Serotonergic Actions and Interactions on the SCN Circadian Pacemaker: In Vitro Investigations

The phase of the mammalian circadian pacemaker located in the suprachiasmatic nuclei (SCN) is controlled by a multitude of stimuli. While phase control is undoubtedly dominated by photic input, the serotonergic input from the raphe nuclei also influences SCN clock phase. In this article I review the evidence for serotonergic modulation of the SCN pacemaker, and the cellular mechanisms underlying these effects, obtained from in vitro experiments performed during the past decade. Serotonin can advance the SCN pacemaker when applied during the subjective day, and delay the pacemaker when applied during the subjective night. The daytime advances appear due to stimulation of 5HT₇ receptors, activation of adenylate cyclase and protein kinase A, and opening of K⁺ channels. The synthesis of new proteins may also be critical for these phase shifts. Serotonergic phase advances can be inhibited by a variety of other modulatory inputs to the SCN, including neuropeptide Y, melatonin, and glutamate. Together, these data demonstrate that SCN circadian pacemaker phase is controlled by a complex interplay between multiple afferent stimuli, and that serotonin plays a critical role in this process.     

GM1 Ganglioside: In Vivo and In Vitro Trophic Actions on Central Neurotransmitter Systems

Abstract: There is now substantial evidence that GM1 ganglioside is effective in partially correcting the consequences of neuroinjury in a number of in vivo and in vitro model systems. Although the molecular mechanism(s) and the substrates for the neurotrophic activity of the gangliosides are not fully understood, the published experimental work suggests that GM1 has antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. This review focuses attention on studies reporting that GM1 restores neuronal integrity and function in the brain of lesioned young as well as aged animals. Critical analysis of these studies can provide guidance for future ganglioside research and may point to novel approaches for treating neuroinjury and a variety of degenerative conditions, including aging.     

Gemcitabine: Actions and Interactions

Abstract

Gemcitabine is a nucleoside analog that acts by multiple mechanisms. Incorporation of the triphosphate and subsequent inhibition of DNA replication and repair appears to be the major mode of action. Ribonucleotide reductase is inhibited by gemcitabine diphosphate, an activity that leads to metabolic self-potentiating effects.     

Behavioral and Serotonergic Regulation of Circadian Rhythms

Endogenous depression is often accompanied by alterations in core parameters of circadian rhythms, and antidepressant treatments, including serotonergic drugs, sleep deprivation and exercise, alter circadian phase or period in humans or animal models. Antidepressants may act in part through the circadian system, and behavioral antidepressants through a common serotonergic path to the clock. This review evaluates the evidence from animal models that serotonin (5-HT) mediates phase-shifting effects of behavioral stimuli on circadian rhythms. In rodents, 'exercise' stimulated during the rest phase of the rest-activity cycle induces large phase shifts of circadian rhythms. These shifts can be mimicked by short-term sleep deprivation without intense activity. During wheel running or sleep deprivation, 5-HT release in the suprachiasmatic nucleus (SCN) circadian clock is significantly elevated. Lesions of 5-HT afferents to the SCN attenuate phase shifts or entrainment induced by activity in response to some stimuli (e.g., triazolam injections in hamsters, treadmill running in mice) but not others (e.g., novel wheel confinement in hamsters). Antagonists selective to 5HT_{1, 2} or ₇ receptors do not attenuate shifts induced by wheel running, although 5-HT_2/7 antagonists do partially block shifts to saline injections. 5-HT agonists (e.g., 8-OH-DPAT) induce large shifts in vitro, but much smaller shifts in vivo, particularly if administered directly to the SCN. Procedures for inducing 5-HT supersensitivity in vivo result in larger shifts to 8-OH-DPAT. 5-HT stimuli may affect the clock by direct and indirect pathways, particularly through the thalamic intergeniculate leaflet, and the role of these pathways may differ across species. At the level of the SCN, 5-HT likely acts through 5-HT₇ receptors on neurons and possibly also glial cells. These receptors may be useful targets for the development of antidepressant drugs. In aggregate, the literature provides mixed support for the hypothesis that exercise or behavioral arousal shift the circadian clock by a 5-HT pathway; the role of indirect pathways, interactions with other transmitters, cellular adaptations to denervation, glial cells, and species differences remain to be more fully clarified. Serotonergic and behavioral stimuli provide an intriguing route to elucidate the circadian clockworks and their possible role in depression.     

Behavioral and Serotonergic Regulation of Circadian Rhythms

Endogenous depression is often accompanied by alterations in core parameters of circadian rhythms, and antidepressant treatments, including serotonergic drugs, sleep deprivation and exercise, alter circadian phase or period in humans or animal models. Antidepressants may act in part through the circadian system, and behavioral antidepressants through a common serotonergic path to the clock. This review evaluates the evidence from animal models that serotonin (5-HT) mediates phase-shifting effects of behavioral stimuli on circadian rhythms. In rodents, 'exercise' stimulated during the rest phase of the rest-activity cycle induces large phase shifts of circadian rhythms. These shifts can be mimicked by short-term sleep deprivation without intense activity. During wheel running or sleep deprivation, 5-HT release in the suprachiasmatic nucleus (SCN) circadian clock is significantly elevated. Lesions of 5-HT afferents to the SCN attenuate phase shifts or entrainment induced by activity in response to some stimuli (e.g., triazolam injections in hamsters, treadmill running in mice) but not others (e.g., novel wheel confinement in hamsters). Antagonists selective to 5HT_{1, 2} or ₇ receptors do not attenuate shifts induced by wheel running, although 5-HT_2/7 antagonists do partially block shifts to saline injections. 5-HT agonists (e.g., 8-OH-DPAT) induce large shifts in vitro, but much smaller shifts in vivo, particularly if administered directly to the SCN. Procedures for inducing 5-HT supersensitivity in vivo result in larger shifts to 8-OH-DPAT. 5-HT stimuli may affect the clock by direct and indirect pathways, particularly through the thalamic intergeniculate leaflet, and the role of these pathways may differ across species. At the level of the SCN, 5-HT likely acts through 5-HT₇ receptors on neurons and possibly also glial cells. These receptors may be useful targets for the development of antidepressant drugs. In aggregate, the literature provides mixed support for the hypothesis that exercise or behavioral arousal shift the circadian clock by a 5-HT pathway; the role of indirect pathways, interactions with other transmitters, cellular adaptations to denervation, glial cells, and species differences remain to be more fully clarified. Serotonergic and behavioral stimuli provide an intriguing route to elucidate the circadian clockworks and their possible role in depression.     

Balamuthia mandrillaris: In Vitro Interactions with Selected Protozoa and Algae

Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one‐on‐one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp.     

Electrophysiology of the Circadian Pacemaker in Mammals

The neurons of the mammalian suprachiasmatic nuclei (SCN) control circadian rhythms in molecular, physiological, endocrine, and behavioral functions. In the SCN, circadian rhythms are generated at the level of individual neurons. The last decade has provided a wealth of information on the genetic basis for circadian rhythm generation. In comparison, a modest but growing number of studies have investigated how the molecular rhythm is translated into neuronal function. Neuronal attributes have been measured at the cellular and tissue level with a variety of electrophysiological techniques. We have summarized electrophysiological research on neurons that constitute the SCN in an attempt to provide a comprehensive view on the current state of the art.     

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors ^1,2 and providing growth promoting adhesion molecules ³ and extracellular matrix molecules ⁴. In addition they myelinate the demyelinated axons at the site of injury ⁵.However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes ^6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules ^8-13. In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer ^14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) ^16,17, FGF/Heparin ¹⁸, Eph/Ephrins¹⁹.This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.Download video file.^{(64M, mov)}     

Circadian disruption and SCN control of energy metabolism

In this review we first present the anatomical pathways used by the suprachiasmatic nuclei to enforce its rhythmicity onto the body, especially its energy homeostatic system. The experimental data show that by activating the orexin system at the start of the active phase, the biological clock not only ensures that we wake up on time, but also that our glucose metabolism and cardiovascular system are prepared for increased activity. The drawback of such a highly integrated system, however, becomes visible when our daily lives are not fully synchronized with the environment. Thus, in addition to increased physical activity and decreased intake of high-energy food, also a well-lighted and fully resonating biological clock may help to withstand the increasing "diabetogenic" pressure of today's 24/7 society.     

Plasmodium Sporozoite Interactions with Macrophages In Vitro: a Videomicroscopic Analysis

ABSTRACT. Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Quantifying the Rhythm of KaiB-C Interaction for In Vitro Cyanobacterial Circadian Clock

An oscillator consisting of KaiA, KaiB, and KaiC proteins comprises the core of cyanobacterial circadian clock. While one key reaction in this process-KaiC phosphorylation-has been extensively investigated and modeled, other key processes, such as the interactions among Kai proteins, are not understood well. Specifically, different experimental techniques have yielded inconsistent views about Kai A, B, and C interactions. Here, we first propose a mathematical model of cyanobacterial circadian clock that explains the recently observed dynamics of the four phospho-states of KaiC as well as the interactions among the three Kai proteins. Simulations of the model show that the interaction between KaiB and KaiC oscillates with the same period as the phosphorylation of KaiC, but displays a phase delay of ～8 hr relative to the total phosphorylated KaiC. Secondly, this prediction on KaiB-C interaction are evaluated using a novel FRET (Fluorescence Resonance Energy Transfer)-based assay by tagging fluorescent proteins Cerulean and Venus to KaiC and KaiB, respectively, and reconstituting fluorescent protein-labeled in vitro clock. The data show that the KaiB∶KaiC interaction indeed oscillates with ～24 hr periodicity and ～8 hr phase delay relative to KaiC phosphorylation, consistent with model prediction. Moreover, it is noteworthy that our model indicates that the interlinked positive and negative feedback loops are the underlying mechanism for oscillation, with the serine phosphorylated-state (the "S-state") of KaiC being a hub for the feedback loops. Because the kinetics of the KaiB-C interaction faithfully follows that of the S-state, the FRET measurement may provide an important real-time probe in quantitative study of the cyanobacterial circadian clock.     

Circadian Pacemaker Neurons Change Synaptic Contacts across the Day

1. Download : Download high-res image (143KB)
2. Download : Download full-size image

     

Pediatric Suppositories of Sulpiride Solid Dispersion for Treatment of Tourette Syndrome: In Vitro and In Vivo Investigations

Pharmaceutical development was adopted in the current study to propose a pediatric rectal formulation of sulpiride as a substitute to the available oral or parenteral formulations in the management of Tourette syndrome (TS). The goal was to formulate a product that is easy to use, stable, and highly bioavailable and to achieve a rapid clinical efficacy. Towards this aim, sulpiride solid dispersion (SD) with tartaric acid at a weight ratio of 1:0.25 was incorporated into different suppository bases, namely witepsol W25, witepsol H15, witepsol E75, suppocire NA, suppocire A, glycerogelatin, and polyethylene glycols. The formulae were evaluated in vitro using different pharmacotechnical methods such as visual, melting, weight and content uniformities, drug release, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR), and X-ray diffraction (XRD) analyses. In vivo bioavailability was also assessed in rabbits to compare the bioavailability of either raw sulpiride-incorporated or its SD-incorporated witepsol H15-based suppositories to its oral suspension (reference). Sulpiride SD-incorporated witepsol H15 formulation showed acceptable in vitro characteristics with a bioavailability of 117% relative to oral dosing, which excel that in humans (27% after dosing of oral product). In addition, the proposed formula not only passed the 6-month stability study but also proposed a promising scale-up approach. Hence, it showed a great potential for pediatric product development to manage TS in rural areas.KEY WORDS: bioavailability, pediatric rectal suppositories, solid dispersion, sulpiride, tartaric acid     

Attractive Interactions among Intermediate Filaments Determine Network Mechanics In Vitro

Mechanical and structural properties of K8/K18 and vimentin intermediate filament (IF) networks have been investigated using bulk mechanical rheometry and optical microrheology including diffusing wave spectroscopy and multiple particle tracking. A high elastic modulus G₀ at low protein concentration c, a weak concentration dependency of G₀ (G₀∼c^0.5±0.1) and pronounced strain stiffening are found for these systems even without external crossbridgers. Strong attractive interactions among filaments are required to maintain these characteristic mechanical features, which have also been reported for various other IF networks. Filament assembly, the persistence length of the filaments and the network mesh size remain essentially unaffected when a nonionic surfactant is added, but strain stiffening is completely suppressed, G₀ drops by orders of magnitude and exhibits a scaling G₀∼c^1.9±0.2 in agreement with microrheological measurements and as expected for entangled networks of semi-flexible polymers. Tailless K8Δ/K18ΔT and various other tailless filament networks do not exhibit strain stiffening, but still show high G₀ values. Therefore, two binding sites are proposed to exist in IF networks. A weaker one mediated by hydrophobic amino acid clusters in the central rod prevents stretched filaments between adjacent cross-links from thermal equilibration and thus provides the high G₀ values. Another strong one facilitating strain stiffening is located in the tail domain with its high fraction of hydrophobic amino acid sequences. Strain stiffening is less pronounced for vimentin than for K8/K18 due to electrostatic repulsion forces partly compensating the strong attraction at filament contact points.     

Perturbing Circadian Oscillations in an In Vitro Suprachiasmatic Nucleus With Magnetic Stimulation

Many neurological disorders are associated with abnormal oscillatory dynamics. The suprachiasmatic nucleus (SCN) is responsible for the timing and synchronization of physiological processes. We performed experiments on PERIOD2::LUCIFERASE transgenic “knock-in” mice. In these mice, a gene that is expressed in a circadian pattern is fused to an inserted gene that codes for luciferase, which is a bioluminescent enzyme. A one-time 3 min magnetic stimulation (MS) was applied to excised slices of the SCN. The MS consisted of a 50-mT field that was turned on and off 4,500 times. The rise time and fall time of the field were 75 μs. A photon count that extended over the full 5 days that the slice remained viable, subsequently revealed how the MS affected the circadian cycle. The MS was applied at points in the circadian cycle that correspond to either maximal or minimal bioluminescence. It was found that both the amplitude and period of the endogenous circadian oscillation are affected by MS and that the effects strongly depend on where in the circadian cycle the stimulation was applied. Our MS dose is in the same range as clinically applied doses, and our findings imply that transcranial MS may be instrumental in remedying disorders that originate in circadian rhythm abnormalities. Bioelectromagnetics. 2020;41:63–72 © 2019 Wiley Periodicals, Inc.     

Interactions of Anaerobic Bacteria with Dental Stem Cells: An In Vitro Study

Background

In patients with periodontitis, it is highly likely that local (progenitor) cells encounter pathogenic bacteria. The purpose of this in vitro study was to elucidate how human dental follicle stem cells (hDFSC) react towards a direct challenge with anaerobic periodontal pathogens under their natural oxygen-free atmosphere. HDFSC were compared to human bone marrow mesenchymal stem cells (hBMSC) and differentiated primary human gingival fibroblasts (hGiF), as well as permanent gingival carcinoma cells (Ca9-22).

Methodology/Principal Findings

The different cell types were investigated in a co-culture system with Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). The viability of the cells and pathogens under anaerobic conditions, as well as interactions in terms of adherence and internalization, were examined. Additionally, the release of pro-inflammatory interleukin-8 (IL-8) and anti-inflammatory interleukin-10 (IL-10) was quantified via enzyme-linked immunosorbent assay. The bacteria adhered less efficiently to hDFSC compared to Ca9-22 (P. gingivalis: 0.18% adherence to hDFSC; 3.1% adherence to Ca9-22). Similar results were observed for host cell internalization (F. nucleatum: 0.002% internalization into hDFSC; 0.09% internalization into Ca9-22). Statistically significantly less IL-8 was secreted from hDFSC after stimulation with F. nucleatum and P. gingivalis in comparison with hGiF (F. nucleatum: 2080.0 pg/ml – hGiF; 19.7 pg/ml – hDFSC). The IL-10 response of the differentiated cells was found to be low in relation to their pro-inflammatory IL-8 response.

Conclusions/Significance

The results indicate that dental stem cells are less prone to interactions with pathogenic bacteria than differentiated cells in an anaerobic environment. Moreover, during bacterial challenge, the stem cell immune response seems to be more towards an anti-inflammatory reaction. For a potential future therapeutic use of hDFSC, these findings support the idea of a save application.