Contributions of cholinergic receptor muscarinic 1 and CYP1A2 gene variants on the effects of plasma ratio of clozapine/N-desmethylclozapine on working memory in schizophrenia

Background: Clozapine has heterogenous efficacy in enhancing working memory in schizophrenia. We have previously hypothesized that this is due to opposing effects of clozapine and its metabolite, N-desmethylclozapine, at the muscarinic M1 receptor and demonstrated that a lower clozapine/ N-desmethylclozapine ratio is associated with better working memory than clozapine or N-desmethylclozapine levels alone. Aims: In this study, we expanded the above hypothesis to explore whether genetic variation in the cholinergic receptor muscarinic 1 gene, encoding the M1 receptor, affects the relationship between clozapine/ N-desmethylclozapine and working memory. Further, we explored whether CYP1A2 gene variants affect the ratio of clozapine/ N-desmethylclozapine and by this, working memory performance. Methods: We evaluated two functionally significant single nucleotide polymorphisms, rs1942499 and rs2075748, in cholinergic receptor muscarinic 1, with the haplotype T-A associated with lower transcriptional activity than the haplotype C-G. Further, we examined CYP1A2 *1F, with *1F/*1F conferring high inducibility in the presence of smoking. Results: In a sample of 30 patients with schizophrenia on clozapine monotherapy, clozapine/ N-desmethylclozapine was correlated with working memory only in non-carriers of the haplotype T-A of the cholinergic receptor muscarinic 1 gene. Interaction of CYP1A2 genotype and smoking status significantly affected clozapine concentrations, but there were no significant effects of CYP1A2 genotype and smoking status on the relationship between clozapine/ N-desmethylclozapine on working memory. Conclusions: Our finding that the relationship between clozapine/ N-desmethylclozapine and working memory is specific to patients with potentially higher transcription of M1 receptor (i.e. non-carriers of the haplotype T-A of cholinergic receptor muscarinic 1) supports a cholinergic mechanism underlying this relationship.

Amyloid Beta Peptide (Aβ1-42) Reverses the Cholinergic Control of Monocytic IL-1β Release

Amyloid-β peptide (Aβ1-42), the cleavage product of the evolutionary highly conserved amyloid precursor protein, presumably plays a pathogenic role in Alzheimer’s disease. Aβ1-42 can induce the secretion of the pro-inflammatory cytokine intereukin-1β (IL-1β) in immune cells within and out of the nervous system. Known interaction partners of Aβ1-42 are α7 nicotinic acetylcholine receptors (nAChRs). The physiological functions of Aβ1-42 are, however, not fully understood. Recently, we identified a cholinergic mechanism that controls monocytic release of IL-1β by canonical and non-canonical agonists of nAChRs containing subunits α7, α9, and/or α10. Here, we tested the hypothesis that Aβ1-42 modulates this inhibitory cholinergic mechanism. Lipopolysaccharide-primed monocytic U937 cells and human mononuclear leukocytes were stimulated with the P2X7 receptor agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate triethylammonium salt (BzATP) in the presence or absence of nAChR agonists and Aβ1-42. IL-1β concentrations were measured in the supernatant. Aβ1-42 dose-dependently (IC50 = 2.54 µM) reversed the inhibitory effect of canonical and non-canonical nicotinic agonists on BzATP-mediated IL-1β-release by monocytic cells, whereas reverse Aβ42-1 was ineffective. In conclusion, we discovered a novel pro-inflammatory Aβ1-42 function that enables monocytic IL-1β release in the presence of nAChR agonists. These findings provide evidence for a novel physiological function of Aβ1-42 in the context of sterile systemic inflammation.

Perspectives on the use of electrostimulation with the device “VEB”® in the management of disorders related to COVID-19

Background. One of the symptoms of COVID-19 is the so-called "cytokine storm". Its pathogenesis is that the initial release by lymphocytes and macrophages of proinflammatory cytokines in the classical immune response to SARS-CoV-2 is significantly enhanced and maintained due to excessive adrenergic stimulation of the immune cells. The proinflammatory adrenergic mechanism of the "cytokine storm" can be offset by the activation of the anti-inflammatory cholinergic mechanism by non-invasive stimulation of the vagus nerve. In 2015, a generator for electrotherapy and stimulation oh human nerve centers was created, called “VEB-1”®. Preliminary observation of volunteers revealed a modulating effect of a four-day course of electrical stimulation on the parameters of electroencephalogram, metabolism, as well as gas-discharge visualization (GDV). We hypothesized that changes in EEG parameters may be accompanied by a vagotonic shift of the sympatho-vagus balance, favorable for calming the “cytokine storm”. The main purpose of this study was to find out. In addition, concomitant changes in EEG, immunity, GDV, etc. due to the use of the devices "VEB-1"® and recently designed "VEB-2" had to be detected. Material and research methods. The object of observation were 18 volunteers: 11 women 33-62 y and 7 men 29-62 y (Mean±SD: 51±12 y) without clinical diagnose but with dysfunction of neuro-endocrine-immune complex and metabolism. In the morning registered HRV (“CardioLab+HRV”, “KhAI-Medica”, Kharkiv, UA), EEG (“NeuroCom Standard”, “KhAI-Medica”, Kharkiv, UA), kirlianogram by the method of GDV (“GDV Chamber”, “Biotechprogress”, SPb, RF), electroconductivity of skin in three pairs of points of acupuncture (“Medissa”), electrokinetic index of buccal epithelium ("Biotest", Kharkiv State University), as well as some parameters of immunity and metabolism. After the initial testing, an electrical stimulation session was performed with a “VEB-1”® or a “VEB-2” devices. The next morning after completing the four-day course, retesting was performed. Results. The effects of electrical stimulation can be divided into the following networks. Regarding EEG, this is a leveling of right-hand lateralization and normalizing decrease in the increased of the amplitude of the θ-rhythm and its spectral power density (SPD) at the loci F3, F7, F8, T3, T4, T6, P3, O1 and O2; further increase of SPD of δ-rhythm in loci F3, F4, T6, P3 and O1 as well as further decrease of SPD F4-α; reversion of the increased level of entropy in loci Fp1, F4, C3 and P3 to the lowered level. Regarding HRV, it is a vagotonic shift of sympatho-vagus balance due to a decrease in elevated levels of sympathetic tone markers and an increase in decreased levels of vagus tone markers, but without normalization. Neurotropic effects are accompanied by favorable changes in a number of immune parameters and a tendency to decrease the level of C-Reactive Protein. Regarding GDV, it is almost complete normalization of the initially increased GDI Area in the frontal projection and third Chakra Energy; normalizing decrease in the initially increased Energy of second and seventh Chakras; normalizing right-hand shift of more or less pronounced left-sided Asymmetry of first and third Chakra. These effects should be clearly interpreted as physiologically beneficial. The effects on these parameters are almost equally pronounced in people of both sexes when using both devices. Conclusion. Vagotonic and immunotropic effects of our device give us a reason to offer it for further research on the leveling of “cytokine storm” in patients with COVID-19.

Perspectives on the use of electrostimulation with the device “VEB”® in the management of disorders related to COVID-19

Background. One of the symptoms of COVID-19 is the so-called "cytokine storm". Its pathogenesis is that the initial release by lymphocytes and macrophages of proinflammatory cytokines in the classical immune response to SARS-CoV-2 is significantly enhanced and maintained due to excessive adrenergic stimulation of the immune cells. The proinflammatory adrenergic mechanism of the "cytokine storm" can be offset by the activation of the anti-inflammatory cholinergic mechanism by non-invasive stimulation of the vagus nuclei (VNS). In 2015, a generator for electrotherapy and stimulation oh human nerve centers was created, called “VEB-1”. Preliminary observation of volunteers revealed a modulating effect of a four-day course of electrical stimulation on the parameters of electroencephalogram, metabolism, as well as gas-discharge visualization (GDV).. This report launches a series of articles on a comparative study of the course effects of the devices "VEB-1" and recently designed "VEB-2" on the sympathetic-vagal balance as key link of pathogenesis of "cytokine storm". Material and research methods. The object of observation were 18 volunteers: 11 women 33-62 y and 7 men 29-62 y (Mean±SD: 51±12 y) without clinical diagnose but with dysfunction of neuro-endocrine-immune complex and metabolism. In the morning registered HRV (“CardioLab+HRV”, “KhAI-Medica”, Kharkiv, UA), EEG (“NeuroCom Standard”, “KhAI-Medica”, Kharkiv, UA), kirlianogram by the method of GDV (“GDV Chamber”, “Biotechprogress”, SPb, RF), electroconductivity of skin in points of acupuncture (“Medissa”), electrokinetic index of buccal epithelium ("Biotest", Kharkiv State University), as well as some parameters of immunity and metabolism. After the initial testing, an electrical stimulation session was performed with a VEB-1 or a VEB-2 devices. The next morning after completing the four-day course, retesting was performed. Results. The effects of electrical stimulation can be divided into the following networks. Regarding EEG, this is a leveling of right-hand lateralization and normalizing decrease in the increased of the amplitude of the θ-rhythm and its spectral power density (SPD) at the loci F3, F7, F8, T3, T4, T6, P3, O1 and O2; further increase of SPD of δ-rhythm in loci F3, F4, T6, P3 and O1 as well as further decrease of SPD F4-α; reversion of the increased level of entropy in loci Fp1, F4, C3 and P3 to the lowered level. Regarding HRV, it is a vagotonic shift of sympatho-vagus balance due to a decrease in elevated levels of sympathetic tone markers and an increase in decreased levels of vagus tone markers, but without normalization. Neurotropic effects are accompanied by favorable changes in a number of immune parameters and a tendency to decrease the level of C-Reactive Protein. Regarding GDV, it is almost complete normalization of the initially increased GDI Area in the frontal projection and third Chakra Energy; normalizing decrease in the initially increased Energy of second and seventh Chakras; normalizing right-hand shift of more or less pronounced left-sided Asymmetry of first and third Chakra. These effects should be clearly interpreted as physiologically beneficial. The effects on these parameters are almost equally pronounced in people of both sexes when using both devices. Conclusion. Vagotonic and immunotropic effects of our device give us a reason to offer it for further research on the leveling of cytokine storm in COVID-19.

A novel role for cortical acetylcholine in object memory updating

Reactivated long-term memories can become labile and sensitive to modification. Memories in this destabilized state can be weakened or strengthened, but there is limited research characterizing the mechanisms underlying retrieval-induced qualitative updates (i.e., information integration). We have previously implicated cholinergic transmission in object memory destabilization. Here we present a novel rodent paradigm developed to assess the role of this cholinergic mechanism in qualitative memory updating. The post-reactivation object memory modification (PROMM) task exposes rats to contextual information following object memory reactivation. Subsequent object exploratory performance suggests that the contextual information is integrated with the original memory in a reactivation- and time-dependent manner. This effect is blocked by interference with M1 muscarinic receptors and several downstream signals in perirhinal cortex. These findings therefore demonstrate a hitherto unacknowledged cognitive function for acetylcholine with important implications for understanding the dynamic nature of long-term memory storage in the normal and aging brain.

Carbachol-Induced Reduction in the Activity of Adult Male Zebra Finch RA Projection Neurons

Cholinergic mechanism is involved in motor behavior. In songbirds, the robust nucleus of the arcopallium (RA) is a song premotor nucleus in the pallium and receives cholinergic inputs from the basal forebrain. The activity of projection neurons in RA determines song motor behavior. Although many evidences suggest that cholinergic system is implicated in song production, the cholinergic modulation of RA is not clear until now. In the present study, the electrophysiological effects of carbachol, a nonselective cholinergic receptor agonist, were investigated on the RA projection neurons of adult male zebra finches through whole-cell patch-clamp techniques in vitro. Our results show that carbachol produced a significant decrease in the spontaneous and evoked action potential (AP) firing frequency of RA projection neurons, accompanying a hyperpolarization of the membrane potential, an increase in the evoked AP latency, afterhyperpolarization (AHP) peak amplitude, and AHP time to peak, and a decrease in the membrane input resistance, membrane time constant, and membrane capacitance. These results indicate that carbachol reduces the activity of RA projection neurons by hyperpolarizing the resting membrane potential and increasing the AHP and the membrane conductance, suggesting that the cholinergic modulation of RA may play an important role in song production.

Cholinergic mechanisms of high-frequency stimulation in entopeduncular nucleus

Chronic, high-frequency (>100 Hz) electrical stimulation, known as deep brain stimulation (DBS), of the internal segment of the globus pallidus (GPi) is a highly effective therapy for Parkinson's disease (PD) and dystonia. Despite some understanding of how it works acutely in PD models, there remain questions about its mechanisms of action. Several hypotheses have been proposed, such as depolarization blockade, activation of inhibitory synapses, depletion of neurotransmitters, and/or disruption/alteration of network oscillations. In this study we investigated the cellular mechanisms of high-frequency stimulation (HFS) in entopeduncular nucleus (EP; rat equivalent of GPi) neurons using whole cell patch-clamp recordings. We found that HFS applied inside the EP nucleus induced a prolonged afterdepolarization that was dependent on stimulation frequency, pulse duration, and current amplitude. The high frequencies (>100 Hz) and pulse widths (>0.15 ms) used clinically for dystonia DBS could reliably induce these afterdepolarizations, which persisted under blockade of ionotropic glutamate (kynurenic acid, 2 mM), GABAA (picrotoxin, 50 μM), GABAB (CGP 55845, 1 μM), and acetylcholine nicotinic receptors (DHβE, 2 μM). However, this effect was blocked by atropine (2 μM; nonselective muscarinic antagonist) or tetrodotoxin (0.5 μM). Finally, the muscarinic-dependent afterdepolarizations were sensitive to Ca2+-sensitive nonspecific cationic (CAN) channel blockade. Hence, these data suggest that muscarinic receptor activation during HFS can lead to feedforward excitation through the opening of CAN channels. This study for the first time describes a cholinergic mechanism of HFS in EP neurons and provides new insight into the underlying mechanisms of DBS.