human central nervous system and enhances brain function. The goal of this scientific study was to determine if rosiridin might shield rats from neurocognitive problems induced by scopolamine. Methods: To track the potential toxicities in rats, the acute toxicity in rats was clarified. Rosiridin at a dose of 10 mg/kg was tested in rats for 14 days. At the conclusion of the investigation, behavioral parameters that were used to identify the rats’ cognitive and motor abilities were evaluated. Several biochemical parameters were estimated using the prepared homogenate, including acetylcholine esterase (AChE), choline acetyltransferase (ChAT), radical scavengers produced by the body (Catalase-CAT, superoxide dismutase-SOD, and reduced glutathione-GSH), indicators of oxidative and nitrative burnout, pro-inflammatory (Interleukins- IL-1β, IL-6, interferon gamma IFN-ꝩ, and tumor necrosis factor-TNF-α), and cell apoptosis caspases 3 and 9. Results and Conclusion: A significant behavioral parameter restoration was seen in the rosiridin-treated group, including reduction in latency time during acquisition and retention trial in the Morris water maze test, and percentage of spontaneous alterations in the y-maze test, when compared to the disease control group that received scopolamine; rosiridin also altered the oxidative stress and neuroinflammatory markers, as well as restoring Ach and ChAT activities and normalizing GSH, SOD, MDA, TNF-α, nitrate, IL-1β, IL-6, IFN-ꝩ, caspases 3 and 9 levels. The results imply that rosiridin limits the effect of scopolamine on rat cognitive function.

The basal forebrain cholinergic system is involved in cognitive processes, but the role of the basal forebrain cholinergic system in depression is unknown. We investigated whether a lesion of cholinergic neurons in the horizontal limb of the diagonal band of Broca (HDB) produces depressive-like behavior and whether fluoxetine or ketamine inhibits such depressive-like behaviors. Here, in rats, we used 192 IgG-saporin to eliminate the cholinergic neurons of the HDB and evaluated depressive-like behaviors using a preference test for sucrose solution and the forced swimming test. Fourteen days after the injection of 192 IgG-saporin into the HDB, the rats exhibited a significantly fewer number of choline acetyltransferase positive cell density in HDB, accompanied with neuronal loss in the entire hippocampus. Meanwhile, these rats significantly reduced preference for sucrose solution, increased immobility time in the forced swimming test, reduced locomotor activity, decreased context dependent memory in fear conditioning and the time spent in the open arms of the plus-maze. A single dose of ketamine (10 mg/kg) increased the sucrose solution consumption, reduced the immobility time in the forced swim test (FST), and increased locomotor activity compared to vehicle-treated rats. Moreover, in rats that were continuously treated with fluoxetine (10 mg/kg/day for 11 days), the sucrose solution consumption increased, the immobility time in the FST decreased, and locomotor activity increased compared to vehicle-treated rats. The present results demonstrate that a lesion of HDB cholinergic neurons results in depressive-like and anxiety-like behaviors and that antidepressants such as fluoxetine or ketamine, can reverse these depressive-like behaviors but not anxiety-like behaviors, and suggest that a lesion of HDB cholinergic neurons and followed hippocampus damage may be involved in the pathogenesis of depression.

Binge drinking and alcohol abuse are common during adolescence and cause both cognitive deficits and lasting cholinergic pathology in the adult basal forebrain. Acetylcholine is anti-inflammatory and studies using the preclinical adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2 day on/2 day off from postnatal day [P]25 to P54) model of human adolescent binge drinking report decreased basal forebrain cholinergic neurons (BFCNs) and induction of proinflammatory genes that persist long into adulthood. Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying mechanisms contributing to the persistent loss of BFCNs are unknown. We report that treatment with the cholinesterase inhibitor galantamine (4.0 mg/kg, i.p.) administered during AIE (i.e., P25–P54) or following the conclusion of AIE (i.e., P57–P72) recovered the persistent loss of cholinergic neuron phenotype markers (i.e., ChAT, TrkA, and p75NTR) and somal shrinkage of residual ChAT + neurons known to persist in AIE-exposed adults. Galantamine treatment also recovered the AIE-increased expression of the proinflammatory receptors TLR4 and RAGE, the endogenous TLR4/RAGE agonist HMGB1, and the transcription activation marker pNF-κB p65. Interestingly, we find BFCNs express TLR4 and RAGE, and that AIE treatment increased pNF-κB p65 expression in adult ChAT + IR neurons, consistent with intracellular HMGB1-TLR4/RAGE signaling within BFCNs. AIE increased epigenetic transcription silencing markers (i.e., H3K9me2 and H3K9me3) in the adult basal forebrain and H3K9me2 occupancy at cholinergic phenotype gene promoters (i.e., ChAT and TrkA). The finding of no AIE-induced changes in total basal forebrain NeuN + neurons with galantamine reversal of AIE-induced ChAT + neuron loss, TLR4/RAGE-pNF-κB p65 signals, and epigenetic transcription silencing markers suggests that AIE does not cause cell death, but rather the loss of the cholinergic phenotype. Together, these data suggest that AIE induces HMGB1-TLR4/RAGE-pNF-κB p65 signals, causing the loss of cholinergic phenotype (i.e., ChAT, TrkA, and p75NTR) through epigenetic histone transcription silencing that result in the loss of the BFCN phenotype that can be prevented and restored by galantamine.

Binge drinking and alcohol abuse are common during adolescence and cause both cognitive deficits and lasting cholinergic pathology in the adult basal forebrain. Acetylcholine is anti-inflammatory and studies using the preclinical adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2 day on/2 day off from postnatal day [P]25 to P54) model of human adolescent binge drinking report decreased basal forebrain cholinergic neurons (BFCNs) and induction of proinflammatory genes that persist long into adulthood. Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying mechanisms contributing to the persistent loss of BFCNs are unknown. We report that treatment with the cholinesterase inhibitor galantamine (4.0 mg/kg, i.p.) administered during AIE (i.e., P25–P54) or following the conclusion of AIE (i.e., P57–P72) recovered the persistent loss of cholinergic neuron phenotype markers (i.e., ChAT, TrkA, and p75NTR) and somal shrinkage of residual ChAT + neurons known to persist in AIE-exposed adults. Galantamine treatment also recovered the AIE-increased expression of the proinflammatory receptors TLR4 and RAGE, the endogenous TLR4/RAGE agonist HMGB1, and the transcription activation marker pNF-κB p65. Interestingly, we find BFCNs express TLR4 and RAGE, and that AIE treatment increased pNF-κB p65 expression in adult ChAT + IR neurons, consistent with intracellular HMGB1-TLR4/RAGE signaling within BFCNs. AIE increased epigenetic transcription silencing markers (i.e., H3K9me2 and H3K9me3) in the adult basal forebrain and H3K9me2 occupancy at cholinergic phenotype gene promoters (i.e., ChAT and TrkA). The finding of no AIE-induced changes in total basal forebrain NeuN + neurons with galantamine reversal of AIE-induced ChAT + neuron loss, TLR4/RAGE-pNF-κB p65 signals, and epigenetic transcription silencing markers suggests that AIE does not cause cell death, but rather the loss of the cholinergic phenotype. Together, these data suggest that AIE induces HMGB1-TLR4/RAGE-pNF-κB p65 signals, causing the loss of cholinergic phenotype (i.e., ChAT, TrkA, and p75NTR) through epigenetic histone transcription silencing that result in the loss of the BFCN phenotype that can be prevented and restored by galantamine. [ABSTRACT FROM AUTHOR]

The basal forebrain cholinergic system is involved in cognitive processes, but the role of the basal forebrain cholinergic system in depression is unknown. We investigated whether a lesion of cholinergic neurons in the horizontal limb of the diagonal band of Broca (HDB) produces depressive-like behavior and whether fluoxetine or ketamine inhibits such depressive-like behaviors. Here, in rats, we used 192 IgG-saporin to eliminate the cholinergic neurons of the HDB and evaluated depressive-like behaviors using a preference test for sucrose solution and the forced swimming test. Fourteen days after the injection of 192 IgG-saporin into the HDB, the rats exhibited a significantly fewer number of choline acetyltransferase positive cell density in HDB, accompanied with neuronal loss in the entire hippocampus. Meanwhile, these rats significantly reduced preference for sucrose solution, increased immobility time in the forced swimming test, reduced locomotor activity, decreased context dependent memory in fear conditioning and the time spent in the open arms of the plus-maze. A single dose of ketamine (10 mg/kg) increased the sucrose solution consumption, reduced the immobility time in the forced swim test (FST), and increased locomotor activity compared to vehicle-treated rats. Moreover, in rats that were continuously treated with fluoxetine (10 mg/kg/day for 11 days), the sucrose solution consumption increased, the immobility time in the FST decreased, and locomotor activity increased compared to vehicle-treated rats. The present results demonstrate that a lesion of HDB cholinergic neurons results in depressive-like and anxiety-like behaviors and that antidepressants such as fluoxetine or ketamine, can reverse these depressive-like behaviors but not anxiety-like behaviors, and suggest that a lesion of HDB cholinergic neurons and followed hippocampus damage may be involved in the pathogenesis of depression. [ABSTRACT FROM AUTHOR]

The cholinergic system is present in both bacteria and mammals and regulates inflammation during bacterial respiratory infections through neuronal and non-neuronal production of acetylcholine (ACh) and its receptors. However, the presence of this system during the immunopathogenesis of pulmonary tuberculosis (TB) in vivo and in its causative agent Mycobacterium tuberculosis (Mtb) has not been studied. Therefore, we used an experimental model of progressive pulmonary TB in BALB/c mice to quantify pulmonary ACh using high-performance liquid chromatography during the course of the disease. In addition, we performed immunohistochemistry in lung tissue to determine the cellular expression of cholinergic system components, and then administered nicotinic receptor (nAChR) antagonists to validate their effect on lung bacterial burden, inflammation, and pro-inflammatory cytokines. Finally, we subjected Mtb cultures to colorimetric analysis to reveal the production of ACh and the effect of ACh and nAChR antagonists on Mtb growth. Our results show high concentrations of ACh and expression of its synthesizing enzyme choline acetyltransferase (ChAT) during early infection in lung epithelial cells and macrophages. During late progressive TB, lung ACh upregulation was even higher and coincided with ChAT and α7 nAChR subunit expression in immune cells. Moreover, the administration of nAChR antagonists increased pro-inflammatory cytokines, reduced bacillary loads and synergized with antibiotic therapy in multidrug resistant TB. Finally, in vitro studies revealed that the bacteria is capable of producing nanomolar concentrations of ACh in liquid culture. In addition, the administration of ACh and nicotinic antagonists to Mtb cultures induced or inhibited bacterial proliferation, respectively. These results suggest that Mtb possesses a cholinergic system and upregulates the lung non-neuronal cholinergic system, particularly during late progressive TB. The upregulation of the cholinergic system during infection could aid both bacterial growth and immunomodulation within the lung to favor disease progression. Furthermore, the therapeutic efficacy of modulating this system suggests that it could be a target for treating the disease. [ABSTRACT FROM AUTHOR]

Cardiac reflexes originating from sensory receptors in the heart ensure blood supply to vital tissues and organs in the face of constantly changing demands. Atrial volume receptors are mechanically sensitive vagal afferents which relay to the medulla and hypothalamus, affecting vasopressin release and renal sympathetic activity. To date, two anatomically distinct sensory endings have been identified which may subserve cardiac mechanosensation: end‐nets and flower‐spray endings. To map the distribution of atrial receptors in the subendocardial space, we have double‐labelled rat right atrial whole mounts for neurofilament heavy chain (NFH) and synaptic vesicle protein 2 (SV2) and generated high‐resolution maps of the rat subendocardial neural plexus at the cavo‐atrial region. In order to elucidate the nature of these fibres, double labelling with synaptophysin (SYN) and either NFH, calcitonin gene‐related peptide (CGRP), choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) was performed. The findings show that subendocardial nerve nets are denser at the superior cavo‐atrial junction than the mid‐atrial region. Adluminal plexuses had the finest diameters and stained positively for synaptic vesicles (SV2 and SYN), CGRP and TH. These plexuses may represent sympathetic post‐ganglionic fibres and/or sensory afferents. The latter are candidate substrates for type B volume receptors which are excited by stretch during atrial filling. Deeper nerve fibres appeared coarser and may be cholinergic (positive staining for ChAT). Flower‐spray endings were never observed using immunohistochemistry but were delineated clearly with the intravital stain methylene blue. We suggest that differing nerve fibre structures form the basis by which atrial deformation and hence atrial filling is reflected to the brain. [ABSTRACT FROM AUTHOR]

The inferior colliculus processes nearly all ascending auditory information. Most collicular cells respond to sound, and for a majority of these cells, the responses can be modulated by acetylcholine (ACh). The cholinergic effects are varied and, for the most part, the underlying mechanisms are unknown. The major source of cholinergic input to the inferior colliculus is the pedunculopontine tegmental nucleus (PPT), part of the pontomesencephalic tegmentum known for projections to the thalamus and roles in arousal and the sleep-wake cycle. Characterization of PPT inputs to the inferior colliculus has been complicated by the mixed neurotransmitter population within the PPT. Using selective viral-tract tracing techniques in a ChAT-Cre Long Evans rat, the present study characterizes the distribution and targets of cholinergic projections from PPT to the inferior colliculus. Following the deposit of viral vector in one PPT, cholinergic axons studded with boutons were present bilaterally in the inferior colliculus, with the greater density of axons and boutons ipsilateral to the injection site. On both sides, cholinergic axons were present throughout the inferior colliculus, distributing boutons to the central nucleus, lateral cortex, and dorsal cortex. In each inferior colliculus (IC) subdivision, the cholinergic PPT axons appear to contact both GABAergic and glutamatergic neurons. These findings suggest cholinergic projections from the PPT have a widespread influence over the IC, likely affecting many aspects of midbrain auditory processing. Moreover, the effects are likely to be mediated by direct cholinergic actions on both excitatory and inhibitory circuits in the inferior colliculus.

Aim: This study aimed to analyze the neuroprotective effect of Ocimum sanctum Linn. ethanolic extract (OSE) on human embryonic kidney-293 (HEK-293) cells as the in vitro model of neurodegenerative diseases. Materials and Methods: In this research, HEK-293 cells divided into five groups consisting of normal and healthy cells (NT), cells treated with Camptothecin 500 μM as the negative control, cells treated with trimethyltin 10 μM (TMT), cells treated with OSE 75 μg/ml, and cells pre-treated with OSE 75 μg/ml then induced by TMT 10 μM (OSE+TMT). MTT assay and phase contrast microscopy were applied to observe the cell viability quantitatively and morphological after Ocimum sanctum Linn extract treatment. Finally, the reverse transcription polymerase chain reaction was employed to study the expression of choline acetyltransferase (ChAT). Results: The MTT assay and phase contrast microscopy showed that OSE pre-treatment significantly increased the viability of TMT-induced apoptotic cells and maintained cell viability of the normal HEK-293 cells. Expression of ChAT markedly reduced on TMT treatment group, but OSE administration stabilized ChAT expression in TMT-induced HEK-293 cells. Conclusion: This present study proved that OSE administration has neuroprotective effect by increased HEK-293 cells viability and maintain ChAT expression.

Objective: The present study investigates the effect of Tiliacora triandra leaf extract on spatial learning, memory, and learning flexibility as well as hippocampal choline acetyltransferase (ChAT) activity in mice. Materials and Methods: Thirty male ICR mice were randomly divided into three groups including 10% Tween 80, T. triandra 300 mg/kg and T. triandra 600 mg/kg. All administrations were done orally for 18 consecutive days. Spatial learning, memory and learning flexibility were assessed using the Morris water maze. ChAT activity and hippocampal neuronal cell number were assessed by immunohistochemistry and histological methods, respectively. Results: The results demonstrated that T. triandra leaf extract (300 and 600 mg/kg) significantly enhances spatial learning and learning flexibility. Only 300 mg/kg of T. triandra significantly improved the spatial memory. The hippocampal ChAT activity and total hippocampal cell number were significantly increased in T. triandra-treated groups. Conclusion: The present study indicated that T. triandra leaf extract improves the spatial learning, memory and learning flexibility, exerts neuroprotective effects on hippocampal neurons and maintains ChAT activity in this brain area.

Abstract Background Cholinergic hypothesis of Alzheimer’s disease (AD) is based on the findings that a reduced and/or perturbed cholinergic activity in the central nervous system correlates with cognitive decline in patients with Alzheimer’s disease. The hypothesis resulted in the development of centrally-acting agents potentiating cholinergic neurotransmission; these drugs, however, only slowed down the cognitive decline and could not prevent it. Consequently, the perturbation of the central cholinergic signalling has been accepted as a part of the Alzheimer’s aetiology but not necessarily the primary cause of the disease. In the present study we have focused on the rs3810950 polymorphism of ChAT (choline acetyltransferase) gene that has not been studied in Czech population before. Methods We carried out an association study to test for a relationship between the rs3810950 polymorphism and Alzheimer’s disease in a group of 1186 persons; 759 patients with Alzheimer’s disease and 427 control subjects. Furthermore, we performed molecular modelling of the terminal domain (1st-126th amino acid residue) of one of the ChAT isoforms (M) to visualise in silico whether the rs3810950 polymorphism (A120T) can change any features of the tertiary structure of the protein which would have a potential to alter its function. Results The AA genotype of CHAT was associated with a 1.25 times higher risk of AD (p

Our previous study revealed that early application of electrical field stimulation (EFS) with the anode at the lesion and the cathode distal to the lesion reduced injury potential, inhibited secondary injury and was neuroprotective in the dorsal corticospinal tract after spinal cord injury (SCI). The objective of this study was to further evaluate the effect of EFS on protection of anterior horn motoneurons and their target musculature after SCI and its mechanism. Rats were randomized into three equal groups. The EFS group received EFS for 30 minutes immediately after injury at T10. SCI group rats were only subjected to SCI and sham group rats were only subjected to laminectomy. Luxol fast blue staining demonstrated that spinal cord tissue in the injury center was better protected; cross-sectional area and perimeter of injured tissue were significantly smaller in the EFS group than in the SCI group. Immunofluorescence and transmission electron microscopy showed that the number of spinal cord anterior horn motoneurons was greater and the number of abnormal neurons reduced in the EFS group compared with the SCI group. Wet weight and cross-sectional area of vastus lateralis muscles were smaller in the SCI group to in the sham group. However, EFS improved muscle atrophy and behavioral examination showed that EFS significantly increased the angle in the inclined plane test and Tarlov’s motor grading score. The above results confirm that early EFS can effectively impede spinal cord anterior horn motoneuron loss, promote motor function recovery and reduce muscle atrophy in rats after SCI.