Spectroscopic Determination of Acetylcholine (ACh): A Representative Review.

Acetylcholine (ACh) is one of the most crucial neurotransmitters of the cholinergic system found in vertebrates and invertebrates and is responsible for many processes in living organisms. Disturbances in ACh transmission are closely related to dementia in Alzheimer's and Parkinson's disease. ACh in biological samples is most often determined using chromatographic techniques, radioenzymatic assays, enzyme-linked immunosorbent assay (ELISA), or potentiometric methods. An alternative way to detect and determine acetylcholine is applying spectroscopic techniques, due to low limits of detection and quantification, which is not possible with the methods mentioned above. In this review article, we described a detailed overview of different spectroscopic methods used to determine ACh with a collection of validation parameters as a perspective tool for routine analysis, especially in basic research on animal models on central nervous system. In addition, there is a discussion of examples of other biological materials from clinical and preclinical studies to give the whole spectrum of spectroscopic methods application. Descriptions of the developed chemical sensors, as well as the use of flow technology, were also presented. It is worth emphasizing the inclusion in the article of multi-component analysis referring to other neurotransmitters, as well as the description of the tested biological samples and extraction procedures. The motivation to use spectroscopic techniques to conduct this type of analysis and future perspectives in this field are briefly discussed.

Alzheimer's disease: Molecular aspects and treatment opportunities using herbal drugs.

Alzheimer's disease (AD), also called senile dementia, is the most common neurological disorder. Around 50 million people, mostly of advanced age, are suffering from dementia worldwide and this is expected to reach 100-130 million between 2040 and 2050. AD is characterized by impaired glutamatergic and cholinergic neurotransmission, which is associated with clinical and pathological symptoms. AD is characterized clinically by loss of cognition and memory impairment and pathologically by senile plaques formed by Amyloid ß deposits or neurofibrillary tangles (NFT) consisting of aggregated tau proteins. Amyloid ß deposits are responsible for glutamatergic dysfunction that develops NMDA dependent Ca2+ influx into postsynaptic neurons generating slow excitotoxicity process leading to oxidative stress and finally impaired cognition and neuronal loss. Amyloid decreases acetylcholine release, synthesis and neuronal transport. The decreased levels of neurotransmitter acetylcholine, neuronal loss, tau aggregation, amyloid ß plaques, increased oxidative stress, neuroinflammation, bio-metal dyshomeostasis, autophagy, cell cycle dysregulation, mitochondrial dysfunction, and endoplasmic reticulum dysfunction are the factors responsible for the pathogenesis of AD. Acetylcholinesterase, NMDA, Glutamate, BACE1, 5HT6, and RAGE (Receptors for Advanced Glycation End products) are receptors targeted in treatment of AD. The FDA approved acetylcholinesterase inhibitors Donepezil, Galantamine and Rivastigmine and N-methyl-D-aspartate antagonist Memantine provide symptomatic relief. Different therapies such as amyloid ß therapies, tau-based therapies, neurotransmitter-based therapies, autophagy-based therapies, multi-target therapeutic strategies, and gene therapy modify the natural course of the disease. Herbal and food intake is also important as preventive strategy and recently focus has also been placed on herbal drugs for treatment. This review focuses on the molecular aspects, pathogenesis and recent studies that signifies the potential of medicinal plants and their extracts or chemical constituents for the treatment of degenerative symptoms related to AD.

Basal forebrain cholinergic signalling: development, connectivity and roles in cognition.

Acetylcholine plays an essential role in fundamental aspects of cognition. Studies that have mapped the activity and functional connectivity of cholinergic neurons have shown that the axons of basal forebrain cholinergic neurons innervate the pallium with far more topographical and functional organization than was historically appreciated. Together with the results of studies using new probes that allow release of acetylcholine to be detected with high spatial and temporal resolution, these findings have implicated cholinergic networks in 'binding' diverse behaviours that contribute to cognition. Here, we review recent findings on the developmental origins, connectivity and function of cholinergic neurons, and explore the participation of cholinergic signalling in the encoding of cognition-related behaviours.

Age-related changes in basal forebrain afferent activation in response to food paired stimuli.

The basal forebrain contains a phenotypically-diverse assembly of neurons, including those using acetylcholine as their neurotransmitter. This basal forebrain cholinergic system projects to the entire neocortical mantle as well as subcortical limbic structures that include the hippocampus and amygdala. Basal forebrain pathology, including cholinergic dysfunction, is thought to underlie the cognitive impairments associated with several age-related neurodegenerative conditions, including Alzheimer's disease. Basal forebrain dysfunction may stem, in part, from a failure of normal afferent regulation of cholinergic and other neurons in this area. However, little is understood regarding how aging, alone, affects the functional regulation of basal forebrain afferents in the context of motivated behavior. Here, we used neuronal tract-tracing combined with motivationally salient stimuli in an aged rodent model to examine how aging alters activity in basal forebrain inputs arising from several cortical, limbic and brainstem structures. Young rats showed greater stimulus-associated activation of basal forebrain inputs arising from prelimbic cortex, nucleus accumbens and the ventral tegmental area compared with aged rats. Aged rats also showed increased latency to respond to palatable food presentation compared to young animals. Changes in activation of intrinsic basal forebrain cell populations or afferents were also observed as a function of age or experimental condition. These data further demonstrate that age-related changes in basal forebrain activation and related behavioral and cognitive functions reflect a failure of afferent regulation of this important brain region.

Muscarinic receptors in the rat ovary are involved in follicular development but not in steroid secretion.

Acetylcholine (ACh) may be involved in the regulation of ovarian functions. A previous systemic study in rats showed that a 4-week, intrabursal local delivery of the ACh-esterase blocker Huperzine-A increased intraovarian ACh levels and changed ovarian follicular development, as evidenced by increased healthy antral follicle numbers and corpora lutea, as well as enhanced fertility. To further characterize the ovarian cholinergic system in the rat, we studied whether innervation may contribute to intraovarian ACh. We explored the cellular distribution of three muscarinic receptors (MRs; M1, M3, and M5), analyzed the involvement of MRs in ovarian steroidogenesis, and examined their roles in ovarian follicular development in normal conditions and in animals exposed to stressful conditions by employing the muscarinic antagonist, atropine. Denervation studies decreased ovarian norepinephrine, but ovarian ACh was not affected, evidencing a local, nonneuronal source of ACh. M1 was located on granulosa cells (GCs), especially in large antral follicles. M5 was associated with the ovarian vascular system and only traces of M3 were found. Ex vivo ovary organo-typic incubations showed that the MR agonist Carbachol did not modify steroid production or expression of steroid biosynthetic enzymes. Intrabursal, in vivo application of atropine (an MR antagonist) for 4 weeks, however, increased atresia of the secondary follicles. The results support the existence of an intraovarian cholinergic system in the rat ovary, located mainly in follicular GCs, which is not involved in steroid production but rather via MRs exerts trophic functions and regulates follicular atresia.

Mechanisms of the end-plate potential noise and its implication for the neuromuscular junction anatomy.

We construct a compact model to mimic the membrane voltage response to the concentration of acetylcholine ([ACh]) which is mediated by the stochastic gating of acetylcholine (ACh) receptors. The patterns of the voltage depolarization against [ACh] as well as the accompanying voltage noises are presented. The mechanism of the voltage fluctuation that caused by the stochastic gating of receptors is explained. We consider that our results explain the frequently observed "end-plate (potential) noise" in physiology and electromyographic literature. These results, together with the requirements of evolution pressure on the motor units, explain reasonably the anatomical structure of the neuromuscular junction.

Differential roles of prelimbic and infralimbic cholinergic neurotransmissions in control of cardiovascular responses to restraint stress in rats.

Previous studies showed a prominent role of the medial prefrontal cortex (mPFC), especially the prelimbic (PL) and infralimbic (IL) subregions, in behavioral and physiological responses to stressful stimuli. Nevertheless, the local neurochemical mechanisms involved are not completely understood. In this sense, previous studies identified cholinergic terminals within the mPFC, and stressful stimuli increased local acetylcholine release. Despite these pieces of evidence, the specific role of cholinergic neurotransmission in different subregions of the mPFC controlling the cardiovascular responses to stress has never been systematically evaluated. Therefore, the purpose of this study was to investigate the involvement of cholinergic neurotransmission present within PL and IL in cardiovascular responses to an acute session of restraint stress in rats. For this, rats received bilateral microinjection of the choline uptake inhibitor hemicholinium-3 before exposure to restraint stress. The arterial pressure and heart rate (HR) increases and the decrease in tail skin temperature as an indirect measurement of sympathetically-mediated cutaneous vasoconstriction were recorded throughout the restraint stress session. The results showed that the depletion of acetylcholine within the PL caused by local microinjection of hemicholinium-3 decreased the tachycardia to restraint stress, but without affecting the pressor response and the drop in tail skin temperature. Conversely, IL treatment with hemicholinium-3 decreased the restraint-evoked pressor response and the sympathetically-mediated cutaneous vasoconstriction without interfering with the HR response. Taken together, these results indicate functional differences of cholinergic neurotransmission within the PL and IL in control of cardiovascular and autonomic responses to stressful stimuli.

Acetylcholine-gated current translates wake neuronal firing rate information into a spike timing-based code in Non-REM sleep, stabilizing neural network dynamics during memory consolidation.

Sleep is critical for memory consolidation, although the exact mechanisms mediating this process are unknown. Combining reduced network models and analysis of in vivo recordings, we tested the hypothesis that neuromodulatory changes in acetylcholine (ACh) levels during non-rapid eye movement (NREM) sleep mediate stabilization of network-wide firing patterns, with temporal order of neurons' firing dependent on their mean firing rate during wake. In both reduced models and in vivo recordings from mouse hippocampus, we find that the relative order of firing among neurons during NREM sleep reflects their relative firing rates during prior wake. Our modeling results show that this remapping of wake-associated, firing frequency-based representations is based on NREM-associated changes in neuronal excitability mediated by ACh-gated potassium current. We also show that learning-dependent reordering of sequential firing during NREM sleep, together with spike timing-dependent plasticity (STDP), reconfigures neuronal firing rates across the network. This rescaling of firing rates has been reported in multiple brain circuits across periods of sleep. Our model and experimental data both suggest that this effect is amplified in neural circuits following learning. Together our data suggest that sleep may bias neural networks from firing rate-based towards phase-based information encoding to consolidate memories.

Theta-gamma coupling emerges from spatially heterogeneous cholinergic neuromodulation.

Theta and gamma rhythms and their cross-frequency coupling play critical roles in perception, attention, learning, and memory. Available data suggest that forebrain acetylcholine (ACh) signaling promotes theta-gamma coupling, although the mechanism has not been identified. Recent evidence suggests that cholinergic signaling is both temporally and spatially constrained, in contrast to the traditional notion of slow, spatially homogeneous, and diffuse neuromodulation. Here, we find that spatially constrained cholinergic stimulation can generate theta-modulated gamma rhythms. Using biophysically-based excitatory-inhibitory (E-I) neural network models, we simulate the effects of ACh on neural excitability by varying the conductance of a muscarinic receptor-regulated K+ current. In E-I networks with local excitatory connectivity and global inhibitory connectivity, we demonstrate that theta-gamma-coupled firing patterns emerge in ACh modulated network regions. Stable gamma-modulated firing arises within regions with high ACh signaling, while theta or mixed theta-gamma activity occurs at the peripheries of these regions. High gamma activity also alternates between different high-ACh regions, at theta frequency. Our results are the first to indicate a causal role for spatially heterogenous ACh signaling in the emergence of localized theta-gamma rhythmicity. Our findings also provide novel insights into mechanisms by which ACh signaling supports the brain region-specific attentional processing of sensory information.

Activity of Selected Group of Monoterpenes in Alzheimer's Disease Symptoms in Experimental Model Studies-A Non-Systematic Review.

Alzheimer's disease (AD) is the leading cause of dementia and cognitive function impairment. The multi-faced character of AD requires new drug solutions based on substances that incorporate a wide range of activities. Antioxidants, AChE/BChE inhibitors, BACE1, or anti-amyloid platelet aggregation substances are most desirable because they improve cognition with minimal side effects. Plant secondary metabolites, used in traditional medicine and pharmacy, are promising. Among these are the monoterpenes-low-molecular compounds with anti-inflammatory, antioxidant, enzyme inhibitory, analgesic, sedative, as well as other biological properties. The presented review focuses on the pathophysiology of AD and a selected group of anti-neurodegenerative monoterpenes and monoterpenoids for which possible mechanisms of action have been explained. The main body of the article focuses on monoterpenes that have shown improved memory and learning, anxiolytic and sleep-regulating effects as determined by in vitro and in silico tests-followed by validation in in vivo models.

Role of Choline in Ocular Diseases.

Choline is essential for maintaining the structure and function of cells in humans. Choline plays an important role in eye health and disease. It is a precursor of acetylcholine, a neurotransmitter of the parasympathetic nervous system, and it is involved in the production and secretion of tears by the lacrimal glands. It also contributes to the stability of the cells and tears on the ocular surface and is involved in retinal development and differentiation. Choline deficiency is associated with retinal hemorrhage, glaucoma, and dry eye syndrome. Choline supplementation may be effective for treating these diseases.

The Property-Based Practical Applications and Solutions of Genetically Encoded Acetylcholine and Monoamine Sensors.

Neuromodulatory communication among various neurons and non-neuronal cells mediates myriad physiological and pathologic processes, yet defining regulatory and functional features of neuromodulatory transmission remains challenging because of limitations of available monitoring tools. Recently developed genetically encoded neuromodulatory transmitter sensors, when combined with superresolution and/or deconvolution microscopy, allow the first visualization of neuromodulatory transmission with nanoscale or microscale spatiotemporal resolution. In vitro and in vivo experiments have validated several high-performing sensors to have the qualities necessary for demarcating fundamental synaptic properties of neuromodulatory transmission, and initial analysis has unveiled unexpected fine control and precision of neuromodulation. These new findings underscore the importance of synaptic dynamics in synapse-, subcellular-, and circuit-specific neuromodulation, as well as the prospect of genetically encoded transmitter sensors in expanding our knowledge of various behaviors and diseases, including Alzheimer's disease, sleeping disorders, tumorigenesis, and many others.

The mammalian efferent vestibular system utilizes cholinergic mechanisms to excite primary vestibular afferents.

Electrical stimulation of the mammalian efferent vestibular system (EVS) predominantly excites primary vestibular afferents along two distinct time scales. Although roles for acetylcholine (ACh) have been demonstrated in other vertebrates, synaptic mechanisms underlying mammalian EVS actions are not well-characterized. To determine if activation of ACh receptors account for efferent-mediated afferent excitation in mammals, we recorded afferent activity from the superior vestibular nerve of anesthetized C57BL/6 mice while stimulating EVS neurons in the brainstem, before and after administration of cholinergic antagonists. Using a normalized coefficient of variation (CV*), we broadly classified vestibular afferents as regularly- (CV* < 0.1) or irregularly-discharging (CV* > 0.1) and characterized their responses to midline or ipsilateral EVS stimulation. Afferent responses to efferent stimulation were predominantly excitatory, grew in amplitude with increasing CV*, and consisted of fast and slow components that could be identified by differences in rise time and post-stimulus duration. Both efferent-mediated excitatory components were larger in irregular afferents with ipsilateral EVS stimulation. Our pharmacological data show, for the first time in mammals, that muscarinic AChR antagonists block efferent-mediated slow excitation whereas the nicotinic AChR antagonist DHßE selectively blocks efferent-mediated fast excitation, while leaving the efferent-mediated slow component intact. These data confirm that mammalian EVS actions are predominantly cholinergic.

Extrasynaptic acetylcholine signaling through a muscarinic receptor regulates cell migration.

Acetylcholine (ACh) promotes various cell migrations in vitro, but there are few investigations into this nonsynaptic role of ACh signaling in vivo. Here we investigate the function of a muscarinic receptor on an epithelial cell migration in Caenorhabditis elegans We show that the migratory gonad leader cell, the linker cell (LC), uses an M1/M3/M5-like muscarinic ACh receptor GAR-3 to receive extrasynaptic ACh signaling from cholinergic neurons for its migration. Either the loss of the GAR-3 receptor in the LC or the inhibition of ACh release from cholinergic neurons resulted in migratory path defects. The overactivation of the GAR-3 muscarinic receptor caused the LC to reverse its orientation through its downstream effectors Gαq/egl-30, PLCß/egl-8, and TRIO/unc-73 This reversal response only occurred in the fourth larval stage, which corresponds to the developmental time when the GAR-3::yellow fluorescent protein receptor in the membrane relocalizes from a uniform to an asymmetric distribution. These findings suggest a role for the GAR-3 muscarinic receptor in determining the direction of LC migration.

Endogenous Cholinergic Signaling Modulates Sound-Evoked Responses of the Medial Nucleus of the Trapezoid Body.

The medial nucleus of trapezoid body (MNTB) is a major source of inhibition in auditory brainstem circuitry. The MNTB projects well-timed inhibitory output to principal sound-localization nuclei in the superior olive (SOC) as well as other computationally important centers. Acoustic information is conveyed to MNTB neurons through a single calyx of Held excitatory synapse arising from the cochlear nucleus. The encoding efficacy of this large synapse depends on its activity rate, which is primarily determined by sound intensity and stimulus frequency. However, MNTB activity rate is additionally influenced by inhibition and possibly neuromodulatory inputs, albeit their functional role is unclear. Happe and Morley (2004) discovered prominent expression of α7 nAChRs in rat SOC, suggesting possible engagement of ACh-mediated modulation of neural activity in the MNTB. However, the existence and nature of this putative modulation have never been physiologically demonstrated. We probed nicotinic cholinergic influences on acoustic responses of MNTB neurons from adult gerbils (Meriones unguiculatus) of either sex. We recorded tone-evoked MNTB single-neuron activity in vivo using extracellular single-unit recording. Piggyback multibarrel electrodes enabled pharmacological manipulation of nAChRs by reversibly applying antagonists to two receptor types, α7 and α4ß2. We observed that tone-evoked responses are dependent on ACh modulation by both nAChR subtypes. Spontaneous activity was not affected by antagonist application. Functionally, we demonstrate that ACh contributes to sustaining high discharge rates and enhances signal encoding efficacy. Additionally, we report anatomic evidence revealing novel cholinergic projections to MNTB arising from pontine and superior olivary nuclei.SIGNIFICANCE STATEMENT This study is the first to physiologically probe how acetylcholine, a pervasive neuromodulator in the brain, influences the encoding of acoustic information by the medial nucleus of trapezoid body, the most prominent source of inhibition in brainstem sound-localization circuitry. We demonstrate that this cholinergic input enhances neural discrimination of tones from noise stimuli, which may contribute to processing important acoustic signals, such as speech. Additionally, we describe novel anatomic projections providing cholinergic input to the MNTB. Together, these findings shed new light on the contribution of neuromodulation to fundamental computational processes in auditory brainstem circuitry and to a more holistic understanding of modulatory influences in sensory processing.

The optomotor response of aging zebrafish reveals a complex relationship between visual motion characteristics and cholinergic system.

Understanding the principles underlying age-related changes in motion perception is paramount for improving the quality of life and health of older adults. However, the mechanisms underlying age-related alterations in this aspect of vision, which is essential for survival in a dynamic world, still remain unclear. Using optomotor responses to drifting gratings, we investigated age-related changes in motion detection of adult zebrafish (wild-type/AB-strain and achesb55/+ mutants with decreased levels of acetylcholinesterase). Our results pointed out negative optomotor responses that significantly depend on the spatial frequency and contrast level of stimulation, providing supporting evidence for the visual motion-driven aspect of this behavior mainly exhibited by adult zebrafish. Although there were no significant main effects of age and genotype, we found a significant three-way interaction between contrast level, age, and genotype. In the contrast domain, the changes in optomotor responses and thus in the detection of motion direction were age- and genotype-specific. Accordingly, these behavioral findings suggest a strong but complicated relationship between visual motion characteristics and the cholinergic system during neural aging.

Neurobiochemical Cross-talk Between COVID-19 and Alzheimer's Disease.

COVID-19, the global threat to humanity, shares etiological cofactors with multiple diseases including Alzheimer's disease (AD). Understanding the common links between COVID-19 and AD would harness strategizing therapeutic approaches against both. Considering the urgency of formulating COVID-19 medication, its AD association and manifestations have been reviewed here, putting emphasis on memory and learning disruption. COVID-19 and AD share common links with respect to angiotensin-converting enzyme 2 (ACE2) receptors and pro-inflammatory markers such as interleukin-1 (IL-1), IL-6, cytoskeleton-associated protein 4 (CKAP4), galectin-9 (GAL-9 or Gal-9), and APOE4 allele. Common etiological factors and common manifestations described in this review would aid in developing therapeutic strategies for both COVID-19 and AD and thus impact on eradicating the ongoing global threat. Thus, people suffering from COVID-19 or who have come round of it as well as people at risk of developing AD or already suffering from AD, would be benefitted.

Establishing Human Male and Female Models of Cholinergic Neurons via Neurokine-Mediated Differentiation of LA-N-2 and LA-N-5 Neuroblastoma Cells.

Cholinergic neurons control numerous primate-specific and sexually dimorphic brain functions. Here, we present our differentiation protocol for the closely related human female and male neuroblastoma-originated cell lines LA-N-2 and LA-N-5. Pro-cholinergic differentiation (with upregulation of choline acetyltransferase) of both lines can be achieved using neurokines such as ciliary neurotrophic factor (CNTF). Comparative RNA sequencing and mass spectrometry analyses between those two cell lines, supported by experimental intervention, will deepen our understanding of cholinergic systems in human psychiatric and neurologic disease. For complete details on the use and execution of this protocol, please refer to Lobentanzer et al. (2019).

Polysynaptic inhibition between striatal cholinergic interneurons shapes their network activity patterns in a dopamine-dependent manner.

Striatal activity is dynamically modulated by acetylcholine and dopamine, both of which are essential for basal ganglia function. Synchronized pauses in the activity of striatal cholinergic interneurons (ChINs) are correlated with elevated activity of midbrain dopaminergic neurons, whereas synchronous firing of ChINs induces local release of dopamine. The mechanisms underlying ChIN synchronization and its interplay with dopamine release are not fully understood. Here we show that polysynaptic inhibition between ChINs is a robust network motif and instrumental in shaping the network activity of ChINs. Action potentials in ChINs evoke large inhibitory responses in multiple neighboring ChINs, strong enough to suppress their tonic activity. Using a combination of optogenetics and chemogenetics we show the involvement of striatal tyrosine hydroxylase-expressing interneurons in mediating this inhibition. Inhibition between ChINs is attenuated by dopaminergic midbrain afferents acting presynaptically on D2 receptors. Our results present a novel form of interaction between striatal dopamine and acetylcholine dynamics.

Effects of basal forebrain stimulation on the vibrotactile responses of neurons from the hindpaw representation in the rat SI cortex.

Basal forebrain (BF) cholinergic system is important for attention and modulates sensory processing. We focused on the hindpaw representation in rat primary somatosensory cortex (S1), which receives inputs related to mechanoreceptors identical to those in human glabrous skin. Spike data were recorded from S1 tactile neurons (n = 87) with (ON condition: 0.5-ms bipolar current pulses at 100 Hz; amplitude 50 µA, duration 0.5 s at each trial) and without (OFF condition) electrical stimulation of BF in anesthetized rats. We expected that prior activation of BF would induce changes in the vibrotactile responses of neurons during sinusoidal (5, 40, and 250 Hz) mechanical stimulation of the glabrous skin. The experiment consisted of sequential OFF-ON conditions in two-time blocks separated by 30 min to test possible remaining effects. Average firing rates (AFRs) and vector strengths of spike phases (VS) were analyzed for different neuron types [regular spiking (RS) and fast spiking (FS)] in different cortical layers (III-VI). Immediate effect of BF activation was only significant by increasing synchronization to 5-Hz vibrotactile stimulus within the second block. Regardless of frequency, ON-OFF paired VS differences were significantly higher in the second block compared to the first, more prominent for RS neurons, and in general for neurons in layers III and VI. No such effects could be found on AFRs. The results suggest that cholinergic activation induces some changes in the hindpaw area, enabling relatively higher increases in synchronization to vibrotactile inputs with subsequent BF modulation. In addition, this modulation depends on neuron type and layer, which may be related to detailed projection pattern from BF.