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Cognitive impairment frequently presents as a prevalent consequence following stroke, imposing significant burdens on patients, families, and society. The objective of this study was to assess the effectiveness and underlying mechanism of nerve growth factor (NGF) in treating post-stroke cognitive dysfunction in rats with cerebral ischemia–reperfusion injury (MCAO/R) through delivery into the brain using specific mode electroacupuncture stimulation (SMES). From the 28th day after modeling, the rats were treated with NGF mediated by SMES, and the cognitive function of the rats was observed after treatment. Learning and memory ability were evaluated using behavioral tests. The impact of SMES on blood–brain barrier (BBB) permeability, the underlying mechanism of cognitive enhancement in rats with MCAO/R, including transmission electron microscopy, enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, and TUNEL staining. We reported that SMES demonstrates a safe and efficient ability to open the BBB during the cerebral ischemia repair phase, facilitating the delivery of NGF to the brain by the p65-VEGFA-TJs pathway. By Figdraw [ABSTRACT FROM AUTHOR]

Neuronal gene expression in the brain dynamically responds to synaptic activity. The interplay among synaptic activity, gene expression, and synaptic plasticity has crucial implications for understanding the pathophysiology of diseases such as Alzheimer's disease and epilepsy. These diseases are marked by synaptic dysfunction that affects the expression patterns of neuroprotective genes that are incompletely understood. In our study, we developed a cellular model of synaptic activity using human cholinergic neurons derived from SH-SY5Y cell differentiation. Depolarization induction modulates the expression of neurotrophic genes and synaptic markers, indicating a potential role in synaptic plasticity regulation. This hypothesis is further supported by the induction kinetics of various long non-coding RNAs, including primate-specific ones. Our experimental model showcases the utility of SH-SY5Y cells in elucidating the molecular mechanisms underlying synaptic plasticity in human cellular systems. [ABSTRACT FROM AUTHOR]

Introduction: The pedunculopontine nucleus (PPN) plays a role in coordinating complex behaviors and adapting to changing environmental conditions. The specific role of cholinergic neurons in PPN function is not well understood, but their ascending connectivity with basal ganglia and thalamus suggests involvement in adaptive functions. Methods: We used a chemogenetic approach in ChAT::Cre rats to explore the specific contribution of PPN cholinergic neurons to behavioral flexibility, focusing on the adaptation to shifting reward contingencies in a Reversal Learning Task. Rats were first trained in a non-probabilistic reversal learning task, followed by a probabilistic phase to challenge their adaptive strategies under varying reward conditions. Results: Motor functions were evaluated to confirm that behavioral observations were not confounded by motor deficits. We found that inhibition of PPN cholinergic neurons did not affect performance in the non-probabilistic condition but significantly altered the rats' ability to adapt to the probabilistic condition. Under chemogenetic inhibition, the rats showed a marked deficiency in utilizing previous trial outcomes for decision-making and an increased sensitivity to negative outcomes. Logistic regression and Q-learning models revealed that suppression of PPN cholinergic activity impaired the adaptation of decision-making strategies. Discussion: Our results highlight the role of PPN cholinergic neurons in dynamically updating action-outcome expectations and adapting to new contingencies. The observed impairments in decision-making under PPN cholinergic inhibition align with cognitive deficits associated with cholinergic dysfunction in neurodegenerative disorders. These findings suggest that cholinergic neurons in the PPN are essential for maximizing rewards through the flexible updating of behavioral strategies. [ABSTRACT FROM AUTHOR]

BACKGROUND & AIMS: Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease. METHODS: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. RESULTS: Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages. CONCLUSIONS: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment.

This review is an attempt to compile existing hypotheses on the mechanisms underlying the initiation and progression of Alzheimer's disease (AD), starting from sensory impairments observed in AD and concluding with molecular events that are typically associated with the disease. These events include spreading of amyloid plaques and tangles of hyperphosphorylated tau and formation of Hirano and Biondi bodies as well as the development of oxidative stress. We have detailed the degenerative changes that occur in several neuronal populations, including the cholinergic neurons in the nucleus basalis of Meynert, the histaminergic neurons in the tuberomammillary nucleus, the serotonergic neurons in the raphe nuclei, and the noradrenergic neurons in the locus coeruleus. Furthermore, we discuss the potential role of iron accumulation in the brains of subjects with AD in the disease progression which served as a basis for the idea that iron chelation in the brain may mitigate oxidative stress and decelerate disease development. We also draw attention to possible role of sympathetic system and, more specifically, noradrenergic neurons of the superior cervical ganglion in triggering of the disease. We also explore the alternative possibility of compensatory protective changes that may occur in these neurons to support cholinergic function in the forebrain of subjects with AD. [ABSTRACT FROM AUTHOR]

The control of the respiratory rhythm and airway motor activity is essential for life. Accumulating evidence indicates that the postinspiratory complex (PiCo) is crucial for generating behaviors that occur during the postinspiratory phase, including expiratory laryngeal activity and swallowing. Located in the ventromedial medulla, PiCo is defined by neurons co‐expressing two neurotransmitter markers (ChAT and Vglut2/Slc17a6). Here, we mapped the input–output connections of these neurons using viral tracers and intersectional viral‐genetic tools. PiCo neurons were specifically targeted by focal injection of a doubly conditional Cre‐ and FlpO‐dependent AAV8 viral marker (AAV8‐Con/Fon‐TVA‐mCherry) into the left PiCo of adult ChatCre/wt: Vglut2FlpO/wt mice, for anterograde axonal tracing. These experiments revealed projections to various brain regions, including the Cu, nucleus of the solitary tract (NTS), Amb, X, XII, Sp5, RMg, intermediate reticular nucleus (IRt), lateral reticular nucleus (LRt), pre‐Bötzinger complex (preBötC), contralateral PiCo, laterodorsal tegmental nucleus (LDTg), pedunculopontine tegmental nucleus (PPTg), periaqueductal gray matter (PAG), Kölliker–Fuse (KF), PB, and external cortex of the inferior colliculus (ECIC). A rabies virus (RV) retrograde transsynaptic approach was taken with EnvA‐pseudotyped G‐deleted (RV‐SAD‐G‐GFP) to similarly target PiCo neurons in ChatCre/wt: Vglut2FlpO/wt mice, following prior injections of helper AAVs (a mixture of AAV‐Ef1a‐Con/Fon oG and viral vector AAV8‐Con/Fon‐TVA‐mCherry). This combined approach revealed prominent synaptic inputs to PiCo neurons from NTS, IRt, and A1/C1. Although PiCo neurons project axons to the contralateral PiCo area, this approach did not detect direct contralateral connections. We suggest that PiCo serves as a critical integration site, projecting and receiving neuronal connections implicated in breathing, arousal, swallowing, and autonomic regulation. [ABSTRACT FROM AUTHOR]

Background: Perfluorooctane sulfonic acid (PFOS), a widely used industrial chemical, was reported to induce memory and learning process dysfunction. Some studies tried to reveal the mechanisms that mediate these effects, but how they are produced is still unknown. Basal forebrain cholinergic neurons (BFCN) maintain cognitive function and their selective neurodegeneration induces cognitive decline, as observed in Alzheimer's disease. PFOS was reported to disrupt cholinergic and glutamatergic transmissions and thyroid hormone action, which regulate cognitive processes and maintain BFCN viability. Objective/Methods: To evaluate PFOS neurodegenerative effects on BFCN and the mechanisms that mediate them, SN56 cells (a neuroblastoma cholinergic cell line from the basal forebrain) were treated with PFOS (0.1 µM to 40 µM) with or without thyroxine (T3; 15 nM), MK-801 (20 µM) or acetylcholine (ACh; 10 µM). Results: In the present study, we found that PFOS treatment (1 or 14 days) decreased thyroid receptor α (TRα) activity by decreasing its protein levels and increased T3 metabolism through increased deiodinase 3 (D3) levels. Further, we observed that PFOS treatment disrupted cholinergic transmission by decreasing ACh content through decreased choline acetyltransferase (ChAT) activity and protein levels and through decreasing muscarinic receptor 1 (M1R) binding and protein levels. PFOS also disrupted glutamatergic transmission by decreasing glutamate content through increased glutaminase activity and protein levels and through decreasing N-methyl-D-aspartate receptor subunit 1 (NMDAR1); effects mediated through M1R disruption. All these effects were mediated through decreased T3 activity and T3 supplementation partially restored to the normal state. Conclusions: These findings may assist in understanding how PFOS induces neurodegeneration, and the mechanisms involved, especially in BFCN, to explain the process that could lead to cognitive dysfunction and provide new therapeutic tools to treat and prevent its neurotoxic effects. [ABSTRACT FROM AUTHOR]

Septo-hippocampal pathway, crucial for physiological functions and involved in epilepsy. Clinical monitoring during epileptogenesis is complicated. We aim to evaluate tissue changes after lesioning the medial septum (MS) of normal rats and assess how the depletion of specific neuronal populations alters the animals' behavior and susceptibility to establishing a pilocarpine-induced status epilepticus. Male Sprague–Dawley rats were injected into the MS with vehicle or saporins (to deplete GABAergic or cholinergic neurons; n = 16 per group). Thirty-two animals were used for diffusion tensor imaging (DTI); scanned before surgery and 14 and 49 days post-injection. Fractional anisotropy and apparent diffusion coefficient were evaluated in the fimbria, dorsal hippocampus, ventral hippocampus, dorso-medial thalamus, and amygdala. Between scans 2 and 3, animals were submitted to diverse behavioral tasks. Stainings were used to analyze tissue alterations. Twenty-four different animals received pilocarpine to evaluate the latency and severity of the status epilepticus 2 weeks after surgery. Additionally, eight different animals were only used to evaluate the neuronal damage inflicted on the MS 1 week after the molecular surgery. Progressive changes in DTI parameters in both white and gray matter structures of the four evaluated groups were observed. Behaviorally, the GAT1-saporin injection impacted spatial memory formation, while 192-IgG-saporin triggered anxiety-like behaviors. Histologically, the GABAergic toxin also induced aberrant mossy fiber sprouting, tissue damage, and neuronal death. Regarding the pilocarpine-induced status epilepticus, this agent provoked an increased mortality rate. Selective septo-hippocampal modulation impacts the integrity of limbic regions crucial for certain behavioral skills and could represent a precursor for epilepsy development. [ABSTRACT FROM AUTHOR]

Background: The cholinergic hypothesis is one of the main theories that describe the pathogenesis of Alzheimer's disease (AD). Cholinergic neurons degenerate early and are severely damaged in AD. Despite extensive research, the causes of cholinergic neuron damage and the underlying molecular changes remain unclear. Objective: This study aimed to explore the characteristics and transcriptomic changes in cholinergic neurons derived from human induced pluripotent stem cells (iPSCs) with APP mutation. Methods: Peripheral blood mononuclear cells from patients with AD and healthy individuals were reprogrammed into iPSCs. The iPSCs were differentiated into cholinergic neurons. Cholinergic neurons were stained, neurotoxically tested, and electrophysiologically and transcriptomically analyzed. Results: The iPSCs-derived cholinergic neurons from a patient with AD carrying a mutation in APP displayed enhanced susceptibility to Aβ1-42-induced neurotoxicity, characterized by severe neurotoxic effects, such as cell body coagulation and neurite fragmentation. Cholinergic neurons exhibited electrophysiological impairments and neuronal death after 21 days of culture in the AD group. Transcriptome analysis disclosed 883 differentially expressed genes (DEGs, 420 upregulated and 463 downregulated) participating in several signaling pathways implicated in AD pathogenesis. To assess the reliability of RNA sequencing, the expression of 16 target DEGs was validated using qPCR. Finally, the expression of the 8 core genes in different cell types of brain was analyzed by the AlzData database. Conclusions: In this study, iPSCs-derived cholinergic neurons from AD patients with APP mutations exhibit characteristics reminiscent of neurodegenerative disease. Transcriptome analysis revealed the corresponding DEGs and pathways, providing potential biomarkers and therapeutic targets for advancing AD research. [ABSTRACT FROM AUTHOR]

The recent increase in the use of nicotine products by teenagers has revealed an urgent need to better understand the impact of nicotine on the adolescent brain. Here, we sought to examine the actions of extracellular ATP as a neurotransmitter and to investigate whether ATP and nicotinic signaling interact during adolescence. With the GRABATP (G-protein-coupled receptor activation-based ATP sensor), we first demonstrated that nicotine induces extracellular ATP release in the medial habenula, a brain region involved in nicotine aversion and withdrawal. Using patch-clamp electrophysiology, we then demonstrated that activation of the ATP receptors P2X or P2Y1 increases the neuronal firing of cholinergic neurons. Surprisingly, contrasting interactive effects were observed with nicotine exposure. For the P2X receptor, activation had no observable effect on acute nicotine-mediated activity, but during abstinence after 10 d of nicotine exposure, coexposure to nicotine and the P2X agonist potentiated neuronal activity in female, but not male, neurons. For P2Y1 signaling, a potentiated effect of the agonist and nicotine was observed with acute exposure, but not following extended nicotine exposure. These data reveal a complex interactive effect between nicotinic and ATP signaling in the adolescent brain and provide mechanistic insights into extracellular ATP signaling with sex-specific alterations of neuronal responses based on prior drug exposure.SIGNIFICANCE STATEMENT In these studies, it was discovered that nicotine induces extracellular ATP release in the medial habenula and subsequent activation of the ATP purinergic receptors increases habenular cholinergic neuronal firing in the adolescent brain. Interestingly, following extended nicotine exposure, nicotine was found to alter the interplay between purinergic and nicotinic signaling in a sex-specific manner. Together, these studies provide a novel understanding for the role of extracellular ATP in mediating habenular activity and reveal how nicotine exposure during adolescence alters these signaling mechanisms, which has important implications given the high incidence of e-cigarette/vape use by youth.

Alzheimers disease (AD) is a chronic neurological condition characterized by the severe loss of cholinergic neurons. Currently, the incomplete understanding of the loss of neurons has prevented curative treatments for familial AD (FAD). Therefore, modeling FAD in vitro is essential for studying cholinergic vulnerability. Moreover, to expedite the discovery of disease-modifying therapies that delay the onset and slow the progression of AD, we depend on trustworthy disease models. Although highly informative, induced pluripotent stem cell (iPSCs)-derived cholinergic neurons (ChNs) are time-consuming, not cost-effective, and labor-intensive. Other sources for AD modeling are urgently needed. Wild-type and presenilin (PSEN)1 p.E280A fibroblast-derived iPSCs, menstrual blood-derived menstrual stromal cells (MenSCs), and umbilical cord-derived Wharton Jellys mesenchymal stromal cells (WJ-MSCs) were cultured in Cholinergic-N-Run and Fast-N-Spheres V2 medium to obtain WT and PSEN 1 E280A cholinergic-like neurons (ChLNs, 2D) and cerebroid spheroids (CSs, 3D), respectively, and to evaluate whether ChLNs/CSs can reproduce FAD pathology. We found that irrespective of tissue source, ChLNs/CSs successfully recapitulated the AD phenotype. PSEN 1 E280A ChLNs/CSs show accumulation of iAPPβ fragments, produce eAβ42, present TAU phosphorylation, display OS markers (e.g., oxDJ-1, p-JUN), show loss of ΔΨm, exhibit cell death markers (e.g., TP53, PUMA, CASP3), and demonstrate dysfunctional Ca2+ influx response to ACh stimuli. However, PSEN 1 E280A 2D and 3D cells derived from MenSCs and WJ-MSCs can reproduce FAD neuropathology more efficiently and faster (11 days) than ChLNs derived from mutant iPSCs (35 days). Mechanistically, MenSCs and WJ-MSCs are equivalent cell types to iPSCs for reproducing FAD in vitro.

Cholinergic degeneration is significant in Lewy body disease, including Parkinson's disease, dementia with Lewy bodies, and isolated REM sleep behaviour disorder. Extensive research has demonstrated cholinergic alterations in the CNS of these disorders. More recently, studies have revealed cholinergic denervation in organs that receive parasympathetic denervation. This enables a comprehensive review of cholinergic changes in Lewy body disease, encompassing both central and peripheral regions, various disease stages and diagnostic categories. Across studies, brain regions affected in Lewy body dementia show equal or greater levels of cholinergic impairment compared to the brain regions affected in Lewy body disease without dementia. This observation suggests a continuum of cholinergic alterations between these disorders. Patients without dementia exhibit relative sparing of limbic regions, whereas occipital and superior temporal regions appear to be affected to a similar extent in patients with and without dementia. This implies that posterior cholinergic cell groups in the basal forebrain are affected in the early stages of Lewy body disorders, while more anterior regions are typically affected later in the disease progression. The topographical changes observed in patients affected by comorbid Alzheimer pathology may reflect a combination of changes seen in pure forms of Lewy body disease and those seen in Alzheimer's disease. This suggests that Alzheimer co-pathology is important to understand cholinergic degeneration in Lewy body disease. Thalamic cholinergic innervation is more affected in Lewy body patients with dementia compared to those without dementia, and this may contribute to the distinct clinical presentations observed in these groups. In patients with Alzheimer's disease, the thalamus is variably affected, suggesting a different sequential involvement of cholinergic cell groups in Alzheimer's disease compared to Lewy body disease. Patients with isolated REM sleep behaviour disorder demonstrate cholinergic denervation in abdominal organs that receive parasympathetic innervation from the dorsal motor nucleus of the vagus, similar to patients who experienced this sleep disorder in their prodrome. This implies that REM sleep behaviour disorder is important for understanding peripheral cholinergic changes in both prodromal and manifest phases of Lewy body disease. In conclusion, cholinergic changes in Lewy body disease carry implications for understanding phenotypes and the influence of Alzheimer co-pathology, delineating subtypes and pathological spreading routes, and for developing tailored treatments targeting the cholinergic system. [ABSTRACT FROM AUTHOR]

Disruptions of the circadian rhythm have shown that it can lead to significant health risks. Factors such as rapid travel across different time zones, increased exposure to artificial light, and the consumption of substances like caffeine contribute to this disruption. Understanding these disruptions and its causes is crucial for improving our health and in finding solutions. [ABSTRACT FROM AUTHOR]

IntroductionThe pedunculopontine nucleus (PPN) plays a role in coordinating complex behaviors and adapting to changing environmental conditions. The specific role of cholinergic neurons in PPN function is not well understood, but their ascending connectivity with basal ganglia and thalamus suggests involvement in adaptive functions.MethodsWe used a chemogenetic approach in ChAT::Cre rats to explore the specific contribution of PPN cholinergic neurons to behavioral flexibility, focusing on the adaptation to shifting reward contingencies in a Reversal Learning Task. Rats were first trained in a non-probabilistic reversal learning task, followed by a probabilistic phase to challenge their adaptive strategies under varying reward conditions.ResultsMotor functions were evaluated to confirm that behavioral observations were not confounded by motor deficits. We found that inhibition of PPN cholinergic neurons did not affect performance in the non-probabilistic condition but significantly altered the rats’ ability to adapt to the probabilistic condition. Under chemogenetic inhibition, the rats showed a marked deficiency in utilizing previous trial outcomes for decision-making and an increased sensitivity to negative outcomes. Logistic regression and Q-learning models revealed that suppression of PPN cholinergic activity impaired the adaptation of decision-making strategies.DiscussionOur results highlight the role of PPN cholinergic neurons in dynamically updating action-outcome expectations and adapting to new contingencies. The observed impairments in decision-making under PPN cholinergic inhibition align with cognitive deficits associated with cholinergic dysfunction in neurodegenerative disorders. These findings suggest that cholinergic neurons in the PPN are essential for maximizing rewards through the flexible updating of behavioral strategies.

Alzheimer's disease (AD) is characterized by a loss of neurons in the cortex and subcortical regions. Previously, we showed that the progressive degeneration of subcortical monoaminergic (MAergic) neurons seen in human AD is recapitulated in the APPswe/PS1ΔE9 (APP/PS) transgenic mouse model. Because degeneration of cholinergic (Ach) neurons is also a prominent feature of AD, we examined the integrity of the Ach system in the APP/PS model. The overall density of Ach fibers is reduced in APP/PS1 mice at 12 and 18 months of age but not at 4 months of age. Analysis of basal forebrain Ach neurons shows no loss of Ach neurons in the APP/PS model. Thus, since MAergic systems show overt cell loss at 18 months of age, the Ach system is less vulnerable to neurodegeneration in the APP/PS1 model. We also examined whether the proximity to Aβ deposition affected the degeneration of Ach and 5-HT afferents. We found that the areas closer to the edges of compact Aβ deposits exhibit a more severe loss of afferents than the areas that are more distal to Aβ deposits. Collectively, the results indicate that the APP/PS model recapitulates the degeneration of multiple subcortical neurotransmitter systems, including the Ach system. In addition, the results indicate that Aβ deposits cause global as well as local toxicity to subcortical afferents. [ABSTRACT FROM AUTHOR]

Background & Aims: Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease. Methods: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. Results: Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages. Conclusions: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.

Background: Perfluorooctane sulfonic acid (PFOS), a widely used industrial chemical, was reported to induce memory and learning process dysfunction. Some studies tried to reveal the mechanisms that mediate these effects, but how they are produced is still unknown. Basal forebrain cholinergic neurons (BFCN) maintain cognitive function and their selective neurodegeneration induces cognitive decline, as observed in Alzheimer’s disease. PFOS was reported to disrupt cholinergic and glutamatergic transmissions and thyroid hormone action, which regulate cognitive processes and maintain BFCN viability. Objective/Methods: To evaluate PFOS neurodegenerative effects on BFCN and the mechanisms that mediate them, SN56 cells (a neuroblastoma cholinergic cell line from the basal forebrain) were treated with PFOS (0.1 µM to 40 µM) with or without thyroxine (T3; 15 nM), MK-801 (20 µM) or acetylcholine (ACh; 10 µM). Results: In the present study, we found that PFOS treatment (1 or 14 days) decreased thyroid receptor α (TRα) activity by decreasing its protein levels and increased T3 metabolism through increased deiodinase 3 (D3) levels. Further, we observed that PFOS treatment disrupted cholinergic transmission by decreasing ACh content through decreased choline acetyltransferase (ChAT) activity and protein levels and through decreasing muscarinic receptor 1 (M1R) binding and protein levels. PFOS also disrupted glutamatergic transmission by decreasing glutamate content through increased glutaminase activity and protein levels and through decreasing N-methyl-D-aspartate receptor subunit 1 (NMDAR1); effects mediated through M1R disruption. All these effects were mediated through decreased T3 activity and T3 supplementation partially restored to the normal state. Conclusions: These findings may assist in understanding how PFOS induces neurodegeneration, and the mechanisms involved, especially in BFCN, to explain the process that could lead to cognitive dysfunction and provide new therapeutic tools to treat and prevent its neurotoxic effects.

Background Insulin is a peptide hormone that regulates post-prandial physiology, and it is well known that insulin controls homeostasis at least in part via the central nervous system. In particular, insulin alters the activity of neurons within the autonomic nervous system. However, currently available data are mostly from unidentified brainstem neurons of the dorsal motor nucleus of the vagus nerve (DMV). Methods In this study, we used several genetically engineered mouse models to label distinct populations of neurons within the brainstem and the spinal cord for whole-cell patch clamp recordings and to assess several in vivo metabolic functions. Results We first confirmed that insulin directly inhibited cholinergic (parasympathetic preganglionic) neurons in the DMV. We also found inhibitory effects of insulin on both the excitatory and inhibitory postsynaptic currents recorded in DMV cholinergic neurons. In addition, GABAergic neurons of the DMV and nucleus tractus solitarius were inhibited by insulin. However, insulin had no effects on the cholinergic sympathetic preganglionic neurons of the spinal cord. Finally, we obtained results suggesting that the insulin-induced inhibition of parasympathetic preganglionic neurons may not play a critical role in the regulation of glucose homeostasis and gastrointestinal motility. Conclusion Our results demonstrate that insulin inhibits parasympathetic neuronal circuitry in the brainstem, while not affecting sympathetic neuronal activity in the spinal cord.

Background and Purpose: Glial cell‐derived neurotrophic factor (GDNF) maintains gut homeostasis. Dopamine promotes GDNF release in astrocytes. We investigated the regulation by dopamine of colonic GDNF secretion. Experimental Approach: D1 receptor knockout (D1R−/−) mice, adeno‐associated viral 9‐short hairpin RNA carrying D2 receptor (AAV9‐shD2R)‐treated mice, 6‐hydroxydopamine treated (6‐OHDA) rats and primary enteric glial cells (EGCs) culture were used. Incubation fluid from colonic submucosal plexus and longitudinal muscle myenteric plexus were collected for GDNF and ACh measurements. Key Results: D2 receptor‐immunoreactivity (IR), but not D1 receptor‐IR, was observed on EGCs. Both D1 receptor‐IR and D2 receptor‐IR were co‐localized on cholinergic neurons. Low concentrations of dopamine induced colonic GDNF secretion in a concentration‐dependent manner, which was mimicked by the D1 receptor agonist SKF38393, inhibited by TTX and atropine and eliminated in D1R−/− mice. SKF38393‐induced colonic ACh release was absent in D1R−/− mice. High concentrations of dopamine suppressed colonic GDNF secretion, which was mimicked by the D2 receptor agonist quinpirole, and absent in AAV‐shD2R‐treated mice. Quinpirole decreased GDNF secretion by reducing intracellular Ca2+ levels in primary cultured EGCs. Carbachol (ACh analogue) promoted the release of GDNF. Quinpirole inhibited colonic ACh release, which was eliminated in the AAV9‐shD2R‐treated mice. 6‐OHDA treated rats with low ACh and high dopamine content showed decreased GDNF content and increased mucosal permeability in the colon. Conclusion and Implications: Low concentrations of dopamine promote colonic GDNF secretion via D1 receptors on cholinergic neurons, whereas high concentrations of dopamine inhibit GDNF secretion via D2 receptors on EGCs and/or cholinergic neurons. [ABSTRACT FROM AUTHOR]

Dementia with Lewy bodies is characterized by a high burden of autonomic dysfunction and Lewy pathology in peripheral organs and components of the sympathetic and parasympathetic nervous system. Parasympathetic terminals may be quantified with 18F-fluoroetoxybenzovesamicol, a PET tracer that binds to the vesicular acetylcholine transporter in cholinergic presynaptic terminals. Parasympathetic imaging may be useful for diagnostics, improving our understanding of autonomic dysfunction and for clarifying the spatiotemporal relationship of neuronal degeneration in prodromal disease. Therefore, we aimed to investigate the cholinergic parasympathetic integrity in peripheral organs and central autonomic regions of subjects with dementia with Lewy bodies and its association with subjective and objective measures of autonomic dysfunction. We hypothesized that organs with known parasympathetic innervation, especially the pancreas and colon, would have impaired cholinergic integrity. To achieve these aims, we conducted a cross-sectional comparison study including 23 newly diagnosed non-diabetic subjects with dementia with Lewy bodies (74 ± 6 years, 83% male) and 21 elderly control subjects (74 ± 6 years, 67% male). We obtained whole-body images to quantify PET uptake in peripheral organs and brain images to quantify PET uptake in regions of the brainstem and hypothalamus. Autonomic dysfunction was assessed with questionnaires and measurements of orthostatic blood pressure. Subjects with dementia with Lewy bodies displayed reduced cholinergic tracer uptake in the pancreas (32% reduction, P = 0.0003) and colon (19% reduction, P = 0.0048), but not in organs with little or no parasympathetic innervation. Tracer uptake in a region of the medulla oblongata overlapping the dorsal motor nucleus of the vagus correlated with autonomic symptoms (rs = −0.54, P = 0.0077) and changes in orthostatic blood pressure (rs = 0.76, P < 0.0001). Tracer uptake in the pedunculopontine region correlated with autonomic symptoms (rs = −0.52, P = 0.0104) and a measure of non-motor symptoms (rs = −0.47, P = 0.0230). In conclusion, our findings provide the first imaging-based evidence of impaired cholinergic integrity of the pancreas and colon in dementia with Lewy bodies. The observed changes may reflect parasympathetic denervation, implying that this process is initiated well before the point of diagnosis. The findings also support that cholinergic denervation in the brainstem contributes to dysautonomia. [ABSTRACT FROM AUTHOR]

Rett syndrome is a rare genetic neurodevelopmental disease, affecting 1 in over 10 000 females born worldwide, caused by de novo mutations in the X-chromosome-located methyl-CpG-binding protein 2 (MeCP2) gene. Despite the great effort put forth by the scientific community, a therapy for this devastating disease is still needed. Here, we tested the therapeutic effects of a painless mutein of the nerve growth factor (NGF), called human NGF painless (hNGFp), via a non-invasive intranasal delivery in female MeCP2 +/− mice. Of note, previous work had demonstrated a broad biodistribution of hNGFp in the mouse brain by the nasal delivery route. We report that (i) the long-term lifelong treatment of MeCP2 +/− mice with hNGFp, starting at 2 months of age, increased the chance of survival while also greatly improving behavioural parameters. Furthermore, when we assessed the phenotypic changes brought forth by (ii) a short-term 1-month-long hNGFp-treatment, starting at 3 months of age (right after the initial presentation of symptoms), we observed the rescue of a well known neuronal target population of NGF, cholinergic neurons in the medial septum. Moreover, we reveal a deficit in microglial morphology in MeCP2 +/− mice, completely reversed in treated animals. This effect on microglia is in line with reports showing microglia to be a TrkA-dependent non-neuronal target cell population of NGF in the brain. To understand the immunomodulatory activity of hNGFp, we analysed the cytokine profile after hNGFp treatment in MeCP2 +/− mice, to discover that the treatment recovered the altered expression of key neuroimmune-communication molecules, such as fractalkine. The overall conclusion is that hNGFp delivered intranasally can ameliorate symptoms in the MeCP2 +/− model of Rett syndrome, by exerting strong neuroprotection with a dual mechanism of action: directly on target neurons and indirectly via microglia. [ABSTRACT FROM AUTHOR]

The pedunculopontine tegmental nucleus (PPTg) plays a vital role in sleep/wake states. There are three main kinds of heterogeneous neurons involved: cholinergic, glutamatergic, and gamma-aminobutyric acidergic (GABAergic) neurons. However, the precise roles of cholinergic, glutamatergic and GABAergic PPTg cell groups in regulating sleep–wake are unknown. Recent work suggests that the cholinergic, glutamatergic, and GABAergic neurons of the PPTg may activate the main arousal-promoting nucleus, thus exerting their wakefulness effects. We review the related projection pathways and functions of various neurons of the PPTg, especially the mechanisms of the PPTg in sleep–wake, thus providing new perspectives for research of sleep–wake mechanisms. [ABSTRACT FROM AUTHOR]

The interplay of the enteric nervous system (ENS) and SIP syncytium (smooth muscle cells–interstitial cells of Cajal–PDGFRα+ cells) plays an important role in the regulation of gastrointestinal (GI) motility. This study aimed to investigate the dynamic regulatory mechanisms of the ENS-SIP system on colon motility during postnatal development. Colonic samples of postnatal 1-week-old (PW1), 3-week-old (PW3), and 5-week-old (PW5) mice were characterized by RNA sequencing, qPCR, Western blotting, isometric force recordings (IFR), and colonic motor complex (CMC) force measurements. Our study showed that the transcriptional expression of Pdgfrα, c-Kit, P2ry1, Nos1, and Slc18a3, and the protein expression of nNOS, c-Kit, and ANO1 significantly increased with age from PW1 to PW5. In PW1 and PW3 mice, colonic migrating movement was not fully developed. In PW5 mice, rhythmic CMCs were recorded, similar to the CMC pattern described previously in adult mice. The inhibition of nNOS revealed excitatory and non-propulsive responses which are normally suppressed due to ongoing nitrergic inhibition. During postnatal development, molecular data demonstrated the establishment and expansion of ICC and PDGFRα+ cells, along with nitrergic and cholinergic nerves and purinergic receptors. Our findings are important for understanding the role of the SIP syncytium in generating and establishing CMCs in postnatal, developing murine colons. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) vectors are currently the most efficient option for intracranial gene therapies to treat neurodegenerative disease. Increased efficacy and safety will depend upon robust and specific expression of therapeutic genes into target cell-types within the human brain. In this study, we set out with two objectives: (1) to identify capsids with broader transduction of the striatum upon intracranial injection in mice and (2) to test a truncated human choline acetyltransferase (ChAT) promoter that would allow efficient and selective transduction of cholinergic neurons. We compared AAV9 and an engineered capsid, AAV-S, to mediate widespread reporter gene expression throughout the striatum. We observed that AAV-S transduced a significantly greater area of the injected hemisphere primarily in the rostral direction compared with AAV9 (CAG promoter). We tested AAV9 vectors packaging a reporter gene expression cassette driven by either the ChAT or CAG promoter. Specificity of transgene expression of ChAT neurons over other cells was 7-fold higher, and efficiency was 3-fold higher for the ChAT promoter compared with the CAG promoter. The AAV-ChAT transgene expression cassette should be a useful tool for the study of cholinergic neurons in mice, and the broader transduction area of AAV-S warrants further evaluation of this capsid.

Abstract Background Based on their anatomical location, rostral projections of nuclei are classified as ascending circuits, while caudal projections are classified as descending circuits. Upper brainstem neurons participate in complex information processing and specific sub-populations preferentially project to participating ascending or descending circuits. Cholinergic neurons in the upper brainstem have extensive collateralizations in both ascending and descending circuits; however, their single-cell projection patterns remain unclear because of the lack of comprehensive characterization of individual neurons. Results By combining fluorescent micro-optical sectional tomography with sparse labeling, we acquired a high-resolution whole-brain dataset of pontine-tegmental cholinergic neurons (PTCNs) and reconstructed their detailed morphology using semi-automatic reconstruction methods. As the main source of acetylcholine in some subcortical areas, individual PTCNs had abundant axons with lengths up to 60 cm and 5000 terminals and innervated multiple brain regions from the spinal cord to the cortex in both hemispheres. Based on various collaterals in the ascending and descending circuits, individual PTCNs were grouped into four subtypes. The morphology of cholinergic neurons in the pedunculopontine nucleus was more divergent, whereas the laterodorsal tegmental nucleus neurons contained richer axonal branches and dendrites. In the ascending circuits, individual PTCNs innervated the thalamus in three different patterns and projected to the cortex via two separate pathways. Moreover, PTCNs targeting the ventral tegmental area and substantia nigra had abundant collaterals in the pontine reticular nuclei, and these two circuits contributed oppositely to locomotion. Conclusions Our results suggest that individual PTCNs have abundant axons, and most project to various collaterals in the ascending and descending circuits simultaneously. They target regions with multiple patterns, such as the thalamus and cortex. These results provide a detailed organizational characterization of cholinergic neurons to understand the connexional logic of the upper brainstem.

The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. In our research, we investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. We applied GAL4 × UAS binary system to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies’ learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15–60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure.

[Figure: see text].

BackgroundWe previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus.MethodsWe developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5.Key resultsIn the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer.Conclusions and inferencesThe intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Basal forebrain cholinergic neurons (BFCNs) modulate cognitive functions such as attention, learning and memory. The NGF/TrkA pathway plays an important role in the development and function of BFCNs, although two mouse models conditionally deleting TrkA expression in the central nervous system (CNS) have shown contradictory results. To shed light into this discrepancy, we used a mouse model with a gain-of-function in TrkA receptor signaling. Our results indicate that enhanced TrkA signaling did not alter hippocampal cholinergic innervation, general locomotion or anxiety-related behaviors, but it increases ChAT expression, the number of cholinergic neurons at early postnatal stages and, mutant mice showed impaired motor learning and memory functions. These data demonstrate that proper functioning of the cholinergic system in CNS requires a balanced NGF/TrkA signaling. [ABSTRACT FROM AUTHOR]

[Display omitted] • The initial electrophysiological characterization of pallidal cholinergic neurons was performed. • Their firing resembles that of some type of basal forebrain cholinergic neurons. • Dopamine receptors activity sustain their membrane resting potential and excitability. • The blockade of D 2 -class receptors hyperpolarizes them and reduces their excitability. • The blockade of D 2 -class receptors changes their induced firing pattern. We employed the whole-cell patch-clamp method and ChAT-Cre mice to study the electrophysiological attributes of cholinergic neurons in the external globus pallidus. Most neurons were inactive, although approximately 20% displayed spontaneous firing, including burst firing. The resting membrane potential, the whole neuron input resistance, the membrane time constant and the total neuron membrane capacitance were also characterized. The current–voltage relationship showed time-independent inward rectification without a "sag". Firing induced by current injections had a brief initial fast adaptation followed by tonic firing with minimal accommodation. Intensity-frequency plots exhibited maximal average firing rates of about 10 Hz. These traits are similar to those of some cholinergic neurons in the basal forebrain. Also, we examined their dopamine sensitivity by acutely blocking dopamine receptors. This action demonstrated that the membrane potential, excitability, and firing pattern of pallidal cholinergic neurons rely on the constitutive activity of dopamine receptors, primarily D 2 -class receptors. The blockade of these receptors induced a resting membrane potential hyperpolarization, a decrease in firing for the same stimulus, the disappearance of fast adaptation, and the emergence of a depolarization block. This shift in physiological characteristics was evident even when the hyperpolarization was corrected with D.C. current. Neither the currents that generate the action potentials nor those from synaptic inputs were responsible. Instead, our findings suggest, that subthreshold slow ion currents, that require further investigation, are the target of this novel dopaminergic signaling. [ABSTRACT FROM AUTHOR]

Cholinergic changes play a fundamental role in the natural history of dementia with Lewy bodies and Lewy body disease in general. Despite important achievements in the field of cholinergic research, significant challenges remain. We conducted a study with four main objectives: (i) to examine the integrity of cholinergic terminals in newly diagnosed dementia with Lewy bodies; (ii) to disentangle the cholinergic contribution to dementia by comparing cholinergic changes in Lewy body patients with and without dementia; (iii) to investigate the in vivo relationship between cholinergic terminal loss and atrophy of cholinergic cell clusters in the basal forebrain at different stages of Lewy body disease; and (iv) to test whether any asymmetrical degeneration in cholinergic terminals would correlate with motor dysfunction and hypometabolism. To achieve these objectives, we conducted a comparative cross-sectional study of 25 newly diagnosed dementia with Lewy bodies patients (age 74 ± 5 years, 84% male), 15 healthy control subjects (age 75 ± 6 years, 67% male) and 15 Parkinson's disease patients without dementia (age 70 ± 7 years, 60% male). All participants underwent 18F-fluoroetoxybenzovesamicol PET and high-resolution structural MRI. In addition, we collected clinical 18F-fluorodeoxyglucose PET images. Brain images were normalized to standard space and regional tracer uptake and volumetric indices of basal forebrain degeneration were extracted. Patients with dementia showed spatially distinct reductions in cholinergic terminals across the cerebral cortex, limbic system, thalamus and brainstem. Also, cholinergic terminal binding in cortical and limbic regions correlated quantitatively and spatially with atrophy of the basal forebrain. In contrast, patients without dementia showed decreased cholinergic terminal binding in the cerebral cortex despite preserved basal forebrain volumes. In patients with dementia, cholinergic terminal reductions were most severe in limbic regions and least severe in occipital regions compared to those without dementia. Interhemispheric asymmetry of cholinergic terminals correlated with asymmetry of brain metabolism and lateralized motor function. In conclusion, this study provides robust evidence for severe cholinergic terminal loss in newly diagnosed dementia with Lewy bodies, which correlates with structural imaging measures of cholinergic basal forebrain degeneration. In patients without dementia, our findings suggest that loss of cholinergic terminal function occurs 'before' neuronal cell degeneration. Moreover, the study supports that degeneration of the cholinergic system is important for brain metabolism and may be linked with degeneration in other transmitter systems. Our findings have implications for understanding how cholinergic system pathology contributes to the clinical features of Lewy body disease, changes in brain metabolism and disease progression patterns. [ABSTRACT FROM AUTHOR]

Synapses are dynamic connections that underlie essential functions of the nervous system. The addition, removal, and maintenance of synapses govern the flow of information in neural circuits throughout the lifetime of an animal. While extensive studies have elucidated many intrinsic mechanisms that neurons employ to modulate their connections, increasing evidence supports the roles of non-neuronal cells, such as glia, in synapse maintenance and circuit function. We previously showed that C. elegans epidermis regulates synapses through ZIG-10, a cell-adhesion protein of the immunoglobulin domain superfamily. Here we identified a member of the Pals1/MPP5 family, MAGU-2, that functions in the epidermis to modulate phagocytosis and the number of synapses by regulating ZIG-10 localization. Furthermore, we used light and electron microscopy to show that this epidermal mechanism removes neuronal membranes from the neuromuscular junction, dependent on the conserved phagocytic receptor CED-1. Together, our study shows that C. elegans epidermis constrains synaptic connectivity, in a manner similar to astrocytes and microglia in mammals, allowing optimized output of neural circuits.

Williams syndrome (WS) is a rare neurodevelopmental disorder caused by the hemideletion of approximately 25-28 genes at 7q11.23. Its unusual social and cognitive phenotype is most strikingly characterized by the disinhibition of social behavior, in addition to reduced global IQ, with a relative sparing of language ability. Hypersociality and increased social approach behavior in WS may represent a unique inability to inhibit responses to specific social stimuli, which is likely associated with abnormalities of frontostriatal circuitry. The striatum is characterized by a diversity of interneuron subtypes, including inhibitory parvalbumin-positive interneurons (PV+) and excitatory cholinergic interneurons (Ch+). Animal model research has identified an important role for these specialized cells in regulating social approach behavior. Previous research in humans identified a depletion of interneuron subtypes associated with neuropsychiatric disorders. Here, we examined the density of PV+ and Ch+ interneurons in the striatum of 13 WS and neurotypical (NT) subjects. We found a significant reduction in the density of Ch+ interneurons in the medial caudate nucleus and nucleus accumbens, important regions receiving cortical afferents from the orbitofrontal and ventromedial prefrontal cortex, and circuitry involved in language and reward systems. No significant difference in the distribution of PV+ interneurons was found. The pattern of decreased Ch+ interneuron densities in WS differs from patterns of interneuron depletion found in other disorders.

Nerve growth factor (NGF) gene therapy rescues and stimulates cholinergic neurons, which degenerate in Alzheimer's disease (AD). In a recent clinical trial for AD, intraparenchymal adeno-associated virus serotype 2 (AAV2)-NGF delivery was safe but did not improve cognition. Before concluding that NGF gene therapy is ineffective, it must be shown that AAV2-NGF successfully engaged the target cholinergic neurons of the basal forebrain. In this study, patients with clinically diagnosed early- to middle-stage AD received a total dose of 2 × 1011 vector genomes of AAV2-NGF by stereotactic injection of the nucleus basalis of Meynert. After a mean survival of 4.0 years, AAV2-NGF targeting, spread, and expression were assessed by immunolabeling of NGF and the low-affinity NGF receptor p75 at 15 delivery sites in 3 autopsied patients. NGF gene expression persisted for at least 7 years at sites of AAV2-NGF injection. However, the mean distance of AAV2-NGF spread was only 0.96 ± 0.34 mm. NGF did not directly reach cholinergic neurons at any of the 15 injection sites due to limited spread and inaccurate stereotactic targeting. Because AAV2-NGF did not directly engage the target cholinergic neurons, we cannot conclude that growth factor gene therapy is ineffective for AD. Upcoming clinical trials for AD will utilize real-time magnetic resonance imaging guidance and convection-enhanced delivery to improve the targeting and spread of growth factor gene delivery.

Interictal spikes (IIS) are a common type of abnormal electrical activity in Alzheimer's disease (AD) and preclinical models. The brain regions where IIS are largest are not known but are important because such data would suggest sites that contribute to IIS generation. Because hippocampus and cortex exhibit altered excitability in AD models, we asked which areas dominate the activity during IIS along the cortical-CA1-dentate gyrus (DG) dorso-ventral axis. Because medial septal (MS) cholinergic neurons are overactive when IIS typically occur, we also tested the novel hypothesis that silencing the MS cholinergic neurons selectively would reduce IIS.We used mice that simulate aspects of AD: Tg2576 mice, presenilin 2 (PS2) knockout mice and Ts65Dn mice. To selectively silence MS cholinergic neurons, Tg2576 mice were bred with choline-acetyltransferase (ChAT)-Cre mice and offspring were injected in the MS with AAV encoding inhibitory designer receptors exclusively activated by designer drugs (DREADDs). We recorded local field potentials along the cortical-CA1-DG axis using silicon probes during wakefulness, slow-wave sleep (SWS) and rapid eye movement (REM) sleep.We detected IIS in all transgenic or knockout mice but not age-matched controls. IIS were detectable throughout the cortical-CA1-DG axis and occurred primarily during REM sleep. In all 3 mouse lines, IIS amplitudes were significantly greater in the DG granule cell layer vs. CA1 pyramidal layer or overlying cortex. Current source density analysis showed robust and early current sources in the DG, and additional sources in CA1 and the cortex also. Selective chemogenetic silencing of MS cholinergic neurons significantly reduced IIS rate during REM sleep without affecting the overall duration, number of REM bouts, latency to REM sleep, or theta power during REM. Notably, two control interventions showed no effects.Consistent maximal amplitude and strong current sources of IIS in the DG suggest that the DG is remarkably active during IIS. In addition, selectively reducing MS cholinergic tone, at times when MS is hyperactive, could be a new strategy to reduce IIS in AD.

Alzheimer's disease (AD) features the loss of cholinergic neurons in the mesopontine area. There is no available approach to repair the damaged cholinergic neurons. Neural stem cell (NSC)‐based therapy is a promising strategy for the treatment of AD. However, it is a challenge to direct the NSCs to specifically differentiate into cholinergic neurons. Herein, calcium folate (CaFO) nanoparticles are synthesized through a facile nanoprecipitation approach for promoting the differentiation of NSCs into functional cholinergic neurons. After uptake by NSCs, the CaFO nanoparticles are distributed in the lysosomes (pH< 5.5) and can be decomposed into Ca2+ and folic acid in the acidic environment. The Ca2+ can accelerate the differentiation rate of NSCs while the folic acid can direct the NSCs to differentiate into cholinergic neurons. The in vitro experiments demonstrate that under the stimulation of CaFO nanoparticles, the NSCs differentiate into functional cholinergic neurons within 5 d. Animal experiments prove that the CaFO nanoparticles also promote the neuronal differentiation of NSCs in vivo, leading to the improvement in the cognitive memory ability of AD mice. This study provides a new strategy to induce the quick differentiation of NSCs into functional cholinergic neurons, which is promising for the treatment of AD. [ABSTRACT FROM AUTHOR]

Background: Based on their anatomical location, rostral projections of nuclei are classified as ascending circuits, while caudal projections are classified as descending circuits. Upper brainstem neurons participate in complex information processing and specific sub-populations preferentially project to participating ascending or descending circuits. Cholinergic neurons in the upper brainstem have extensive collateralizations in both ascending and descending circuits; however, their single-cell projection patterns remain unclear because of the lack of comprehensive characterization of individual neurons. Results: By combining fluorescent micro-optical sectional tomography with sparse labeling, we acquired a high-resolution whole-brain dataset of pontine-tegmental cholinergic neurons (PTCNs) and reconstructed their detailed morphology using semi-automatic reconstruction methods. As the main source of acetylcholine in some subcortical areas, individual PTCNs had abundant axons with lengths up to 60 cm and 5000 terminals and innervated multiple brain regions from the spinal cord to the cortex in both hemispheres. Based on various collaterals in the ascending and descending circuits, individual PTCNs were grouped into four subtypes. The morphology of cholinergic neurons in the pedunculopontine nucleus was more divergent, whereas the laterodorsal tegmental nucleus neurons contained richer axonal branches and dendrites. In the ascending circuits, individual PTCNs innervated the thalamus in three different patterns and projected to the cortex via two separate pathways. Moreover, PTCNs targeting the ventral tegmental area and substantia nigra had abundant collaterals in the pontine reticular nuclei, and these two circuits contributed oppositely to locomotion. Conclusions: Our results suggest that individual PTCNs have abundant axons, and most project to various collaterals in the ascending and descending circuits simultaneously. They target regions with multiple patterns, such as the thalamus and cortex. These results provide a detailed organizational characterization of cholinergic neurons to understand the connexional logic of the upper brainstem. [ABSTRACT FROM AUTHOR]

Simple Summary: Environmental pollutants have been suggested to be among the possible causes that play a role in the generation of Alzheimer's disease (AD) and other neurodegenerative diseases. Included in these environmental pollutants, we find the highly used plasticizer bisphenol-A, which produces neurodegeneration and cognitive disorders similar to those induced in AD. However, the mechanisms through which this and other environmental pollutants produce these effects are unknown. In AD, as well as in other neurodegenerative diseases that produce cognitive disorders, a selective cholinergic neuronal loss is induced in the brain region of the basal forebrain, which in turn leads to the denervation of the hippocampus and cortex, producing neurodegeneration in these regions and, eventually, cognitive disorders. Our results show the alteration of some mechanisms that could mediate bisphenol-A disruption of synaptic plasticity and neurodegeneration induction in this specific type of basal forebrain neurons. These results may assist to elucidate the processes that mediate the cognition alterations produced by bisphenol-A and other environmental pollutants, which it shares mechanisms with, and could lead to the development of preventive and therapeutic tools to avoid and treat these effects in the population. The widely used plasticizer bisphenol-A (BPA) is well-known for producing neurodegeneration and cognitive disorders, following acute and long-term exposure. Although some of the BPA actions involved in these effects have been unraveled, they are still incompletely known. Basal forebrain cholinergic neurons (BFCN) regulate memory and learning processes and their selective loss, as observed in Alzheimer's disease and other neurodegenerative diseases, leads to cognitive decline. In order to study the BPA neurotoxic effects on BFCN and the mechanisms through which they are induced, 60-day old Wistar rats were used, and a neuroblastoma cholinergic cell line from the basal forebrain (SN56) was used as a basal forebrain cholinergic neuron model. Acute treatment of rats with BPA (40 µg/kg) induced a more pronounced basal forebrain cholinergic neuronal loss. Exposure to BPA, following 1- or 14-days, produced postsynaptic-density-protein-95 (PSD95), synaptophysin, spinophilin, and N-methyl-D-aspartate-receptor-subunit-1 (NMDAR1) synaptic proteins downregulation, an increase in glutamate content through an increase in glutaminase activity, a downregulation in the vesicular-glutamate-transporter-2 (VGLUT2) and in the WNT/β-Catenin pathway, and cell death in SN56 cells. These toxic effects observed in SN56 cells were mediated by overexpression of histone-deacetylase-2 (HDAC2). These results may help to explain the synaptic plasticity, cognitive dysfunction, and neurodegeneration induced by the plasticizer BPA, which could contribute to their prevention. [ABSTRACT FROM AUTHOR]

Aging is a physiological process accompanied by a decline in cognitive performance. The cholinergic neurons of the basal forebrain provide projections to the cortex that are directly engaged in many cognitive processes in mammals. In addition, basal forebrain neurons contribute to the generation of different rhythms in the EEG along the sleep/wakefulness cycle. The aim of this review is to provide an overview of recent advances grouped around the changes in basal forebrain activity during healthy aging. Elucidating the underlying mechanisms of brain function and their decline is especially relevant in today's society as an increasingly aged population faces higher risks of developing neurodegenerative diseases such as Alzheimer's disease. The profound age-related cognitive deficits and neurodegenerative diseases associated with basal forebrain dysfunction highlight the importance of investigating the aging of this brain region. [ABSTRACT FROM AUTHOR]

Basal forebrain cholinergic neurons (BFCNs) modulate cognitive functions such as attention, learning and memory. The NGF/TrkA pathway plays an important role in the development and function of BFCNs, although two mouse models conditionally deleting TrkA expression in the central nervous system (CNS) have shown contradictory results. To shed light into this discrepancy, we used a mouse model with a gain-of-function in TrkA receptor signaling. Our results indicate that enhanced TrkA signaling did not alter hippocampal cholinergic innervation, general locomotion or anxiety-related behaviors, but it increases ChAT expression, the number of cholinergic neurons at early postnatal stages and, mutant mice showed impaired motor learning and memory functions. These data demonstrate that proper functioning of the cholinergic system in CNS requires a balanced NGF/TrkA signaling.

Vesicular glutamate transporters (VGLUTs) mediate the synaptic uptake of glutamate from the cytosol into synaptic vesicles and are considered unambiguous neurochemical markers of glutamate neurons. However, many neurons not classically thought of as glutamatergic also express a VGLUT and co-release glutamate. Using a genetic fate-mapping strategy we found that most cholinergic neurons in the mouse mesopontine tegmentum express VGLUT2 at some point during development, including the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus, and parabigeminal nucleus (PBG), but not the oculomotor nucleus. In contrast, very few of these cholinergic neurons displayed evidence of vesicular GABA transporter expression. Using multiplex fluorescent in situ hybridization, we determined that only PBG cholinergic neurons are also predominantly positive for VGLUT2 mRNA in the adult, with only small numbers of PPTg cholinergic neurons overlapping with VGLUT2 mRNA. Using Cre-dependent viral vectors we confirm these in situ hybridization data, and demonstrate projection patterns of cholinergic and glutamatergic populations. These results demonstrate that most mesopontine cholinergic neurons may transiently express VGLUT2, but that a large majority of PBG neurons retain VGLUT2 expression throughout adulthood, and support a growing body of literature indicating that distinct cholinergic populations have differing potential for GABA or glutamate co-release.

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.

Propensity to relapse, even following long periods of abstinence, is a key feature in substance use disorders. Relapse and relapse-like behaviors are known to be induced, in part, by re-exposure to drug-associated cues. Yet, while many critical nodes in the neural circuitry contributing to relapse have been identified and studied, a full description of the networks driving reinstatement of drug-seeking behaviors is lacking. One area that may provide further insight to the mechanisms of relapse is the habenula complex, an epithalamic region composed of lateral and medial (MHb) substructures, each with unique cell and target populations. Although well conserved across vertebrate species, the functions of the MHb are not well understood. Recent research has demonstrated that the MHb regulates nicotine aversion and withdrawal. However, it remains undetermined whether MHb function is limited to nicotine and aversive stimuli or if MHb circuit regulates responses to other drugs of abuse. Advances in circuit-level manipulations now allow for cell-type and temporally specific manipulations during behavior, specifically in spatially restrictive brain regions, such as the MHb. In this study, we focus on the response of the MHb to reinstatement of cocaine-associated behavior, demonstrating that cocaine-primed reinstatement of conditioned place preference engages habenula circuitry. Using chemogenetics, we demonstrate that MHb activity is sufficient to induce reinstatement behavior. Together, these data identify the MHb as a key hub in the circuitry underlying reinstatement and may serve as a target for regulating relapse-like behaviors.

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the world. The aggregation of both amyloid beta (Aβ) peptides extracellularly and Tau proteins intracellularly plays key roles in the pathological consequences of AD, which lead to cholinergic neurodegeneration and eventually death. Currently, there are no effective methods to stop the progression of AD. Using ex vivo , in vivo and clinical approaches, we investigated the functional effects of plasminogen on the widely used FAD, Aβ42 oligomer or Tau intracranial injection-induced AD mouse model and explored its therapeutic effects on patients with AD. The results show that intravenously injected plasminogen rapidly crosses the blood‒brain barrier (BBB); increases plasmin activity in the brain; colocalizes with and effectively promotes the clearance of Aβ42 peptide and Tau protein deposits ex vivo and in vivo ; increases the choline acetyltransferase (ChAT) level and decreases the acetylcholinesterase (AChE) activity; and improves the memory functions. Clinically, when GMP-level plasminogen was administered to 6 AD patients for 1–2 weeks, their average scores on the Minimum Mental State Examination (MMSE), which is a standard scoring system used to measure the memory loss and cognitive deficits, were extremely significantly improved by 4.2 ± 2.23 points, e.g., an average increase from 15.5 ± 8.22 before treatment to 19.7 ± 7.09 after treatment. The preclinical study and pilot clinical study suggest that plasminogen is effective in treating AD and may be a promising drug candidate. • Plasminogen crosses the blood‒brain barrier (BBB) and increases plasmin activity in the brain. • Plasminogen promotes the clearance of Aβ42 peptide and Tau protein. • Plasminogen improves the memory functions in AD mouse model. • Plasminogen increases the Minimum Mental State Examination (MMSE) score in AD patients. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cholinergic neurons, a type of neurons found in central nervous system, play a vital role in muscle movement and activities. Cholinergic neurons degeneration is the main pathological symptom of neurodegenerative diseases. Among a variety of stem cells, iPSCs have emerged as a promising candidate for transplantation to improve the repair of neuronal lesion sites. However, the establishment of an appropriate induction method to yield large numbers of cholinergic neurons has yet to be determined. Here, we studied the differentiation potential of iPSCs to generate cholinergic neurons by developing a new optimized differentiation protocol. METHODS: The iPSCs were harvested on 6-well matrigel-coated plate and incubated with serum-free DMEM/F12 with 2 % B27 supplement, 20 ng/ml the basic fibroblast growth factor and 20 ng/ml epidermal growth factor for 48 hours. Then, the pre-induced cells were treated in neuronal induction medium supplemented with all-trans retinoic acid, sonic hedgehog, 100 ng/ml glial-derived neurotrophic factor and 200 ng/ml brainderived neurotrophic factor for 7 days. Cell viability during induction stages was tested by MTT assay. Differentiated cells were evaluated with crystal violet staining, immunocytochemistry and real-time PCR. RESULTS: Our results showed that the survival rate of iPSCs leveled out and was similar to that in the control group following the differentiation process. Immunochemistry results revealed that the expression of ChAT was observed in cells in both pre-induction and induction stages with a significantly higher expression level at the induction stage as compared to the pre-induction stage. However, none of these markers was expressed in the iPSCs. Cresyl violet staining confirmed the neuronal phenotype of differentiated cells. The induction group significantly expressed the higher levels of Islet1, Olig2 and HB9, whereas pluripotency markers including those of Oct4 and Nestin plunged. CONCLUSION: Our investigation represents a highly efficient protocol for iPSCs differentiation toward cholinergic neurons which could be used for further preclinical transplantation studies. [ABSTRACT FROM AUTHOR]