Nitric oxide synthase inhibitor attenuates the effects of repeated restraint stress on synaptic transmission in the paraventricular nucleus of the rat hypothalamus

Corticotropin-releasing hormone (CRH)-synthesizing parvocellular neuroendocrine cells (PNCs) of the hypothalamic paraventricular nucleus (PVN) play a key role in the activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Several studies have demonstrated that synaptic inputs to these cells may undergo stress-related enhancement but, on the other hand, it has been reported that exposition to the same stressor for prolonged time periods may induce a progressive reduction in the response of the HPA axis to homotypic stressors. In the present study rats were subjected to 10 min restraint sessions, repeated twice daily for 3 or 7 days. Miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were then recorded from PNCs in ex vivo hypothalamic slice preparations obtained 24 h after the last restraint. Restraint stress repeated over 3 days resulted in increased mean frequency and decreased rise time and decay time constant of mEPSCs, accompanied by a decrease in the excitability of PNCs, however, no such changes were evident in slices obtained from rats subjected to restraint over 7 days. There were no changes in mIPSCs after repeated restraint. Administration of the unspecific nitric oxide synthase (NOS) blocker Nω-Nitro-L-arginine (L-NNA) before each restraint, repeated over 3 days, prevented the occurrence of an increase in mEPSC frequency. However, animals receiving L-NNA and subjected to repeated restraint had similar changes in PNCs membrane excitability and mEPSC kinetics as stressed rats not receiving L-NNA. Comparison of the effects of a single 10 min restraint session followed by either an immediate or delayed (24 h) decapitation revealed an increase in the mean mEPSC frequency and a decrease in the mean mIPSC frequency in slices prepared immediately after restraint, with no apparent effects when slice preparation was delayed by 24 h. These results demonstrate that restraint, lasting 10 min and repeated twice daily for 3 days, induces a selective and long-lasting enhancement of excitatory synaptic input onto PNCs, partially by a NOS-dependent mechanism, and reduces PNC excitability, whereas prolongation of repeated stress for up to 7 days results in an adaptation

Course of fatigue among patients previously hospitalised due to COVID-19

Introduction. Discrepancies exist regarding the clinical course and prognostic factors for post-COVID fatigue. Therefore, our aim was to assess the timely course of fatigue and its possible predictors in patients previously hospitalised due to SARS-CoV-2 infection. Material and methods. Patients and employees of the University Hospital in Krakow were assessed with the use of a validated neuropsychological questionnaire. Included were participants aged 18 or more, previously hospitalised due to COVID-19, who completed questionnaires only once > 3 months after the onset of infection. Individuals were retrospectively asked about the presence of eight symptoms of chronic fatigue syndrome at four timepoints: before COVID-19, within 0–4 weeks, 4–12 weeks, and > 12 weeks post-infection. Results. We enrolled 204 patients [40.2% women, median age 58 (46–66) years] evaluated after a median of 187 (156–220) days from the first positive nasal swab test for SARS-CoV-2. The most common comorbidities were hypertension (44.61%), obesity (36.27%), smoking (28.43%), and hypercholesterolemia (21.08%); none of the patients required mechanical ventilation during hospitalisation. Before COVID-19, 43.62% of patients reported at least one symptom of chronic fatigue. Within 4, 4–12, and > 12 weeks after COVID-19, the prevalence of chronic fatigue was 76.96%, 75.49%, and 66.17%, respectively (all p < 0.001). The frequency of chronic fatigue symptoms decreased within > 12 weeks following the onset of infection but did not return to baseline values, except for self-reported lymph node enlargement. In a multivariable linear regression model, the number of fatigue symptoms was predicted by female sex [β 0.25 (0.12; 0.39), p < 0.001 and 0.26 (0.13; 0.39), p < 0.001 for weeks 0–12 and > 12, respectively], and age [for < 4 weeks, β –0.12 (–0.28; –0.01), p = 0.029]. Conclusions. Most patients previously hospitalised due to COVID-19 suffer from fatigue > 12 weeks after infection onset. The presence of fatigue is predicted by female sex and – only for the acute phase — age

Hyperforin Potentiates Antidepressant-Like Activity of Lanicemine in Mice

N-methyl-D-aspartate receptor (NMDAR) modulators induce rapid and sustained antidepressant like-activity in rodents through a molecular mechanism of action that involves the activation of Ca2+ dependent signaling pathways. Moreover, ketamine, a global NMDAR antagonist is a potent, novel, and atypical drug that has been successfully used to treat major depressive disorder (MDD). However, because ketamine evokes unwanted side effects, alternative strategies have been developed for the treatment of depression. The objective of the present study was to determine the antidepressant effects of either a single dose of hyperforin or lanicemine vs. their combined effects in mice. Hyperforin modulates intracellular Ca2+ levels by activating Ca2+-conducting non-selective canonical transient receptor potential 6 channel (TRPC6) channels. Lanicemine, on the other hand, blocks NMDARs and regulates Ca2+ dependent processes. To evaluate the antidepressant-like activity of hyperforin and lanicemine, a set of in vivo (behavioral) and in vitro methods (western blotting, Ca2+ imaging studies, electrophysiological, and radioligand binding assays) was employed. Combined administration of hyperforin and lanicemine evoked long-lasting antidepressant-like effects in both naïve and chronic corticosterone-treated mice while also enhancing the expression of the synapsin I, GluA1 subunit, and brain derived neurotrophic factor (BDNF) proteins in the frontal cortex. In Ca2+ imaging studies, lanicemine enhanced Ca2+ influx induced by hyperforin. Moreover, compound such as MK-2206 (Akt kinase inhibitor) inhibited the antidepressant-like activity of hyperforin in the tail suspension test (TST). Hyperforin reversed disturbances induced by MK-801 in the novel object recognition (NOR) test and had no effects on NMDA currents and binding to NMDAR. Our results suggest that co-administration of hyperforin and lanicemine induces long-lasting antidepressant effects in mice and that both substances may have different molecular targets

Effects of orexin A on electrophysiologically and neurochemically characterized neurons of the rat nucleus incertus

Jądro niepewne (NI) to grupa neuronów GABAergicznych w pniu mózgu. Jest głównym źródłem nowoodkrytego peptydu – relaksyny-3 (RLN3). Badania potwierdziły udział NI i RLN3 w kontroli apetytu, modulacji wzbudzenia i odpowiedzi stresowej, jak również kontroli hipokampalnego rytmu theta. Wymienione funkcje pokrywają się z funkcjami oreksyn, neuropeptydów syntetyzowanych przez neurony bocznego i przysklepieniowego podwzgórza, unerwiających wiele obszarów mózgowia, w tym pień mózgu. Natywnymi receptorami dla oreksyn są sprzężone z białkiem G receptory OX1R oraz OX2R, których aktywacja z reguły prowadzi do depolaryzacji błony komórkowej poprzez szereg mechanizmów, w tym poprzez zwiększenie błonowych prądów wapniowych.Niniejsza praca jest próbą scharakteryzowania aktywności neuronów jądra niepewnego oraz weryfikacji efektu działania oreksyny A na te komórki. Wykonano rejestracje whole-cell patch clamp oraz barwienia immunohistochemiczne na obecność relaksyny-3 w neuronach NI na skrawkach mózgu szczura. W celu obiektywnego zbadania populacji neuronów NI bez założeń a priori dotyczących grup posłużono się analizą klastrów. Wyłonione trzy podgrupy komórek różniły się szeregiem właściwości elektrofizjologicznych, co bezpośrednio przekładało się na ich wzorzec aktywności. Najwięcej neuronów należało do klastra II i charakteryzowało się najwyższą regularnością i częstotliwością wyładowań. Neurony klastra I wykazywały się najmniejszą regularnością wyładowań oraz pośrednią częstotliwością. Klaster III stanowiły neurony o najniższej częstotliwości i pośredniej regularności wyładowań. Występowały również różnice w proporcjonalnej ilości komórek RLN3-immunoreaktywnych w poszczególnych klastrach.W celu zbadania wpływu oreksyny A (OXA) na komórki NI wykonano rejestracje w obecności tetrodotoksyny (TTX) oraz blokera kanałów wapniowych – chlorku niklu (II). Wykazano, że depolaryzujący efekt OXA na komórki NI był niwelowany w obecności niklu, co wskazuje na zależny od jonów wapnia mechanizm aktywacji receptorów oreksynowych w NI. Wyniki te przyczyniają się do rosnącej wiedzy o neuronach jądra niepewnego oraz pozwalają na lepsze zrozumienie natury interakcji dwóch systemów peptydów – oreksyn i relaksyny-3. Dalsze badania pozwolą na wyjaśnienie udziału NI i systemu relaksyny-3 w neurofizjologicznych mechanizmach związanych z pobieraniem pokarmu, wzbudzeniem i pamięcią przestrzenną.The nucleus incertus (NI) is a group of GABAergic neurons located in the brainstem. It is the main source of a newly discovered peptide - relaxin-3 (RLN3). Research indicated the involvement of the NI relaxin-3 system in appetite control, modulation of arousal and stress responses, as well as regulation of the hippocampal theta rhythm. These functions are similar to the ones of orexins - neuropeptides synthesized by neurons of the lateral and perifornical hypothalamus. Orexin neurons innervate many brain regions, including the brainstem, and activate native receptors OX1R and OX2R, which most often leads to cell membrane depolarization through a number of possible mechanisms, including increases in the membrane calcium current.This research is an attempt to characterize the activity of NI neurons, as well as to verify the effects of orexin A on these cells. Whole-cell patch clamp recordings were performed on rat brain slices, along with immunohistochemical staining for the presence of relaxin-3 in NI neurons. Cluster analysis was performed to ensure objective examination of neuronal populations without a priori assumptions regarding groups. Three clusters were revealed, differing in various electrophysiological properties, which translated directly to distinct patterns of activity. Cluster II neurons were the largest population, characterized by the highest regularity and frequency of firing. Cells belonging to cluster I were the least regular and of medium frequency. Cluster III contained cells with medium regularity of firing and lowest frequency. The differences between clusters were also visible in the relative distribution of cells immunoreactive for RLN3.In order to examine the effects of orexin A on NI neurons, recordings were made using tetrodotoxin (TTX) and the calcium channel blocker nickel (II) chloride. The depolarizing effect of orexin A on NI neurons was abolished in the presence of nickel chloride, which suggests a calcium-dependent mechanism of orexin receptor activation in the NI. These results contribute to the expanding knowledge about NI neurons and allow for better understanding of the nature of interactions between the two peptide systems. Further research will help clarify the NI/RLN3 involvement in neurophysiological mechanisms connected with feeding, arousal and spatial memory

The impact of peptide systems on the functions of the nucleus incertus

Nucleus incertus is the main source of relaxin-3 in the brain. It has widespread connections with many brain structures. Nucleus incertus cells express CRF1 receptors. Relaxin-3 is a newly-discovered peptide belonging to the insulin superfamily. This compound takes part in a range of mechanisms connected with stress, feeding, mnemonic processes and circadian rhythms. Orexins (hypocretins) are peptides expressed by the lateral and perifornical parts of the hypothalamus. Thanks to their diffuse projections, they are part of a system responsible for mediating states of arousal. They also take part in the regulation of feeding and reward seeking behavior. The functional dichotomy of the orexin system on the axis of arousal – reward also has an anatomical character, as there are separate populations of orexin neurons with different functions. Orexins have an excitatory effect on nucleus incertus cells, however the functional character of this excitation remains unknown. Additional experiments are needed to characterize the interactions between orexins and the relaxin-3 system.Nucleus incertus jest głównym źródłem relaksyny-3 w mózgowiu. Posiada on rozległe połączenia z licznymi strukturami. Na jego komórkach występują receptory CRF1. Relaksyna-3 jest nowoodkrytym peptydem należącym do nadrodziny insulinowej. Związek ten bierze udział w szeregu mechanizmów związanych ze stresem, regulacją apetytu, procesami pamięciowymi i rytmami okołodobowymi. Oreksyny (hipokretyny) są peptydami wydzielanymi przez komórki bocznej i przysklepieniowej części podwzgórza. Dzięki swoim rozsianym projekcjom tworzą one część systemu odpowiedzialnego za wzbudzenie organizmu oraz uczestniczą w regulacji apetytu i zachowania związanego z poszukiwaniem nagrody. Dychotomia funkcji oreksyn na osi wzbudzenie – nagroda ma również charakter anatomiczny, ponieważ istnieją rozdzielone populacje neuronów oreksynergicznych pełniących różne funkcje. Oreksyny działają pobudzająco na neurony nucleus incertus, jednak funkcjonalny charakter tego pobudzenia wciąż nie jest do końca znany. Potrzebne są dodatkowe eksperymenty mające na celu ustalenie zależności między oreksynami a systemem relaksyny-3

Formation of an organic film on an electrode via a suspension of redox-active droplets in acidic aqueous solution

Previous electrochemical studies of redox emulsions have been mainly performed in the context of electro-organic synthesis. More recently, this research has been oriented towards applications of emulsions in flow batteries. Such biphasic systems seem to provide a suitable environment for reactions at the liquid–liquid interface. Taking an emulsion consisting of microdroplets of decamethylferrocene solution in a hydrophobic ionic liquid/toluene mixture in acidic aqueous solution as an example, we have demonstrated that an electrochemical redox reaction involving the hydrophobic redox probe occurs at the glassy carbon electrode|organic liquid film interface. This reaction is followed by ion exchange between liquid phases. This effect is explained by the instability of the emulsion. A portion of the organic liquid stays on the electrode surface after transfer to a purely aqueous electrolyte and remains electroactive