Role of Ion Channels in the Chemotransduction and Mechanotransduction in Digestive Function and Feeding Behavior.

The gastrointestinal tract constantly communicates with the environment, receiving and processing a wide range of information. The contents of the gastrointestinal tract and the gastrointestinal tract generate mechanical and chemical signals, which are essential for regulating digestive function and feeding behavior. There are many receptors here that sense intestinal contents, including nutrients, microbes, hormones, and small molecule compounds. In signal transduction, ion channels are indispensable as an essential component that can generate intracellular ionic changes or electrical signals. Ion channels generate electrical activity in numerous neurons and, more importantly, alter the action of non-neurons simply and effectively, and also affect satiety, molecular secretion, intestinal secretion, and motility through mechanisms of peripheral sensation, signaling, and altered cellular function. In this review, we focus on the identity of ion channels in chemosensing and mechanosensing in the gastrointestinal tract.

Chemical and physical influences in bone and cartilage regeneration: a review of literature.

Nowadays several studies demonstrate the influence of chemical and physical stimulation to bone and cartilage exist. The first studies date back to the 50s and for a long time, they did not have a strong impact on clinical practice. In recent times, however, the findings arising from these studies are increasingly used to address clinical problems such as osteoarthritis or non-unions. The aim of this article is to make a review of the literature of the state of the art about physical and chemical influences on bone and cartilage.

Effects of chemical and mechanical stimulation on laryngeal motion during alfaxalone, thiopentone or propofol anaesthesia in healthy dogs.

OBJECTIVE: To compare the effect of chemical and mechanical stimulation on arytenoid cartilage motion during anaesthetic induction with alfaxalone, thiopentone or propofol. STUDY DESIGN: Masked, randomized, crossover study. ANIMALS: A group of eight adult Beagle dogs. METHODS: Anaesthesia was induced with thiopentone (7.5 mg kg-1), propofol (3 mg kg-1) or alfaxalone (1.5 mg kg-1) intravenously (IV), which were concurrently paired with either chemical (doxapram at 2.5 mg kg-1 IV) or mechanical (gentle pressure to the corniculate process of the right arytenoid cartilage using a cotton bud) stimulation for enhanced assessment of laryngeal motion, in random order, with a 1 week wash-out period between treatments. If deemed inadequately anaesthetized, supplemental boli of thiopentone (1.8 mg kg-1), propofol (0.75 mg kg-1) or alfaxalone (0.4 mg kg-1) were administered. Assessment of number of arytenoid motions and vital breaths, among others, was initiated immediately after induction. Chemical (doxapram) and mechanical stimulation were begun 2 minutes after anaesthetic induction. Data were collected at 2, 3 and 5 minutes after anaesthetic induction and the Friedman rank-sum or repeated-measures analysis of variance tests were used when applicable for statistical analysis. RESULTS: The duration of examination time was shorter among treatments combined with chemical stimulation (p=0.001). Examination time during induction was longer for alfaxalone-chemical (8.9 minutes) and -mechanical (10.9 minutes) compared to both induction with thiopentone-chemical (3.8 minutes) and propofol-chemical (4.0 minutes). The median number of arytenoid motions for both thiopentone (67) and propofol (59) induction combined with chemical stimulation was significantly higher in comparison to that of alfaxalone (1), thiopentone (2) and propofol (2), when combined with mechanical stimulation at 3 minutes after induction. CONCLUSION AND CLINICAL RELEVANCE: Among the regimens for assessing laryngeal motion assessed in the present study, combinations of thiopentone or propofol with doxapram are the most effective means of stimulating arytenoid motion and could improve the accuracy of diagnosis of laryngeal paralysis in dogs.

A membrane-based microfluidic device for mechano-chemical cell manipulation.

We introduce a microfluidic device for chemical manipulation and mechanical investigation of circulating cells. The device consists of two crossing microfluidic channels separated by a porous membrane. A chemical compound is flown through the upper "stimulus channel", which diffuses through the membrane into the lower "cell analysis channel", in which cells are mechanically deformed in two sequential narrow constrictions, one before and one after crossing the stimulus channel. Thus, this system permits to measure cell deformability before and after chemical cues are delivered to the cells within one single chip. The validity of the device was tested with monocytic cells stimulated with an actin-disrupting agent (Cytochalasin-D). Furthermore, as proof of principle of the device application, the effect of an anti-inflammatory drug (Pentoxifylline) was tested on monocytic cells activated with Lipopolysaccharides and on monocytes from patients affected by atherosclerosis. The results show that the system can detect differences in cell mechanical deformation after chemical cues are delivered to the cells through the porous membrane. Diffusion of Cytochalasin-D resulted in a considerable decrease in entry time in the narrow constriction and an evident increase in the velocity within the constriction. Pentoxifylline showed to decrease the entry time but not to affect the transit time within the constriction for monocytic cells. Monocytes from patients affected by atherosclerosis were difficult to test in the device due to increased adhesion to the walls of the microfluidic channel. Overall, this analysis shows that the device has potential applications as a cellular assay for analyzing cell-drug interaction.

The mechanical effects of chemical stimuli on neurospheres.

The formulation of more accurate models to describe tissue mechanics necessitates the availability of tools and instruments that can precisely measure the mechanical response of tissues to physical loads and other stimuli. In this regard, neuroscience has trailed other life sciences owing to the unavailability of representative live tissue models and deficiency of experimentation tools. We previously addressed both challenges by employing a novel instrument called the cantilevered-capillary force apparatus (CCFA) to elucidate the mechanical properties of mouse neurospheres under compressive forces. The neurospheres were derived from murine stem cells, and our study was the first of its kind to investigate the viscoelasticity of living neural tissues in vitro. In the current study, we demonstrate the utility of the CCFA as a broadly applicable tool to evaluate tissue mechanics by quantifying the effect that oxidative stress has on the mechanical properties of neurospheres. We treated mouse neurospheres with non-cytotoxic levels of hydrogen peroxide and subsequently evaluated the storage and loss moduli of the tissues under compression and tension. We observed that the neurospheres exhibit viscoelasticity consistent with neural tissue and show that elastic modulus decreases with increasing size of the neurosphere. Our study yields insights for establishing rheological measurements as biomarkers by laying the groundwork for measurement techniques and showing that the influence of a particular treatment may be misinterpreted if the size dependence is ignored.

Electrical and chemical modulation of homogeneous and heterogeneous human-iPSCs-derived neuronal networks on high density arrays.

The delicate "Excitatory/Inhibitory balance" between neurons holds significance in neurodegenerative and neurodevelopmental diseases. With the ultimate goal of creating a faithful in vitro model of the human brain, in this study, we investigated the critical factor of heterogeneity, focusing on the interplay between excitatory glutamatergic (E) and inhibitory GABAergic (I) neurons in neural networks. We used high-density Micro-Electrode Arrays (MEA) with 2304 recording electrodes to investigate two neuronal culture configurations: 100% glutamatergic (100E) and 75% glutamatergic / 25% GABAergic (75E25I) neurons. This allowed us to comprehensively characterize the spontaneous electrophysiological activity exhibited by mature cultures at 56 Days in vitro, a time point in which the GABA shift has already occurred. We explored the impact of heterogeneity also through electrical stimulation, revealing that the 100E configuration responded reliably, while the 75E25I required more parameter tuning for improved responses. Chemical stimulation with BIC showed an increase in terms of firing and bursting activity only in the 75E25I condition, while APV and CNQX induced significant alterations on both dynamics and functional connectivity. Our findings advance understanding of diverse neuron interactions and their role in network activity, offering insights for potential therapeutic interventions in neurological conditions. Overall, this work contributes to the development of a valuable human-based in vitro system for studying physiological and pathological conditions, emphasizing the pivotal role of neuron diversity in neural network dynamics.

Microfluidic Aqueous Two-Phase Focusing of Chemical Species for In Situ Subcellular Stimulation.

Confinement of chemical species in a controllable micrometer-level (several to a dozen micrometers) space in an aqueous environment is essential for precisely manipulating chemical events in subcellular regions. However, rapid diffusion and hard-to-control micrometer-level fluids make it a tough challenge. Here, a versatile open microfluidic method based on an aqueous two-phase system (ATPS) is developed to restrict species inside an open space with micron-level width. Unequal standard chemical potentials of the chemical species in two phases and space-time correspondence in the microfluidic system prevent outward diffusion across the phase interface, retaining the target species inside its preferred phase flow and creating a sharp boundary with a dramatic concentration change. Then, the chemical flow (the preferred phase with target chemical species) is precisely manipulated by a microfluidic probe, which can be compressed to a micron-level width and aimed at an arbitrary position of the sample. As a demonstration of the feasibility and versatility of the strategy, chemical flow is successfully applied to subcellular regions of various kinds of living single cells. Subcellular regions are successfully labeled (cytomembrane and mitochondria) and damaged. Healing-regeneration behaviors of living single cells are triggered by subcellular damage and analyzed. The method is relatively general regarding the species of chemicals and biosamples, which could promote deeper cell research.

The effect of hypergravity, hyperbaric pressure, and hypoxia on osteogenic differentiation of adipose stem cells.

Human adipose stem cells (ASCs) hold great potential for regenerative medicine approaches, including osteogenic regeneration of bone defects, that fail to heal autonomously. Osteogenic differentiation of stem cells is dependent on the stimulation of biophysical factors. In the present study, the effects of hypergravity, hypoxia, and hyperbaric treatment were investigated on adipose stem cell (ASC) metabolic activity, quantified by PrestoBlue conversion, and cell numbers, evaluated by crystal violet staining. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and cresolphthalein staining of calcium deposition. Differentiation was performed for 12 days, which was accompanied by periodical stimulation. Increasing gravity forces up to 50x g did not affect ASC viability, but it enhanced osteogenic markers with a strongest effect between 20 and 30x g. Hyperbaric stimulation at 3 bar decreased ASC cell numbers but increased ALP activity and calcium deposition. Hypoxia at 8 % atmospheric oxygen did not affect ASC proliferation, while cell numbers were reduced at 3 % oxygen. Furthermore, hypoxic conditions produced opposing results on osteogenic markers, as ALP activity increased whereas cresolphthalein staining decreased upon stimulation. These data demonstrated that intermittent short duration of basal physical or chemical impulses interfere with the osteogenic differentiation of ASCs. Our findings could be of specific relevance in ASC based therapies for regenerative medicine and bone tissue engineering approaches.

Switchable Adhesion on Curved Surfaces Mimicking the Coordination of Radial-Oriented Spatular Tips and Motion of Gecko Toes.

Bio-inspired structured adhesives have promising applications in many fields, like biomedicine, robotics, and aerospace. However, achieving robust and switchable adhesion in structured adhesives on non-planar surfaces remains highly challenging. Inspired by the gripping and rolling motions of gecko toes, a strong and switchable adhesive, which comprises a pillar array with radial-oriented spatular tips and is named as PROST, is developed. PROST possesses a robust adhesion on flat surfaces and doubles its adhesion on curved surfaces. Moreover, in situ and fast adhesion switching of PROST on flat/curved surfaces in dry and wet conditions has been realized by solvent stimulation, mimicking the bending locomotion of gecko toes. The work here provides a new strategy for designing controllable adhesion on curved substrates.

On the meta-analysis of hormetic effects.

The evidence for hormetic responses with chemical effects at doses lower than the no-observed-adverse-effect-level (sub-NOAEL) is increasing, creating a need for meta-analyses of sub-NOAEL effects across studies. However, the distinct features of hormetic responses complicate the procedures of meta-analyses aiming to study sub-NOAEL, hormetic effects, and there is no standardized methodology to serve as a guideline. In this piece, a protocol is proposed, which covers the selection of more holistic keywords to be integrated into the literature search queries, the designation of control, and the identification of NOAEL (and thus sub-NOAEL dose responses). It also considers the selection of the response indicators and the incorporation of time and dose as sources of variation. This protocol can serve as a reference point for a harmonized and more robust methodology to meta-analyze sub-NOAEL effects of chemicals on living organisms.

Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans.

Microfluidic devices offer several advantages for C. elegans research, particularly for presenting precise physical and chemical environments, immobilizing animals during imaging, quantifying behavior, and automating screens. However, challenges to their widespread adoption in the field include increased complexity over conventional methods, operational problems (such as clogging, leaks, and bubbles), difficulty in obtaining or fabricating devices, and the need to characterize biological results obtained from new assay formats. Here we describe the preparation and operation of simple, reusable microfluidic devices for quantifying behavioral responses to chemical patterns, and single-use devices to arrange animals for time-lapse microscopy and to measure neuronal activity. We focus on details that eliminate or reduce the frustrations commonly experienced by new users of microfluidic devices.

Dose-Dependent Pain and Pain Radiation after Chemical Stimulation of the Thoracolumbar Fascia and Multifidus Muscle: A Single-Blinded, Cross-Over Study Revealing a Higher Impact of Fascia Stimulation.

Acute low back pain can be experimentally induced by injections of hypertonic saline into deep tissues of the back, such as fascia and muscle. The current study investigated the dose-dependency of peak-pain and spatial extent of concomitant radiating pain following 50, 200 and 800 µL bolus injections of hypertonic saline (5.8%) into the thoracolumbar fascia and multifidus muscle, since data on dose-dependency is lacking in humans. Sixteen healthy subjects rated (11 female, 5 male; 23.3 ± 3.1 years, mean ± SD) intensity and spatial extent of pain. Injections into the fascia resulted in significantly higher peak-pain (+86%, p < 0.001), longer pain durations (p < 0.05), and larger pain areas (+65%, p < 0.02) and were less variable than intramuscular injections. Peak-pain ratings and pain areas were 2−3-fold higher/larger for 200 µL vs. 50 µL. In contrast, peak pain increased only marginally at 800 µL by additional 20%, while pain areas did not increase further at all in both, fascia and muscle. Thus, higher injection volumes did also not compensate the lower sensitivity of muscle. Peak-pain ratings and pain areas correlated between fascia and muscle (r = 0.530, p < 0.001 and r = 0.337, p < 0.02, respectively). Peak-pain ratings and pain areas correlated overall (r = 0.490, p < 0.0001), but a weak correlation remained when the impact of between-tissue differences and different injection volumes were singled out (partial r = 0.261, p < 0.01). This study shows dose-dependent pain responses of deep tissues where an injection volume of 200 µL of hypertonic saline is deemed an adequate stimulus for tissue differentiation. We suggest that pain radiation is not simply an effect of increased peripheral input but may afford an individual disposition for the pain radiation response. Substantially higher pain-sensitivity and wider pain areas support fascia as an important contributor to non-specific low back pain.

Interplay of dopamine and GABA in substantia nigra for the regulation of rapid eye movement sleep in rats.

Substantia nigra (SN) is rich in dopamine (DA)-ergic and GABA-ergic neurons, which project to and receive inputs from locus coeruleus (LC) and pedunculo-pontine tegmentum (PPT) possessing REM-OFF and REM-ON neurons, respectively. Loss of DA-ergic neurons and disturbed REM sleep (REMS) are associated with Parkinson's disease, depression and REMS behavior disorder. GABA-ergic projections from SN act pre-synaptically on the noradrenaline (NA)-ergic terminals coming from the LC-REM-OFF neurons onto the REM-ON neurons in PPT and play a critical role in initiating REMS. However, it was unknown how SN neurons get activated and whether the SN-DA-ergic neurons interact with the SN-GABA-ergic neurons for REMS regulation. In freely moving chronically prepared rats, neurons in SN (bilateral) were stimulated by local microinjection of Glutamate (Glut), sparing the fibers of passage, in the presence and absence of i.p. Haloperidol (Hal, DA-antagonist). In other sets, either Hal or Bicuculine (Bic, GABA-antagonist) alone or simultaneously was microinjected bilaterally into the SN and the effects on sleep-wakefulness were recorded. We observed that Glut in SN significantly increased REMS, which was prevented by Hal. REMS was decreased and increased by Hal and Bic, respectively; while their co-injection neutralized (ineffective) the individual effects. Combining these findings with previous reports suggest that the SN-DA-ergic neurons act on the SN-GABA-ergic to regulate REMS. The results advance our understanding of the neuro-anatomo-chemical connections and pharmaco-physiological regulation of REMS in health and diseases.

Olfactory deficits decrease the time resolution for trigeminal lateralization.

OBJECTIVES: To date the temporal resolution of the detection of almost simultaneously applied intranasal trigeminal stimuli is unknown. The aim of our study was to examine this temporal resolution in an/hyposmic subjects, who are known to have reduced trigeminal sensitivity and compare it with healthy controls. METHODS: Participants were 20 posttraumatic an/hyposmic patients, and 23 healthy controls (matched with regard to sex and age). Olfactory function was tested psychophysically using the Sniffin´ Sticks test battery. Bilateral trigeminal stimulation was carried out using a birhinal high-precision olfactometer. The trigeminal stimulus used was CO2 60% v/v, the interstimulus interval ranged from 28 to 32s, stimulus duration was 200ms. Time-lags tested between right and left side of stimulation were at 40, 80, 120, 160 and 200ms. Subjects raised their left or right hand to indicate the side on which the stimulus had been perceived first. RESULTS: In both groups the accuracy in the trigeminal lateralization task increased with the time-lag but normosmic subjects significantly outperformed an/hyposmics in the 200ms time-lag condition. Normosmics significantly exceeded 50% chance level at the time-lag of 80ms, whereas an/hyposmics were only able to score above chance starting from 120ms time-lag. Lateralization scores significantly decreased with age. CONCLUSIONS: At a time lag of 200ms intranasal trigeminal stimuli can be lateralized. The reduced trigeminal sensitivity in patients with anosmia or hyposmia leads to an increased time lag required for correct perception of intranasal, almost simultaneously, applied stimuli.

Tenocytic extract and mechanical stimulation in a tissue-engineered tendon construct increases cellular proliferation and ECM deposition.

Chemical and mechanical stimulation, when properly utilized, positively influence both the differentiation of in vitro cultured stem cells and the quality of the deposited extracellular matrix (ECM). This study aimed to find if cell-free extract from primary tenocytes can positively affect the development of a tissue-engineered tendon construct, consisting of a human umbilical vein (HUV) seeded with mesenchymal stem cells (MSCs) subjected to cyclical mechanical stimulation. The tenocytic cell-free extract possesses biological material from tendon cells that could potentially influence MSC tenocytic differentiation and construct development. We demonstrate that the addition of tenocytic extract in statically cultured tendon constructs increases ECM deposition and tendon-related gene expression of MSCs. The incorporation of mechanical stimulation (2% strain for 30 min/day at 0.5 cycles/min) with tenocytic extract further improved the MSC seeded HUV constructs by increasing cellularity of the construct by 37% and the ultimate tensile strength by 33% compared to the constructs with only mechanical stimulation after 14 days. Furthermore, the addition of mechanical stimulation to the extract supplementation produced longitudinal ECM fibril alignment along with dense connective tissue, reminiscent of natural tendon.

A multiphase model for chemically- and mechanically- induced cell differentiation in a hollow fibre membrane bioreactor: minimising growth factor consumption.

We present a simplified two-dimensional model of fluid flow, solute transport, and cell distribution in a hollow fibre membrane bioreactor. We consider two cell populations, one undifferentiated and one differentiated, with differentiation stimulated either by growth factor alone, or by both growth factor and fluid shear stress. Two experimental configurations are considered, a 3-layer model in which the cells are seeded in a scaffold throughout the extracapillary space (ECS), and a 4-layer model in which the cell-scaffold construct occupies a layer surrounding the outside of the hollow fibre, only partially filling the ECS. Above this is a region of free-flowing fluid, referred to as the upper fluid layer. Following previous models by the authors (Pearson et al. in Math Med Biol, 2013, Biomech Model Mechanbiol 1-16, 2014a, we employ porous mixture theory to model the dynamics of, and interactions between, the cells, scaffold, and fluid in the cell-scaffold construct. We use this model to determine operating conditions (experiment end time, growth factor inlet concentration, and inlet fluid fluxes) which result in a required percentage of differentiated cells, as well as maximising the differentiated cell yield and minimising the consumption of expensive growth factor.

REM sleep modulation by perifornical orexinergic inputs to the pedunculo-pontine tegmental neurons in rats.

Rapid eye movement sleep (REMS) is regulated by the interaction of the REM-ON and REM-OFF neurons located in the pedunculo-pontine-tegmentum (PPT) and the locus coeruleus (LC), respectively. Many other brain areas, particularly those controlling non-REMS (NREMS) and waking, modulate REMS by modulating these REMS-related neurons. Perifornical (PeF) orexin (Ox)-ergic neurons are reported to increase waking and reduce NREMS as well as REMS; dysfunction of the PeF neurons are related to REMS loss-associated disorders. Hence, we were interested in understanding the neural mechanism of PeF-induced REMS modulation. As a first step we have recently reported that PeF Ox-ergic neurons modulate REMS by influencing the LC neurons (site for REM-OFF neurons). Thereafter, in this in vivo study we have explored the role of PeF inputs on the PPT neurons (site for REM-ON neurons) for the regulation of REMS. Chronic male rats were surgically prepared with implanted bilateral cannulae in PeF and PPT and electrodes for recording sleep-waking patterns. After post-surgical recovery sleep-waking-REMS were recorded when bilateral PeF neurons were stimulated by glutamate and simultaneously bilateral PPT neurons were infused with either saline or orexin receptor1 (OX1R) antagonist. It was observed that PeF stimulation increased waking and decreased NREMS as well as REMS, which were prevented by OX1R antagonist into the PPT. We conclude that the PeF stimulation-induced reduction in REMS was likely to be due to inhibition of REM-ON neurons in the PPT. As waking and NREMS are inversely related, subject to confirmation, the reduction in NREMS could be due to increased waking or vice versa. Based on our findings from this and earlier studies we have proposed a model showing connections between PeF- and PPT-neurons for REMS regulation.

Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans.

Microfluidic devices offer several advantages for C. elegans research, particularly for presenting precise physical and chemical environments, immobilizing animals during imaging, quantifying behavior, and automating screens. However, challenges to their widespread adoption in the field include increased complexity over conventional methods, operational problems (such as clogging, leaks, and bubbles), difficulty in obtaining or fabricating devices, and the need to characterize biological results obtained from new assay formats. Here we describe the preparation and operation of simple, reusable microfluidic devices for quantifying behavioral responses to chemical patterns, and single-use devices to arrange animals for time-lapse microscopy and to measure neuronal activity. We focus on details that eliminate or reduce the frustrations commonly experienced by new users of microfluidic devices.

Indole-2-carboxamide derivatives: a patent evaluation of WO2015036412A1.

INTRODUCTION: Hippocampal neurogenesis in adults is a new and attractive target for the treatment and prevention of neurodegenerative and neuro-psychiatric diseases. Recently, neurogenesis stimulating activity was observed in some of the commonly used small molecule drugs such as antidepressants and atypical antipsychotics. Stimulation of neurogenesis is attractive mainly due to its wide scope of application, ranging from depressions, schizophrenia, dementia, Parkinson`s and Alzheimer`s Disease to various brain injuries. AREAS COVERED: New compounds based on 7-phenyl or 7-pyridinyl-1H-indole-2-carboxamide showed interesting neural stem cell proliferation inducing activity in vitro and were claimed as potential therapeutics for various neurodegenerative and neuropsychiatric diseases as well as brain injuries. The potential of the presented compounds is evaluated with respect to other small molecule neurogenesis inducers in literature. EXPERT OPINION: Nanomolar in vitro activities of presented compounds and their favorable physico-chemical properties, giving a fair chance of good oral bioavailability and sufficient CNS penetration, make these compounds promising drug candidates. The biggest drawback of the presented application is the absence of pharmacokinetics, toxicity and in vivo activity data. On the other hand, the high number of applications in this area (seven published in last two years) indicates that Hoffmann-La Roche takes it seriously.

Perifornical orexinergic neurons modulate REM sleep by influencing locus coeruleus neurons in rats.

Activation of the orexin (OX)-ergic neurons in the perifornical (PeF) area has been reported to induce waking and reduce rapid eye movement sleep (REMS). The activities of OX-ergic neurons are maximum during active waking and they progressively reduce during non-REMS (NREMS) and REMS. Apparently, the locus coeruleus (LC) neurons also behave in a comparable manner as that of the OX-ergic neurons particularly in relation to waking and REMS. Further, as PeF OX-ergic neurons send dense projections to LC, we argued that the former could drive the LC neurons to modulate waking and REMS. Studies in freely moving normally behaving animals where simultaneously neuro-chemo-anatomo-physio-behavioral information could be deciphered would significantly strengthen our understanding on the regulation of REMS. Therefore, in this study in freely behaving chronically prepared rats we stimulated the PeF neurons without or with simultaneous blocking of specific subtypes of OX-ergic receptors in the LC while electrophysiological recording characterizing sleep-waking was continued. Single dose of glutamate stimulation as well as sustained mild electrical stimulation of PeF (both bilateral) significantly increased waking and reduced REMS as compared to baseline. Simultaneous application of OX-receptor1 (OX1R) antagonist bilaterally into the LC prevented PeF stimulation-induced REMS suppression. Also, the effect of electrical stimulation of the PeF was long lasting as compared to that of the glutamate stimulation. Further, sustained electrical stimulation significantly decreased both REMS duration as well as REMS frequency, while glutamate stimulation decreased REMS duration only.