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Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high‐affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis., Acetylcholine (ACh) is synthesized from choline. In cholinergic neurons, choline uptake through high‐affinity choline transporters was considered to be the only pathway for ACh synthesis. In the present study, we report that P2X2 receptors function in a novel pathway of choline transport for ACh synthesis.

Genes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, the cholinergic gene expression in a specific subset of neurons is unknown in insects except for Drosophila melanogaster. In this study, we analysed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. We found multiple enhancer regions that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. We found that dibutyryl-cAMP, an analogue of cAMP, influences the expression of ChAT in B. mori. Tissue-specific expression analysis of transcriptional factors identified potential candidates that control the cholinergic gene locus expression. Our investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect.

Background X-linked dystonia parkinsonism is a generalized, progressive dystonia followed by parkinsonism with onset in adulthood and accompanied by striatal neurodegeneration. Causative mutations are located in a noncoding region of the TATA-box binding protein-associated factor 1 (TAF1) gene and result in aberrant splicing. There are 2 major TAF1 isoforms that may be decreased in symptomatic patients, including the ubiquitously expressed canonical cTAF1 and the neuronal-specific nTAF1. Objective The objective of this study was to determine the behavioral and transcriptomic effects of decreased cTAF1 and/or nTAF1 in vivo. Methods We generated adeno-associated viral (AAV) vectors encoding microRNAs targeting Taf1 in a splice-isoform selective manner. We performed intracerebroventricular viral injections in newborn mice and rats and intrastriatal infusions in 3-week-old rats. The effects of Taf1 knockdown were assayed at 4 months of age with evaluation of motor function, histology, and RNA sequencing of the striatum, followed by its validation. Results We report motor deficits in all cohorts, more pronounced in animals injected at P0, in which we also identified transcriptomic alterations in multiple neuronal pathways, including the cholinergic synapse. In both species, we show a reduced number of striatal cholinergic interneurons and their marker mRNAs after Taf1 knockdown in the newborn. Conclusion This study provides novel information regarding the requirement for TAF1 in the postnatal maintenance of striatal cholinergic neurons, the dysfunction of which is involved in other inherited forms of dystonia. © 2021 International Parkinson and Movement Disorder Society.

The basal forebrain cholinergic neurons (BFCN) provide the primary source of cholinergic innervation of the human cerebral cortex. They are involved in the cognitive processes of learning, memory, and attention. These neurons are differentially vulnerable in various neuropathologic entities that cause dementia. This review summarizes the relevance to BFCN of neuropathologic markers associated with dementias, including the plaques and tangles of Alzheimer's disease (AD), the Lewy bodies of diffuse Lewy body disease, the tauopathy of frontotemporal lobar degeneration (FTLD-TAU) and the TDP-43 proteinopathy of FTLD-TDP. Each of these proteinopathies has a different relationship to BFCN and their corticofugal axons. Available evidence points to early and substantial degeneration of the BFCN in AD and diffuse Lewy body disease. In AD, the major neurodegenerative correlate is accumulation of phosphotau in neurofibrillary tangles. However, these neurons are less vulnerable to the tauopathy of FTLD. An intriguing finding is that the intracellular tau of AD causes destruction of the BFCN, whereas that of FTLD does not. This observation has profound implications for exploring the impact of different species of tauopathy on neuronal survival. The proteinopathy of FTLD-TDP shows virtually no abnormal inclusions within the BFCN. Thus, the BFCN are highly vulnerable to the neurodegenerative effects of tauopathy in AD, resilient to the neurodegenerative effect of tauopathy in FTLD and apparently resistant to the emergence of proteinopathy in FTLD-TDP and perhaps also in Pick's disease. Investigations are beginning to shed light on the potential mechanisms of this differential vulnerability and their implications for therapeutic intervention.

Background In movement disorders such as Parkinson's disease (PD), cholinergic signaling is disrupted by the loss of basal forebrain cholinergic neurons, as well as aberrant activity in striatal cholinergic interneurons (ChIs). Several lines of evidence suggest that gait imbalance, a key disabling symptom of PD, may be driven by alterations in high-level frontal cortical and cortico-striatal processing more typically associated with cognitive dysfunction. Methods Here we describe the corticostriatal circuitry that mediates the cognitive-motor interactions underlying such complex movement control. The ability to navigate dynamic, obstacle-rich environments requires the continuous integration of information about the environment with movement selection and sequencing. The cortical-attentional processing of extero- and interoceptive cues requires modulation by cholinergic activity to guide striatal movement control. Cue-derived information is "transferred" to striatal circuitry primarily via fronto-striatal glutamatergic projections. Result Evidence from parkinsonian fallers and from a rodent model reproducing the dual cholinergic-dopaminergic losses observed in these patients supports the main hypotheses derived from this neuronal circuitry-guided conceptualization of parkinsonian falls. Furthermore, in the striatum, ChIs constitute a particularly critical node for the integration of cortical with midbrain dopaminergic afferents and thus for cues to control movements. Conclusion Procholinergic treatments that enhance or rescue cortical and striatal mechanisms may improve complex movement control in parkinsonian fallers and perhaps also in older persons suffering from gait disorders and a propensity for falls. © 2021 International Parkinson and Movement Disorder Society.

The Nucleus Basalis of Meynert (NBM) is the main source of cholinergic neurons in the basal forebrain to be crucially involved in cognitive functions and whose degeneration correlates with cognitive decline in major degenerative pathologies as Alzheimer's and Parkinson's diseases. However, knowledge concerning NBM neurons derived from human brain is very limited to date. We recently characterized a primary culture of proliferating neuroblasts isolated from the human fetal NBM (hfNBM) as immature cholinergic neurons expressing the machinery to synthetize and release acetylcholine. Here we studied in detail electrophysiological features and cholinergic effects in this cell culture by patch-clamp recordings. Our data demonstrate that atropine-blocked muscarinic receptor activation by acetylcholine or carbachol enhanced IK and reduced INa currents by stimulating Gi -coupled M2 or phospholipase C-coupled M3 receptors, respectively. Inhibition of acetylcholine esterase activity by neostigmine unveiled a spontaneous acetylcholine release from hfNBM neuroblasts that might account for an autocrine/paracrine signaling during human brain development. Present data provide the first description of cholinergic effects in human NBM neurons and point to a role of acetylcholine as an autocrine/paracrine modulator of voltage-dependent channels. Our research could be of relevance in understanding the mechanisms of cholinergic system development and functions in the human brain, either in health or disease.

Cerebellar functions are modulated by cholinergic inputs, the density of which varies among cerebellar regions. Although the prepositus hypoglossi nucleus (PHN), a brainstem structure involved in controlling gaze holding, is known as one of the major sources of these cholinergic inputs, the proportions of cholinergic neurons in PHN projections to distinct cerebellar regions have not been quantitatively analyzed. In this study, we identified PHN neurons projecting to the cerebellum by applying retrograde labeling with dextran-conjugated Alexa 488 in choline acetyltransferase (ChAT)-tdTomato transgenic rats and compared the proportion of cholinergic PHN neurons in the PHN projections to four different regions of the cerebellum, namely the flocculus (FL), the uvula and nodulus (UN), lobules III-V in the vermis (VM), and the hemispheric paramedian lobule and crus 2 (PC). In the PHN, the percentage of cholinergic PHN neurons was lower in the projection to the FL than in the projection to the UN, VM or PC. Preposito-cerebellar neurons, except for preposito-FL neurons, included different proportions of cholinergic neurons at different rostrocaudal positions in the PHN. These results suggest that cholinergic PHN neurons project to not only the vestibulocerebellum but also the anterior vermis and posterior hemisphere and that the proportion of cholinergic neurons among projection neurons from the PHN differs depending on cerebellar target areas and the rostro-caudal regions of the PHN. This study provides insights regarding the involvement of cerebellar cholinergic networks in gaze holding.

Tau accumulation and cholinergic impairment are characteristic pathologies in Alzheimer's disease (AD). However, the causal role of tau accumulation in cholinergic lesion is elusive. Here, we observed an aberrant tau accumulation in the medial septum (MS) of 3xTg and 5xFAD mice, especially in their cholinergic neurons. Overexpressing hTau in mouse MS (MShTau) for 6 months but not 3 months induced spatial memory impairment without changing object recognition and anxiety‐like behavior, indicating a specific and time‐dependent effect of MS‐hTau accumulation on spatial cognitive functions. With increasing hTau accumulation, the MShTau mice showed a time‐dependent cholinergic neuron loss with reduced cholinergic projections to the hippocampus. Intraperitoneal administration of donepezil, a cholinesterase inhibitor, for 1 month ameliorated the MS‐hTau‐induced spatial memory deficits with preservation of MS–hippocampal cholinergic pathway and removal of tau load; and the beneficial effects of donepezil was more prominent at low dose. Proteomics revealed that MS‐hTau accumulation deregulated multiple signaling pathways with numerous differentially expressed proteins (DEPs). Among them, the vacuolar protein sorting‐associated protein 37D (VP37D), an autophagy‐related protein, was significantly reduced in MShTau mice; the reduction of VP37D was restored by donepezil, and the effect was more significant at low dose than high dose. These novel evidences reveal a causal role of tau accumulation in linking MS cholinergic lesion to hippocampus‐dependent spatial cognitive damages as seen in the AD patients, and the new tau‐removal and autophagy‐promoting effects of donepezil may extend its application beyond simple symptom amelioration to potential disease modification., Proposed working model: Medial septum (MS) tau accumulation disrupts MS–hippocampus cholinergic pathway and impairs hippocampus‐associated spatial memory as seen on patients with Alzheimer's disease (AD). In addition to traditional cholinesterase inhibition, low‐dose donepezil exerts novel tau‐removal and autophagy‐promoting effects, efficiently rescuing MS‐htau‐induced memory loss. Our results supplement tau toxicity and the new findings on donepezil may extend its application beyond simple symptom amelioration to potential disease modification; the latter may help physicians to optimize dosing in donepezil treatment on AD.

Background Parasympathetic nerve hypersensitivity contributes to the severity of allergic rhinitis (AR), but the precise mechanism underlying neuroimmune regulation in patients with AR remains unclear. This study investigated the effect of cholinergic nerve inhibition on AR CD4+ T-helper (Th)2-cell polarization and the underlying regulatory mechanism in vitro. Methods An in-vitro neuroimmune coculture model of D-U87 cells and CD4+ T cells was established. D-U87 cells with cholinergic neuron characteristics were used as cholinergic neuron models. CD4+ T cells were derived from peripheral blood monocytes from AR patients (n = 60) and control subjects (n = 40). Th1- and Th2-cell percentages were measured by flow cytometry. Proteins involved in related signaling pathways were analyzed by protein chip assay and Western blotting. Results The Th2-cell percentage among CD4+ T cells from AR patients was significantly increased after coculture with D-U87 cells and was decreased by ipratropium bromide (IB) treatment. In contrast, the Th1-cell percentage among control CD4+ T cells was significantly increased after coculture with D-U87 cells, but was unaltered by IB treatment. Furthermore, phosphorylated Akt (p-Akt) protein levels increased in CD4+ T cells from both controls and AR patients after coculture with D-U87 cells and decreased after IB treatment. However, higher p-Akt levels were observed in cells from AR patients than in cells from control subjects. Moreover, Akt inhibition decreased Th2-cell percentage in AR patients. Conclusion In-vitro cholinergic nerve inhibition with IB decreased AR CD4+ T-cell polarization into Th2 cells partially through an Akt-dependent mechanism.

Nerve growth factor (NGF) is an essential neurotrophic factor for the development and maintenance of the central and the peripheral nervous system. NGF deficiency in the basal forebrain precedes degeneration of basal forebrain cholinergic neurons in Alzheimer's disease, contributing to memory decline. NGF mediates neurotrophic support via its high‐affinity receptor, the tropomyosin‐related kinase A (TrkA) receptor, and mediates mitogenic and differentiation signals via the extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2). However, the molecular mechanisms underlying the different NGF/TrkA/ERK signalling pathways are far from clear. In this study, we have investigated the role of human NGF and three NGF mutants, R100E, W99A and K95A/Q96A, their ability to activate TrkA or ERK1/2, and their ability to induce proliferation or differentiation in human foetal dorsal root ganglion (DRG) neurons or in PC12 cells. We show that the R100E mutant was significantly more potent than NGF itself to induce proliferation and differentiation, and significantly more potent in activation of ERK1/2 in DRG neurons. The W99A and K95A/Q96A mutants, on the other hand, were less effective than the wild‐type protein. An unexpected finding was the high efficacy of the K95A/Q96A mutant to activate TrkA and to induce differentiation of DRG neurons at elevated concentrations. These data demonstrate an NGF mutant with improved neurotrophic properties in primary human neuronal cells. The R100E mutant represents an interesting candidate for further drug development in Alzheimer's disease and other neurodegenerative disorders., The nerve growth factor (NGF) mutant R100E is more potent to induce proliferation, differentiation (anti‐β‐tubulin green staining) and phosphorylation of ERK1/2 (red staining) than wild‐type NGF in human foetal dorsal root ganglion (DRG) neurons after a 4‐day treatment. The NGF double mutant K95A/Q96A is more efficient to induce neuronal differentiation than wild‐type NGF in DRG neurons at high concentrations (>3 μg/ml). We show enhanced functional response in human nerve cells with NGF mutants.

Down syndrome (DS), trisomy 21, is marked by intellectual disability and a premature aging profile including degeneration of the basal forebrain cholinergic neuron (BFCN) projection system, similar to what is seen in Alzheimer’s disease (AD). Although data indicate that perinatal maternal choline supplementation (MCS) alters the structure and function of these neurons in the Ts65Dn mouse model of DS and AD (Ts), how MCS affects the molecular profile of vulnerable BFCNs is unknown. We investigated the genetic signature of BFCNs obtained from Ts and disomic (2N) offspring of Ts65Dn dams maintained on a MCS diet (Ts+, 2N+) or a choline-normal diet (ND) from mating until weaning, then maintained on ND until 4.4–7.5 months of age. Brains were then collected and prepared for choline acetyltransferase (ChAT) immunohistochemistry and laser capture microdissection followed by RNA extraction and custom-designed microarray analysis. Findings revealed upregulation of select transcripts in classes of genes related to the cytoskeleton (Tubb4b), AD (Cav1), cell death (Bcl2), presynaptic (Syngr1), immediate early (Fosb, Arc), G protein signaling (Gabarap, Rgs10), and cholinergic neurotransmission (Chrnb3) in Ts compared to 2N mice, which were normalized with MCS. Moreover, significant downregulation was seen in select transcripts associated with the cytoskeleton (Dync1h1), intracellular signaling (Itpka, Gng3, Mlst8), and cell death (Ccng1) in Ts compared to 2N mice that were normalized with MCS. This study provides valuable insight into mechanisms of genotype-dependent differences and the effects of MCS at the molecular level within a key vulnerable cell type in DS and AD.

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [3 H]choline. Electrical stimulation-evoked pronounced [3 H]ACh release from cholinergic neurons. The estimated quantity of [3 H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre-synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M2 -selective antagonist AF-DX 116, partially inhibited by M3 -selective darifenacin, and minimally by M4 -selective PD 102807. Other subtype-selective antagonists had no effect at subtype-selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [3 H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre-synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [3 H]efflux was also enhanced by ACh uptake inhibitors (100 μM hemicholinium-3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre-synaptic M2 /M3 /M4 mAChRs, AChE and post-synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/.

KEY POINTS: Cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to the retrotrapezoid nucleus (RTN) are considered to be important for sleep–wake state‐dependent control of breathing. The RTN also receives cholinergic input from the postinspiratory complex. Stimulation of the PPTg increases respiratory output under control conditions but not when muscarinic receptors in the RTN are blocked. The data obtained in the present study support the possibility that arousal‐dependent modulation of breathing involves recruitment of cholinergic projections from the PPTg to the RTN. ABSTRACT: The pedunculopontine tegmental nucleus (PPTg) in the mesopontine region has important physiological functions, including breathing control. The PPTg contains a variety of cell types, including cholinergic neurons that project to the rostral aspect of the ventrolateral medulla. In addition, cholinergic signalling in the retrotrapezoid nucleus (RTN), a region that contains neurons that regulate breathing in response to changes in CO(2)/H(+), has been shown to activate chemosensitive neurons and increase inspiratory activity. The present study aimed to identify the source of cholinergic input to the RTN and determine whether cholinergic signalling in this region influences baseline breathing or the ventilatory response to CO(2) in conscious male Wistar rats. Retrograde tracer Fluoro‐Gold injected into the RTN labelled a subset of cholinergic PPTg neurons that presumably project directly to the chemosensitive region of the RTN. In unrestrained awake rats, unilateral injection of the glutamate (10 mm/100 nL) in the PPTg decreased tidal volume (V (T)) but otherwise increased respiratory rate (f (R)) and net respiratory output as indicated by an increase in ventilation (V (E)). All respiratory responses elicited by PPTg stimulation were blunted by prior injection of methyl‐atropine (5 mm/50–75 nL) into the RTN. These results show that stimulation of the PPTg can increase respiratory activity in part by cholinergic activation of chemosensitive elements of the RTN. Based on previous evidence that cholinergic PPTg projections may simultaneously activate expiratory output from the pFRG, we speculate that cholinergic signalling at the level of RTN region could also be involved in breathing regulation.

Background Constipation is commonly seen in patients with Parkinson's disease associated with a loss of dopaminergic neurons in both central and enteric nervous systems. However, the roles of enteric dopaminergic neurons in developing constipation remain to be elucidated. Here, we investigated the roles of enteric dopaminergic neurons in the generation of colonic peristalsis. Methods Cannulated segments of rat proximal colon were situated in the organ bath, abluminally perfused with physiological salt solution and luminally perfused with 0.9% saline. Drugs were applied in the abluminal solution. Changes in diameter along the length of the colonic segment were captured by a video camera and transformed into spatio-temporal maps. Fluorescence immunohistochemistry was also carried out. Key results Blockade of nitrergic neurotransmission prevented oro-aboral propagation of peristaltic waves and caused a colonic constriction without affecting ripples, non-propagating myogenic contractions. Blockade of cholinergic neurotransmission also prevented peristaltic waves but suppressed ripples with a colonic dilatation. Tetrodotoxin (0.6 μM) abolished peristaltic waves and increased ripples with a constriction. SCH 23390 (20 μM), a D1 -like dopamine receptor antagonist, slowed the peristaltic waves and caused a constriction, while GBR 12909 (1 μM), a dopamine reuptake inhibitor, diminished the peristaltic waves with a dilatation. Bath-applied dopamine (3 μM) abolished the peristaltic waves associated with a colonic dilation in an SCH 23390 (5 μM)-sensitive manner. D1 receptor immunoreactivity was co-localized to nitrergic and cholinergic neurons. Conclusions and inferences Dopaminergic neurons appear to facilitate nitrergic neurons via D1 -like receptors to stabilize asynchronous contractile activity resulting in the generation of colonic peristalsis.

To develop a PET imaging agent to visualize brain cholinergic neurons and synaptic changes caused by Alzheimer's disease, (-)- and (+)-o-[11 C]methyl-trans-decalinvesamicol ([11 C]OMDV) were isolated and investigated for differences in not only their binding affinity and selectivity to vesicular acetylcholine transporter (VAChT), but also their in vivo activities. [11 C]OMDV has a high binding affinity for VAChT both in vitro and in vivo. Racemic OMDV and o-trimethylstannyl-trans-decalinvesamicol (OTDV), which are precursors for synthesis of [11 C]OMDV, were separated into (-)-optical isomers ((-)-OMDV and (-)-OTDV) and (+)-optical isomers ((+)-OMDV and (+)-OTDV) by HPLC. In the in vitro binding assay, (-)-OMDV(7.2 nM) showed eight times higher binding affinity (Ki) to VAChT than that of (+)-OMDV(57.5 nM). In the biodistribution study, the blood-brain barrier permeability of both enantiomers ((-)-[11 C]OMDV and (+)-[11 C]OMDV) was similarly high (about 1.0%ID/g) at 2 min post-injection. However, (+)-[11 C]OMDV clearance from the brain was faster than (-)-[11 C]OMDV. In the in vivo blocking study, accumulation of (-)-[11 C]OMDV in the cortex was markedly decreased (approximately 30% of control) by coadministration of vesamicol, and brain uptake of (-)-[11 C]OMDV was not significantly altered by coadministration of (+)-pentazocine or (+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP). PET-CT imaging revealed inhibition of the rat brain uptake of (-)-[11 C]OMDV by coadministration of vesamicol. In conclusion, (-)-[11 C]OMDV, which is an enantiomer of OMDV, selectively binds to VAChT with high affinity in the rat brain in vivo. (-)-[11 C]OMDV may be utilized as a potential PET ligand for studying presynaptic cholinergic neurons in the brain.

Background Cholinergic dysfunction and oxidative stress are the crucial mechanisms of Alzheimer's disease (AD). GAPT, also called GEPT (a combination of several active components extracted from the Chinese herbs ginseng, epimedium, polygala and tuber curcumae) or Jinsiwei, is a patented Chinese herbal compound, has been clinically widely used to improve learning and memory impairment, but whether it can play a neuroprotective role by protecting cholinergic neurons and reducing oxidative stress injury remains unclear. Methods Male ICR mice were intraperitoneally injected with scopolamine (3 mg/kg) to establish a learning and memory disordered model. An LC‐MS method was established to study the chemical compounds and in vivo metabolites of GAPT. After scopolamine injection, a step‐down passive‐avoidance test (SDPA) and a Y maze test were used to estimate learning ability and cognitive function. In addition, ELISA detected the enzymatic activities of acetylcholinesterase (AChE), acetylcholine (ACh), choline acetyltransferase (ChAT), malondialdehyde (MDA), glutathione peroxidase (GPX), and total superoxide dismutase (T‐SOD). The protein expressions of AChE, ChAT, SOD1, and GPX1 were observed by western blot, and the distribution of ChAT, SOD1, and GPX1 was observed by immunohistochemical staining. Results After one‐half or 1 month of intragastric administration, GAPT can ameliorate scopolamine‐induced behavioral changes in learning and memory impaired mice. It can also decrease the activity of MDA and protein expression level of AChE, increase the activity of Ach, and increase activity and protein expression level of ChAT, SOD, and GPX in scopolamine‐treated mice. After one and a half month of intragastric administration of GAPT, echinacoside, salvianolic acid A, ginsenoside Rb1, ginsenoside Rg2, pachymic acid, and beta asarone could be absorbed into mice blood and pass through BBB. Conclusions GAPT can improve the learning and memory ability of scopolamine‐induced mice, and its mechanism may be related to protecting cholinergic neurons and reducing oxidative stress injury., GAPT can improve the learning and memory ability of scopolamine‐induced mice, and its mechanism may be related to protecting cholinergic neurons and reducing oxidative stress injury.

Background Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is commonly found in cereals ingested by humans and animals. Its ingestion is correlated with hepatic, hematologic, renal, splenic, cardiac, gastrointestinal, and neural damages, according to dose, duration of exposure and species. In this work, the effects of the ingestion of DON-contaminated diet at concentrations considered tolerable for human and animal intake were assessed. Methods Male Wistar rats aging 21 days were allotted to five groups that were given, for 42 days, diets contaminated with different concentrations of DON (0, 0.2, 0.75, 1.75, and 2 mg kg-1 of chow). Food ingestion, bodyweight, oxidative status and morphometric analyses of gliocytes, and neurons of jejunal myenteric ganglia were recorded. Key results At these concentrations, there was no food rejection, decrease in bodyweight gain, changes in oxidative status, or loss of either neurons or gliocytes. However, DON decreased gliocyte area, general neuronal population, nitrergic, cholinergic and NADH-diaphorase positive subpopulations and, as a result, ganglion area. Conclusions & inferences It was concluded that, even in the absence of visible effect, DON exposure reduces cell body area of gliocytes and neurons of the myenteric plexus of the rat jejunum.

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus that sends cholinergic, glutamatergic, and gamma-aminobutyric acid (GABA)-ergic projections to the ventral tegmental area (VTA), a key brain region associated with reward information processing and reinforcement learning, and thus, with addiction induced by drugs of abuse, including cocaine. Recent studies have revealed that the LDT, in addition to the VTA, plays important roles in the development and expression of cocaine-induced addiction and stress-induced enhancement of addictive behaviors. Additionally, neuroplasticity induced in LDT cholinergic neurons by repeated cocaine administration critically contributes to these behaviors. Elucidation of the underlying mechanisms of cocaine-induced neuroplasticity in the LDT that influences reward circuit activity may lead to the development of therapeutic strategies to treat cocaine addiction and stress-induced reinstatement of cocaine use. This review summarizes recent progress in the study of the LDT, specifically neuroplasticity in LDT cholinergic neurons induced by cocaine and its functional roles in the development and modulation of addictive behaviors associated with cocaine.

The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin‐like cell adhesion molecules (CAMs) nectin‐2α and nectin‐2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin‐2α, but not nectin‐2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage‐gated A‐type K+ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin‐2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin‐2α at this boundary was not colocalized with the nectin‐2α‐binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin‐2α forms a cell adhesion apparatus different from the Kv4.2‐associated MSs. Genetic ablation of nectin‐2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin‐2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.

Background The etiology of cervical dystonia is unknown. Cholinergic abnormalities have been identified in dystonia animal models and human imaging studies. Some animal models have cholinergic neuronal loss in the striatum and increased acetylcholinesterase activity in the pedunculopontine nucleus. Objectives The objective of this study was to determine the presence of cholinergic abnormalities in the putamen and pedunculopontine nucleus in cervical dystonia human brain donors. Methods Formalin-fixed brain tissues were obtained from 8 cervical dystonia and 7 age-matched control brains (controls). Pedunculopontine nucleus was available in only 6 cervical dystonia and 5 controls. Neurodegeneration was evaluated pathologically in the putamen, pedunculopontine nucleus, and other regions. Cholinergic neurons were detected using choline acetyltransferase immunohistochemistry in the putamen and pedunculopontine nucleus. Putaminal cholinergic neurons were quantified. A total of 6 cervical dystonia patients and 6 age-matched healthy controls underwent diffusion tensor imaging to determine if there were white matter microstructural abnormalities around the pedunculopontine nucleus. Results Decreased or absent choline acetyltransferase staining was identified in all 6 pedunculopontine nucleus samples in cervical dystonia. In contrast, strong choline acetyltransferase staining was present in 4 of 5 pedunculopontine nucleus controls. There were no differences in pedunculopontine nucleus diffusion tensor imaging between cervical dystonia and healthy controls. There was no difference in numbers of putaminal cholinergic neurons between cervical dystonia and controls. Conclusions Our findings suggest that pedunculopontine nucleus choline acetyltransferase deficiency represents a functional cholinergic deficit in cervical dystonia. Structural lesions and confounding neurodegenerative processes were excluded by absence of neuronal loss, gliosis, diffusion tensor imaging abnormalities, and beta-amyloid, tau, and alpha-synuclein pathologies. © 2018 International Parkinson and Movement Disorder Society.

Choline acetyltransferase (ChAT) expressing retinal amacrine cells are present across vertebrates. These interneurons play important roles in the development of retinal projections to the brain and in motion detection, specifically in generating direction-selective responses to moving stimuli. ChAT amacrine cells typically comprise two spatially segregated populations that form circuits in the 'ON' or 'OFF' synaptic layers of the inner retina. This stereotypic arrangement is also found across the adult human retina, with the notable exception that ChAT expression is evident in the ON but not OFF layer of the fovea, a region specialized for high-acuity vision. We thus investigated whether the human fovea exhibits a developmental path for ON and OFF ChAT cells that is retinal location-specific. Our analysis shows that at each retinal location, human ON and OFF ChAT cells differentiate, form their separate synaptic layers, and establish non-random mosaics at about the same time. However, unlike in the adult fovea, ChAT immunostaining is initially robust in both ON and OFF populations, up until at least mid-gestation. ChAT expression in the OFF layer in the fovea is therefore significantly reduced after mid-gestation. OFF ChAT cells in the human fovea and in the retinal periphery thus follow distinct maturational paths.

Objective The cholinergic deficit is thought to underlie progressed cognitive decline in Alzheimer Disease. The lineage reprogramming of somatic cells into cholinergic neurons may provide strategies toward cell-based therapy of neurodegenerative diseases. Methods and results Here, we found that a combination of neuronal transcription factors, including Ascl1, Myt1l, Brn2, Tlx3, and miR124 (5Fs) were capable of directly converting human brain vascular pericytes (HBVPs) into cholinergic neuronal cells. Intriguingly, the inducible effect screening of reprogramming factors showed that a single reprogramming factor, Myt1l, induced cells to exhibit similarly positive staining for Tuj1, MAP2, ChAT, and VAChT upon lentivirus infection with the 5Fs after 30 days. HBVP-converted neurons were rarely labeled even after long-term incubation with BrdU staining, suggesting that induced neurons were directly converted from HBVPs rather than passing through a proliferative state. In addition, the overexpression of Myt1l induced the elevation of Ascl1, Brn2, and Ngn2 levels that contributed to reprogramming. Conclusions Our findings provided proof of the principle that cholinergic neurons could be produced from HBVPs by reprogramming factor-mediated fate instruction. Myt1l was a critical mediator of induced neuron cell reprogramming. HBVPs represent another excellent alternative cell resource for cell-based therapy to treat neurodegenerative disease.

Although there are changes in gene expression and alterations in neuronal density and afferent inputs in the forebrain of trisomic mouse models of Down syndrome (DS) and Alzheimer’s disease (AD), there is a lack of systematic assessments of gene expression and encoded proteins within individual vulnerable cell populations, precluding translational investigations at the molecular and cellular level. Further, no effective treatment exists to combat intellectual disability and basal forebrain cholinergic neurodegeneration seen in DS. To further our understanding of gene expression changes before and following cholinergic degeneration in a well-established mouse model of DS/AD, the Ts65Dn mouse, we assessed RNA expression levels from CA1 pyramidal neurons at two adult ages (~6 months of age and ~11 months of age) in both Ts65Dn and their normal disomic (2N) littermates. We further examined a therapeutic intervention, maternal choline supplementation (MCS), which has been previously shown to lessen dysfunction in spatial cognition and attention, and have protective effects on the survival of basal forebrain cholinergic neurons (BFCNs) in the Ts65Dn mouse model. Results indicate that MCS normalized expression of several genes in key gene ontology categories, including synaptic plasticity, calcium signaling, and AD-associated neurodegeneration related to amyloid-beta peptide (Aβ) clearance. Specifically, normalized expression levels were found for endothelin converting enzyme-2 (Ece2), insulin degrading enzyme (Ide), Dyrk1a, and calcium/calmodulin-dependent protein kinase II (Camk2a), among other relevant genes. Single population expression profiling of vulnerable CA1 pyramidal neurons indicates that MCS is a viable therapeutic for long-term reprogramming of key transcripts involved in neuronal signaling that are dysregulated in the trisomic mouse brain which have translational potential for DS and AD.

Duchenne-like muscular dystrophy (canine dystrophinopathy) is a hereditary degenerative disease characterized by muscle changes similar to those described for Duchenne muscular dystrophy (DMD) and by alterations in the smooth muscles of the gastrointestinal tract. Some authors have suggested that these abnormalities may be associated with intestinal motility. This study analyzed the nitrergic and cholinergic neurons and P2X7 receptor expression in the myenteric plexus of the ileum and distal colon of dogs with muscular dystrophy. Immunohistochemical techniques were used to detect nitric oxide synthase (NOS) and acetylcholine transferase (ChAT) expression and to label all HuC/D- and P2X7 receptor-immunoreactive (IR) neurons. Transmission electron microscopy and basic histology were performed for further analysis. The results showed that nitrergic neurons exhibited a Dogiel type I morphology in the ileum and distal colon. The neuronal profile results showed that there were fewer NOS-, ChAT-, and HuC/D-IR neurons in the ileum than in the distal colon in the dystrophic (DT) dogs. Additionally, there were more NOS-, ChAT- and HuC/D-IR neurons per ganglion in the distal colon than in the ileum. The P2X7 receptor-expressing neurons colocalized with nitrergic and cholinergic neurons. Transmission and light microscopy revealed collagen between the muscle fibers, between the circular and longitudinal muscle layers and within the myenteric ganglia of dogs with muscular dystrophy. These findings provide a morphological description of the myenteric neurons in the ileum and distal colon of these DT dogs and may contribute to a better understanding of the gastrointestinal disorders found in patients with DMD. Anat Rec, 301:673-685, 2018. © 2017 Wiley Periodicals, Inc.

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O-acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.

Acetylcholine (ACh) plays an important role in memory function and has been implicated in aging-related dementia, in which the impairment of hippocampus-dependent learning strongly manifests. Cholinergic neurons densely innervate the hippocampus, mediating the formation of episodic as well as semantic memory. Here, we will review recent findings on acetylcholine's modulation of memory function, with a particular focus on hippocampus-dependent learning, and the circuits involved. In addition, we will discuss the complexity of ACh actions in memory function to better understand the physiological role of ACh in memory. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Alcohol has a profound effect on sleep. However, neuronal substrates mediating sleep-promoting effects of alcohol are unknown. Since the basal forebrain (BF) cholinergic neurons are implicated in the homeostatic regulation of sleep, we hypothesized that the BF cholinergic neurons may have an important role in sleepiness observed after alcohol consumption. 192-IgG-saporin (bilateral BF infusions) was used to selectively lesion BF cholinergic neurons in adult male Sprague-Dawley rats. Standard surgical procedures were used to implant sleep recording electrodes or microdialysis guide cannulas. The first experiment used between-group design [lesion and sham (controls)] and examined effects of BF cholinergic neuronal lesions on alcohol (3 g/Kg; ig) induced sleep promotion. The second experiment used within-group design [lesion (ipsilateral BF) and sham (controls; contralateral BF) in same animal] and local reverse microdialysis infusion of alcohol (300 mM) to examine the effects of cholinergic neuronal lesions on extracellular adenosine in the BF. Alcohol had a robust sleep promoting effect in controls as evidenced by a significant reduction in sleep onset latency and wakefulness; non-rapid eye movement sleep was significantly increased. No such alcohol-induced sleep promotion was observed in lesioned rats with significantly fewer BF cholinergic neurons. Rapid eye movement sleep was minimally affected. Adenosine release was significantly reduced following local infusion of alcohol on the lesion side, with significantly fewer cholinergic neurons as compared with the control side. Based on these results, we suggest that alcohol promotes sleep by increasing extracellular adenosine via its action on cholinergic neurons of the BF. Read the Editorial Highlight for this article on page 620.

The septohippocampal cholinergic neurotransmission has long been implicated in seizures, but little is known about the structural features of this projection system in epileptic brain. We evaluated the effects of experimental epilepsy on the areal density of cholinergic terminals (fiber varicosities) in the dentate gyrus. For this purpose, we used two distinct post-status epilepticus rat models, in which epilepsy was induced with injections of either kainic acid or pilocarpine. To visualize the cholinergic fibers, we used brain sections immunostained for the vesicular acetylcholine transporter. It was found that the density of cholinergic fiber varicosities was higher in epileptic rats versus control rats in the inner and outer zones of the dentate molecular layer, but it was reduced in the dentate hilus. We further evaluated the effects of kainate treatment on the total number, density, and soma volume of septal cholinergic cells, which were visualized in brain sections stained for either vesicular acetylcholine transporter or choline acetyltransferase (ChAT). Both the number of septal cells with cholinergic phenotype and their density were increased in epileptic rats when compared to control rats. The septal cells stained for vesicular acetylcholine transporter, but not for ChAT, have enlarged perikarya in epileptic rats. These results revealed previously unknown details of structural reorganization of the septohippocampal cholinergic system in experimental epilepsy, involving fiber sprouting into the dentate molecular layer and a parallel fiber retraction from the dentate hilus. We hypothesize that epilepsy-related neuroplasticity of septohippocampal cholinergic neurons is capable of increasing neuronal excitability of the dentate gyrus.

The orexin (hypocretin) neurons play an essential role in promoting arousal, and loss of the orexin neurons results in narcolepsy, a condition characterized by chronic sleepiness and cataplexy. The orexin neurons excite wake-promoting neurons in the basal forebrain (BF), and a reciprocal projection from the BF back to the orexin neurons may help promote arousal and motivation. The BF contains at least three different cell types (cholinergic, glutamatergic, and γ-aminobutyric acid (GABA)ergic neurons) across its different regions (medial septum, diagonal band, magnocellular preoptic area, and substantia innominata). Given the neurochemical and anatomical heterogeneity of the BF, we mapped the pattern of BF projections to the orexin neurons across multiple BF regions and neuronal types. We performed conditional anterograde tracing using mice that express Cre recombinase only in neurons producing acetylcholine, glutamate, or GABA. We found that the orexin neurons are heavily apposed by axon terminals of glutamatergic and GABAergic neurons of the substantia innominata (SI) and magnocellular preoptic area, but there was no innervation by the cholinergic neurons. Channelrhodopsin-assisted circuit mapping (CRACM) demonstrated that glutamatergic SI neurons frequently form functional synapses with the orexin neurons, but, surprisingly, functional synapses from SI GABAergic neurons were rare. Considering their strong reciprocal connections, BF and orexin neurons likely work in concert to promote arousal, motivation, and other behaviors. J. Comp. Neurol. 525:1668-1684, 2017. © 2016 Wiley Periodicals, Inc.

Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signalling in the brainstem. We investigated how long-term loss of cholinergic signalling from mesopontine nuclei influence motor behaviours. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1-Cre-flox/flox). VAChTEn1-Cre-flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChTEn1-Cre-flox/flox mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1-Creflox/flox mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. This article is protected by copyright. All rights reserved.

Binge drinking and alcohol abuse are common during adolescence and cause lasting pathology. Preclinical rodent studies using the adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2‐day on/2‐day off from postnatal day [P]25 to P55) model of human adolescent binge drinking report decreased basal forebrain cholinergic (ie, ChAT+) neurons that persist into adulthood (ie, P56‐P220). Recent studies link AIE‐induced neuroimmune activation to cholinergic pathology, but the underlying molecular mechanisms contributing to the persistent loss of basal forebrain ChAT+ neurons are unknown. We report here that the AIE‐induced loss of cholinergic neuron markers (ie, ChAT, TrkA, and p75NTR), cholinergic neuron shrinkage, and increased expression of the neuroimmune marker pNF‐κB p65 are restored by exercise exposure from P56 to P95 after AIE. Our data reveal that persistently reduced expression of cholinergic neuron markers following AIE is because of the loss of the cholinergic neuron phenotype most likely through an epigenetic mechanism involving DNA methylation and histone 3 lysine 9 dimethylation (H3K9me2). Adolescent intermittent ethanol caused a persistent increase in adult H3K9me2 and DNA methylation at promoter regions of Chat and H3K9me2 of Trka, which was restored by wheel running. Exercise also restored the AIE‐induced reversal learning deficits on the Morris water maze. Together, these data suggest that AIE‐induced adult neuroimmune signaling and cognitive deficits are linked to suppression of Chat and Trka gene expression through epigenetic mechanisms that can be restored by exercise. Exercise restoration of the persistent AIE‐induced phenotypic loss of cholinergic neurons via epigenetic modifications is novel mechanism of neuroplasticity., Adolescent intermittent ethanol (AIE), which models human adolescent binge drinking, causes a loss of basal forebrain cholinergic neurons through neuroimmune and epigenetic processes that persists into adulthood. Voluntary wheel running after AIE restored the AIE‐induced neuroimmune and epigenetic processes leading to recovery of basal forebrain cholinergic neurons. Exercise restoration of the persistent AIE‐induced phenotypic loss of cholinergic neurons via epigenetic modifications is novel mechanism of neuroplasticity.

Converging evidence shows that ventral tegmental area (VTA) dopamine neurons receive laterodorsal tegmental nucleus (LDTg) cholinergic and glutamatergic inputs. To test the behavioral consequences of selectively driving the two sources of excitatory LDTg input to the VTA, channelrhodopsin-2 (ChR2) was expressed in LDTg cholinergic neurons of ChAT::Cre mice (ChAT-ChR2 mice) or in LDTg glutamatergic neurons of VGluT2::Cre mice (VGluT2-ChR2 mice). Mice were tested in a 3-chamber place preference apparatus where entry into a light-paired chamber resulted in VTA light stimulation of LDTg-cholinergic or LDTg-glutamatergic axons for the duration of a chamber stay. ChAT-ChR2 mice spent more time in the light-paired chamber and subsequently showed conditioned place preference for the light-paired chamber in the absence of light. VGluT2-ChR2 mice, entered the light-paired chamber significantly more times than a light-unpaired chamber, but remained in the light-paired chamber for short time periods and did not show a conditioned place preference. When each entry into the light-paired chamber resulted in a single train of VTA light stimulation, VGluT2-ChR2 mice entered the light-paired chamber significantly more times than the light-unpaired chamber, but spent approximately equal amounts of time in the two chambers. VTA excitation of LDTg-glutamatergic inputs may be more important for reinforcement of initial chamber entry while VTA excitation of LDTg-cholinergic inputs may be more important for the rewarding effects of chamber stays. We suggest that LDTg-cholinergic and LDTg-glutamatergic inputs to the VTA each contribute to the net rewarding effects of exciting LDTg axons in the VTA.

Cholinergic activation regulates cognitive function, particularly long-term memory consolidation. This Review presents an overview of the anatomical, neurochemical, and pharmacological evidence supporting the cholinergic regulation of Pavlovian contextual and cue-conditioned fear learning and extinction. Basal forebrain cholinergic neurons provide inputs to neocortical regions and subcortical limbic structures such as the hippocampus and amygdala. Pharmacological manipulations of muscarinic and nicotinic receptors support the role of cholinergic processes in the amygdala, hippocampus, and prefrontal cortex in modulating the learning and extinction of contexts or cues associated with threat. Additional evidence from lesion studies and analysis of in vivo acetylcholine release with microdialysis similarly support a critical role of cholinergic neurotransmission in corticoamygdalar or corticohippocampal circuits during acquisition of fear extinction. Although a few studies have suggested a complex role of cholinergic neurotransmission in the cellular plasticity essential for extinction learning, more work is required to elucidate the exact cholinergic mechanisms and physiological role of muscarinic and nicotinic receptors in these fear circuits. Such studies are important for elucidating the role of cholinergic neurotransmission in disorders such as posttraumatic stress disorder that involve deficits in extinction learning as well as for developing novel therapeutic approaches for such disorders. © 2016 Wiley Periodicals, Inc.

Hippocampal network oscillations are important for learning and memory. Theta rhythms are involved in attention, navigation, and memory encoding, whereas sharp wave-ripple complexes are involved in memory consolidation. Cholinergic neurons in the medial septum-diagonal band of Broca (MS-DB) influence both types of hippocampal oscillations, promoting theta rhythms and suppressing sharp wave-ripples. They also receive frequency-dependent hyperpolarizing feedback from hippocamposeptal connections, potentially affecting their role as neuromodulators in the septohippocampal circuit. However, little is known about how the integration properties of cholinergic MS-DB neurons change with hyperpolarization. By potentially altering firing behavior in cholinergic neurons, hyperpolarizing feedback from the hippocampal neurons may, in turn, change hippocampal network activity. To study changes in membrane integration properties in cholinergic neurons in response to hyperpolarizing inputs, we used whole-cell patch-clamp recordings targeting genetically labeled, choline acetyltransferase-positive neurons in mouse brain slices. Hyperpolarization of cholinergic MS-DB neurons resulted in a long-lasting decrease in spike firing rate and input-output gain. Additionally, voltage-clamp measures implicated a slowly inactivating, 4-AP-insensitive, outward K+ conductance. Using a conductance-based model of cholinergic MS-DB neurons, we show that the ability of this conductance to modulate firing rate and gain depends on the expression of an experimentally verified shallow intrinsic spike frequency-voltage relationship. Together, these findings point to a means through which negative feedback from hippocampal neurons can influence the role of cholinergic MS-DB neurons. © 2016 Wiley Periodicals, Inc.

Falls are a leading cause of death in the elderly and, in a majority of patients with Parkinson's disease (PD), the leading levodopa-insensitive cause of hospitalization and long-term care. Falling in PD has been attributed to degeneration of forebrain cholinergic neurons that, in interaction with striatal dopamine losses, impairs the cognitive control of balance, gait, and movement. We previously established an animal model of these dual cholinergic-dopaminergic losses ("DL rats") and a behavioral test system (Michigan Complex Motor Control Task, MCMCT) to measure falls associated with traversing dynamic surfaces and distractors. Because the combined treatment of the acetylcholinesterase inhibitor donepezil and the 5-HT6 receptor antagonist idalopirdine (Lu AE58054) was reported to exhibit synergistic pro-cholinergic activity in rats and improved cognition in patients with moderate Alzheimer's disease, here we assessed the effects of this treatment on MCMCT performance and attention in DL rats. Compared with the vehicle-treated group, the combined treatment greatly reduced (Cohen's d = 0.96) falls in DL rats when traversing dynamic surfaces and when exposed to a passive distractor. However, falls associated with a dual task distractor and sustained attentional performance did not benefit from this treatment. Analyses of the behavior in fall-prone moments suggested that this treatment improved the efficacy and speed of re-instating forward movement after relatively short stoppages. This treatment may reduce fall propensity in PD patients via maintaining planned movement sequences in working memory and improving the vigor of executing such movements following brief periods of freezing of gait.

We have previously found that thalamostriatal axodendritic terminals are reduced as early as 1 month of age in heterozygous Q140 HD mice (Deng et al. [] Neurobiol Dis 60:89-107). Because cholinergic interneurons are a major target of thalamic axodendritic terminals, we examined the VGLUT2-immunolabeled thalamic input to striatal cholinergic interneurons in heterozygous Q140 males at 1 and 4 months of age, using choline acetyltransferase (ChAT) immunolabeling to identify cholinergic interneurons. Although blinded neuron counts showed that ChAT+ perikarya were in normal abundance in Q140 mice, size measurements indicated that they were significantly smaller. Sholl analysis further revealed the dendrites of Q140 ChAT+ interneurons were significantly fewer and shorter. Consistent with the light microscopic data, ultrastructural analysis showed that the number of ChAT+ dendritic profiles per unit area of striatum was significantly decreased in Q140 striata, as was the abundance of VGLUT2+ axodendritic terminals making synaptic contact with ChAT+ dendrites per unit area of striatum. The density of thalamic terminals along individual cholinergic dendrites was, however, largely unaltered, indicating that the reduction in the areal striatal density of axodendritic thalamic terminals on cholinergic neurons was due to their dendritic territory loss. These results show that the abundance of thalamic input to individual striatal cholinergic interneurons is reduced early in the life span of Q140 mice, raising the possibility that this may occur in human HD as well. Because cholinergic interneurons differentially affect striatal direct vs. indirect pathway spiny projection neurons, their reduced thalamic excitatory drive may contribute to early abnormalities in movement in HD. J. Comp. Neurol. 524:3518-3529, 2016. © 2016 Wiley Periodicals, Inc.

Cholinergic dysfunction in the brain is closely related to cognitive impairment including memory loss. In addition to the degeneration of basal forebrain cholinergic neurons, deficits in the cholinergic receptor signaling may also play an important role. In the present study, to examine the cholinergic signaling pathways responsible for the induction of a memory-related postsynaptic protein, a cholinergic agonist carbachol was used to induce the expression of activity-regulated cytoskeleton associated protein (Arc) in primary rat cortical neurons. After pretreating neurons with various antagonists or inhibitors, the levels of carbachol-induced Arc protein expression were detected by Western blot analysis. The results show that carbachol induces Arc protein expression mainly through activating M1 acetylcholine receptors and the downstream phospholipase C pathway, which may lead to the activation of the MAPK/ERK signaling pathway. Importantly, carbachol-mediated M2 receptor activation exerts negative effects on Arc protein expression and thus counteracts the enhanced effects of M1 activation. Furthermore, it is suggested for the first time that M1-mediated enhancement of N-methyl-D-aspartate receptor (NMDAR) responses, leading to Ca(2+) entry through NMDARs, contributes to carbachol-induced Arc protein expression. These findings reveal a more complete cholinergic signaling that is responsible for carbachol-induced Arc protein expression, and thus provide more information for developing treatments that can modulate cholinergic signaling and consequently alleviate cognitive impairment. J. Cell. Physiol. 231: 2428-2438, 2016. © 2016 Wiley Periodicals, Inc.

This study provides the first systematic analysis of the nuclear organization of the neural systems related to sleep and wake in the basal forebrain, diencephalon, midbrain, and pons of the river hippopotamus, one of the closest extant terrestrial relatives of the cetaceans. All nuclei involved in sleep regulation and control found in other mammals, including cetaceans, were present in the river hippopotamus, with no specific nuclei being absent, but novel features of the cholinergic system, including novel nuclei, were present. This qualitative similarity relates to the cholinergic, noradrenergic, serotonergic, and orexinergic systems and is extended to the γ-aminobutyric acid (GABA)ergic elements of these nuclei. Quantitative analysis reveals that the numbers of pontine cholinergic (259,578) and noradrenergic (127,752) neurons, and hypothalamic orexinergic neurons (68,398) are markedly higher than in other large-brained mammals. These features, along with novel cholinergic nuclei in the intralaminar nuclei of the dorsal thalamus and the ventral tegmental area of the midbrain, as well as a major expansion of the hypothalamic cholinergic nuclei and a large laterodorsal tegmental nucleus of the pons that has both parvocellular and magnocellular cholinergic neurons, indicates an unusual sleep phenomenology for the hippopotamus. Our observations indicate that the hippopotamus is likely to be a bihemispheric sleeper that expresses REM sleep. The novel features of the cholinergic system suggest the presence of an undescribed sleep state in the hippopotamus, as well as the possibility that this animal could, more rapidly than other mammals, switch cortical electroencephalographic activity from one state to another. J. Comp. Neurol. 524:2036-2058, 2016. © 2016 Wiley Periodicals, Inc.

Key points The basal forebrain is an important component of the ascending arousal system and may be a key site through which the orexin neurons promote arousal. It has long been known that orexin-A and -B excite basal forebrain cholinergic neurons, but orexin-producing neurons also make the inhibitory peptide dynorphin. Using whole-cell recordings in brain slices, we found that dynorphin-A directly inhibits basal forebrain cholinergic neurons via κ-opioid receptors, and decreases afferent excitatory synaptic input to these neurons. While the effects of dynorphin-A and orexin-A desensitize over multiple applications, co-application of dynorphin-A and orexin-A produces a sustained response that reverses depending on the membrane potential of basal forebrain cholinergic neurons. At −40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at −70 mV the excitatory response to orexin-A prevails. Abstract The basal forebrain (BF) is an essential component of the ascending arousal systems and may be a key site through which the orexin (also known as hypocretin) neurons drive arousal and promote the maintenance of normal wakefulness. All orexin neurons also make dynorphin, and nearly all brain regions innervated by the orexin neurons express kappa opiate receptors, the main receptor for dynorphin. This is remarkable because orexin excites target neurons including BF neurons, but dynorphin has inhibitory effects. We identified the sources of dynorphin input to the magnocellular preoptic nucleus and substantia innominata (MCPO/SI) in mice and determined the effects of dynorphin-A on MCPO/SI cholinergic neurons using patch-clamp recordings in brain slices. We found that the orexin neurons are the main source of dynorphin input to the MCPO/SI region, and dynorphin-A inhibits MCPO/SI cholinergic neurons through κ-opioid receptors by (1) activation of a G protein-coupled inwardly rectifying potassium current, (2) inhibition of a voltage-gated Ca2+ current and (3) presynaptic depression of the glutamatergic input to these neurons. The responses both to dynorphin-A and to orexin-A desensitize, but co-application of dynorphin-A and orexin-A produces a sustained response. In addition, the polarity of the response to the co-application depends on the membrane potential of BF neurons; at −40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at −70 mV the excitatory response to orexin-A prevails. This suggests that depending on their state of activation, BF cholinergic neurons can be excited or inhibited by signals from the orexin neurons.