Slices Of Rats Research Articles

The Effects of Ghrelin on Spike Activity of the Suprachiasmatic Nucleus Neurones of the Rat

In one of the most important non-photic mechanisms of the circadian biological clock synchronization with environmental geophysical 24 h rhythm, information on feeding schedule, composition and calorie content of food is used. Hormone ghrelin, a product of the neuroendocrine oxyntic cells of the gastric mucosa to be a signal molecule within this mechanism. In experiments on sagittal hypothalamic slices of male Wistar rats, the effects of 25 nM ghrelin on spike activity and parameters of spike information coding were investigated. Application of ghrelin induced an increase in spike frequency and a decrease in entropy of interspike interval distribution in 32.1% of the neurones recorded. In 29.6% of the cells, opposite responses in the form of a reduction of activity and an increase in the entropy of interspike interval distribution were observed. Parameters of spike activity of the reminder 38.3% neurones of the suprachiasmatic nucleus did not change. The observed responses of the entropy of interspike interval distribution indicate the appropriate changes in a degree of irregularity of interspike interval induced by ghrelin. Application of selective high-affinity antagonist of GHS-R1a receptor, JMV 2959 (100 nM) did not induce responses of the investigated parameters of spike activity but completely prevented changes of both, spike frequency and entropy of interspike interval distribution observed in the presence of ghrelin. The obtained data show that hormone ghrelin by a direct influence on the suprachiasmatic nucleus in vitro modulates the activity level and spike code of relatively numerous population of neurones of the nucleus, wherein the effects of ghrelin are implemented via GHS-R1a receptors. The results of the current study provide additional evidence in favour of the hypothesis on the involvement of ghrelin in mechanisms of non-photic entrainment of the circadian biological clock in accordance with severity of food motivation and level of metabolism.

Cellular mechanisms of synchronized rhythmic burst generation in the ventromedial hypothalamus.

The ventromedial hypothalamus (VMH) plays an important role in feeding behavior and control of the sympathetic nervous system (SNS). The VMH includes a group of neurons that exhibit strong synchronized rhythmic burst firing (so-called VMH oscillation). This VMH oscillation is glucose inhibited, responsive to feeding-related peptides, and is functionally coupled to outputs of the SNS. However, the details of its rhythm generation and synchronization mechanisms are unknown. In the present study, we investigated cellular mechanisms of VMH oscillation by means of electrophysiological recordings and calcium imaging in juvenile rat slice preparations including the VMH. In the electrophysiological study, we performed membrane potential recording from neurons in the vicinity of pipettes for field potential recording. We found that the rhythmic bursts in the VMH were preserved in low Ca2+/high Mg2+ synaptic transmission blockade solution. During membrane hyperpolarization by current injection, the action potential was largely inhibited, but fluctuation of the membrane potential remained with a frequency similar to that at resting potential level. The electric VMH oscillation disappeared after application of either a gap junction blocker, carbenoxolone (100µM), or a persistent sodium channel blocker, riluzole (20µM). Membrane potentials and input resistances of rhythmic burst neurons in the VMH were not significantly changed during these manipulations. A calcium imaging study revealed that all VMH cells exhibiting synchronized rhythmic activity detected by intracellular calcium increases were silenced following the application of carbenoxolone. These results suggest that VMH oscillation arises from the activation of persistent sodium channels and coupling via gap junctions.

Inducing Long-Term Plasticity of Intrinsic Neuronal Excitability in Neurons of the Dorsal Lateral Geniculate Nucleus.

The dorsal lateral geniculate nucleus (dLGN) has long been held to act as a basic relay for visual information traveling from the retina to cortical areas, but recent findings suggest largely underestimated functional plasticity of dLGN principal cells. However, the cellular mechanisms supporting these changes have not been fully explored. Here, we report a protocol to induce long-term potentiation of intrinsic neuronal excitability (LTP-IE) in dorsal dLGN relay cells from acute brain slices of young rats. Intrinsic plasticity is generally induced in parallel with synaptic plasticity. However, in dLGN neurons, LTP-IE is reliably induced by spiking activity at a frequency of 40 Hz for 10 min. LTP-IE in dLGN relay neurons is long-lasting as it can be followed up to 40 min after the induction protocol. In conclusion, the results of this study provide the first evidence for the induction of intrinsic plasticity in dLGN relay cells, thus further pointing to the role of thalamic neurons in activity-dependent visual plasticity.

Synthesis of higher-B0 CEST Z-spectra from lower-B0 data via deep learning and singular value decomposition.

Chemical exchange saturation transfer (CEST) MRI at 3 T suffers from low specificity due to overlapping CEST effects from multiple metabolites, while higher field strengths (B0) allow for better separation of Z-spectral "peaks," aiding signal interpretation and quantification. However, data acquisition at higher B0 is restricted by equipment access, field inhomogeneity and safety issues. Herein, we aim to synthesize higher-B0 Z-spectra from readily available data acquired with 3 T clinical scanners using a deep learning framework. Trained with simulation data using models based on Bloch-McConnell equations, this framework comprised two deep neural networks (DNNs) and a singular value decomposition (SVD) module. The first DNN identified B0 shifts in Z-spectra and aligned them to correct frequencies. After B0 correction, the lower-B0 Z-spectra were streamlined to the second DNN, casting into the key feature representations of higher-B0 Z-spectra, obtained through SVD truncation. Finally, the complete higher-B0 Z-spectra were recovered from inverse SVD, given the low-rank property of Z-spectra. This study constructed and validated two models, a phosphocreatine (PCr) model and a pseudo-in-vivo one. Each experimental dataset, including PCr phantoms, egg white phantoms, and in vivo rat brains, was sequentially acquired on a 3 T human and a 9.4 T animal scanner. Results demonstrated that the synthetic 9.4 T Z-spectra were almost identical to the experimental ground truth, showing low RMSE (0.11% ± 0.0013% for seven PCr tubes, 1.8% ± 0.2% for three egg white tubes, and 0.79% ± 0.54% for three rat slices) and high R2 (>0.99). The synthesized amide and NOE contrast maps, calculated using the Lorentzian difference, were also well matched with the experiments. Additionally, the synthesis model exhibited robustness to B0 inhomogeneities, noise, and other acquisition imperfections. In conclusion, the proposed framework enables synthesis of higher-B0 Z-spectra from lower-B0 ones, which may facilitate CEST MRI quantification and applications.

The role of serotonin receptor 7 signalling in macrophages for the inflammatory response after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Background The hormone and neurotransmitter serotonin regulates multiple processes, including immune responses and inflammatory signalling. After a cardiac injury, such as myocardial infarction (MI), a precise regulation of the local inflammation is determining the healing process and therefore functional outcome. Immune cells invading the infarcted tissue can be critical modulators, and various immune cells, including macrophages, express different serotonin receptors. Purpose We characterized the importance of serotonin receptor 7 (5-HT7R) signalling in the regulation of macrophage function, which infiltrate the heart after MI and thus can determine the course of cardiac remodelling processes. Methods For investigation of 5-HT7R signalling on a cellular level, we used two in vitro models: primary murine bone marrow-derived macrophages (BMDM) and secondary human THP-1-derived macrophages. To validate the impact of macrophages on intact cardiac tissue, we pre-treated pro-inflammatory M1-like BMDMs with a selective 5-HT7R agonist and then co-cultured them with living cardiac rat slices for up to 48 h (ex vivo) followed by functional analysis. Results We detected 5-HT7R expression in both models during all stages of macrophage polarization at mRNA as well as protein levels. Pro-inflammatory THP-1-derived macrophages displayed significantly enhanced mRNA expression levels of IL-1β and CD80, while pro-inflammatory differentiated M1-like BMDMs showed increased levels of CD38 and TNFα. Pharmacological activation of 5-HT7R with LP-211 reduced phagocytic activity up to 4-fold in THP-1-derived pro-inflammatory macrophages, altered their cytokine secretion profile and inhibited their migration ability (recovered area in scratch assay 1.3 % after LP-211 treatment compared to 6.1 % in control). A reduction in phagocytic activity was also verified in pro-inflammatory BMDMs. Our results also indicate reduced ability of LP-211 treated M1-like BMDMs to infiltrate living cardiac slices 3, 12, 24 and 48 h after seeding. Conclusion 5-HT7R signalling affects the functional properties of pro-inflammatory macrophages, including phagocytic activity, motility and secretion profile. Targeted manipulation of 5-HT7R activity in macrophages represents thus a possibility to modulate their inflammatory profile, which might be a reasonable approach to guide recovery post MI.

ВЛИЯНИЕ НОВЫХ ПРОИЗВОДНЫХ НИКОТИНОВОЙ КИСЛОТЫ НА ГИППОКАМП КРЫС

Experiments on hippocampal slices in rats showed that new nicotine acid derivatives LKhT 8-20 and LKhT 9-20 at a concentration of 5 mM inhibited orthrodromic population responses in field СА1 by 89  1 % and 84  2 %, respectively; in comparison, mexicor (reference preparation) in equal concentration inhibited these responses by 52  4 %. Effectiveness of LKhT 8-20 and LKhT 9-20 exceeded that of mexicor in 1.7 and 1.6 times, respectively; LKhT 8-20 exceeded LKhT 9-20 in 1.1 times. Also, in the presence of L-glutamate (0.75 М) LKhT 8-20 (5 mМ) inhibited the evoked neuron responses by 95  2 % and LKhT 9-20 (5 mМ) by 86  1 %. Therefore, LKhT 8-20 and LKhT 9-20 inhibit synaptic transmission between Schaffer collaterals and pyramidal neurons in rat's hippocampal field СА1. Both compounds are L-glutamergic by nature which reveals itself by almost complete in LKhT 8-20 and to a less degree (primarily) in LKhT 9-20. It may be deduced that hippocampus has an important role in the central effect of these new compounds.

Modulating effect of irisin on the functional state of inhibitory afferent inputs to the suprachiasmatic nucleus from the arcuate nucleus

In vitro experiments on viable hypothalamic slices of male Wistar rats, the modulating effect of the myokine irisin on the parameters of inhibitory responses of neurons in the suprachiasmatic nucleus to stimulation of the arcuate nucleus was studied. In 25% of cases, applications of 4 nM irisin caused a qualitative change in reactions, expressed in the appearance of a new reaction phase, or in the disappearance of a pre-existing inhibitory phase. In remaining cases, there was a quantitative change in the inhibitory response to stimulation in the form of a decrease in its duration. The reactions were characterized by complete reversibility: 15 minutes after “washing” the slice from irisin with artificial cerebrospinal fluid, the parameters of inhibitory reactions did not differ from the initial ones. The results obtained show that in addition to directly influencing the spike activity of neurons of the suprachiasmatic nucleus, irisin has an indirect effect on the circadian biological clock, modulating the functional state of inhibitory afferent inputs from the region of the arcuate nucleus.

Maternal separation modifies spontaneous synaptic activity in the infralimbic cortex of stress-resilient male rats.

Glutamate and GABA signaling systems are necessary to maintain proper function of the central nervous system through excitation/inhibition (E/I) balance. Alteration of this balance in the medial prefrontal cortex (mPFC), as an effect of early-life stress, may lead to the development of anxiety and depressive disorders. Few studies exist in the infralimbic division of the mPFC to understand the effect of early-life stress at different ages, which is the purpose of the present work. Newborn Sprague Dawley male rats were subjected to maternal separation (MS) for two weeks. First, tests measuring anxiety- and depression-like behaviors were performed on adolescent and adult rats subjected to MS (MS-rats). Then, to establish a relationship with behavioral results, electrophysiological recordings were performed in neurons of the infralimbic cortex in acute brain slices of infant, adolescent, and adult rats. In the behavioral tests, there were no significant differences in MS-rats compared to control rats at any age. Moreover, MS had no effect on the passive membrane properties nor neuronal excitability in the infralimbic cortex, whereas spontaneous synaptic activity in infralimbic neurons was altered. The frequency of spontaneous glutamatergic synaptic events increased in infant MS-rats, whereas in adolescent MS-rats both the frequency and the amplitude of spontaneous GABAergic events increased without any effect on glutamatergic synaptic responses. In adult MS-rats, these two parameters decreased in spontaneous GABAergic synaptic events, whereas only the frequency of glutamatergic events decreased. These data suggest that rats subjected to MS did not exhibit behavioral changes and presented an age-dependent E/I imbalance in the infralimbic cortex, possibly due to differential changes in neurotransmitter release and/or receptor expression.

Fully-primed slowly-recovering vesicles mediate presynaptic LTP at neocortical neurons.

Pre- and postsynaptic forms of long-term potentiation (LTP) are candidate synaptic mechanisms underlying learning and memory. At layer 5 pyramidal neurons, LTP increases the initial synaptic strength but also short-term depression during high-frequency transmission. This classical form of presynaptic LTP has been referred to as redistribution of synaptic efficacy. However, the underlying mechanisms remain unclear. We therefore performed whole-cell recordings from layer 5 pyramidal neurons in acute cortical slices of rats and analyzed presynaptic function before and after LTP induction by paired pre- and postsynaptic neuronal activity. LTP was successfully induced in about half of the synaptic connections tested and resulted in increased synaptic short-term depression during high-frequency transmission and a decelerated recovery from short-term depression due to an increased fraction of a slow recovery component. Analysis with a recently established sequential two-step vesicle priming model indicates an increase in the abundance of fully-primed and slowly-recovering vesicles. A systematic analysis of short-term plasticity and synapse-to-synapse variability of synaptic strength at various types of synapses revealed that stronger synapses generally recover more slowly from synaptic short-term depression. Finally, pharmacological stimulation of the cyclic adenosine monophosphate and diacylglycerol signaling pathways, which are both known to promote synaptic vesicle priming, mimicked LTP and slowed the recovery from short-term depression. Our data thus demonstrate that LTP at layer 5 pyramidal neurons increases synaptic strength primarily by enlarging a subpool of fully-primed slowly-recovering vesicles.

Localized cardiomyocyte lipid accumulation is associated with slowed epicardial conduction in rats.

Transmural action potential duration differences and transmural conduction gradients aid the synchronization of left ventricular repolarization, reducing vulnerability to transmural reentry and arrhythmias. A high-fat diet and the associated accumulation of pericardial adipose tissue are linked with conduction slowing and greater arrhythmia vulnerability. It is predicted that cardiac adiposity may more readily influence epicardial conduction (versus endocardial) and disrupt normal transmural activation/repolarization gradients. The aim of this investigation was to determine whether transmural conduction gradients are modified in a rat model of pericardial adiposity. Adult Sprague-Dawley rats were fed control/high-fat diets for 15 wk. Left ventricular 300 µm tangential slices were generated from the endocardium to the epicardium, and conduction was mapped using microelectrode arrays. Slices were then histologically processed to assess fibrosis and cardiomyocyte lipid status. Conduction velocity was significantly greater in epicardial versus endocardial slices in control rats, supporting the concept of a transmural conduction gradient. High-fat diet feeding increased pericardial adiposity and abolished the transmural conduction gradient. Slowed epicardial conduction in epicardial slices strongly correlated with an increase in cardiomyocyte lipid content, but not fibrosis. The positive transmural conduction gradient reported here represents a physiological property of the ventricular activation sequence that likely protects against reentry. The absence of this gradient, secondary to conduction slowing and cardiomyocyte lipid accumulation, specifically in the epicardium, indicates a novel mechanism by which pericardial adiposity may exacerbate ventricular arrhythmias.

Analogue signaling of somatodendritic synaptic activity to axon enhances GABA release in young cerebellar molecular layer interneurons.

Axons are equipped with the digital signaling capacity by which they generate and faithfully propagate action potentials (APs), and also with the analogue signaling capacity by which subthreshold activity in dendrites and soma is transmitted down the axon. Despite intense work, the extent and physiological role for subthreshold synaptic activity reaching the presynaptic boutons has remained elusive because of the technical limitation to record from them. To address this issue, we made simultaneous patch-clamp recordings from the presynaptic varicosities of cerebellar GABAergic interneurons together with their parent soma or postsynaptic target cells in young rat slices and/or primary cultures. Our tour-de-force direct functional dissection indicates that the somatodendritic spontaneous excitatory synaptic potentials are transmitted down the axon for significant distances, depolarizing presynaptic boutons. These analogously transmitted excitatory synaptic potentials augment presynaptic Ca++ influx upon arrival of an immediately following AP through a mechanism that involves a voltage-dependent priming of the Ca++ channels, leading to an increase in GABA release, without any modification in the presynaptic AP waveform or residual Ca++. Our work highlights the role of the axon in synaptic integration.

Serum of COVID-19 patients changes neuroinflammation and mitochondrial homeostasis markers in hippocampus of aged rats.

Patients affected by COVID-19 present mostly with respiratory symptoms but acute neurological symptoms are also commonly observed. Furthermore, a considerable number of individuals develop persistent and often remitting symptoms months after infection, characterizing the condition called long-COVID. Since the pathophysiology of acute and persistent neurological manifestations is not fully established, we evaluated the expression of different genes in hippocampal slices of aged rats exposed to the serum of a post-COVID (sPC) individual and to the serum of patients infected by SARS-CoV-2 [Zeta (sZeta) and Gamma (sGamma) variants]. The expression of proteins related to inflammatory process, redox homeostasis, mitochondrial quality control and glial reactivity was determined. Our data show that the exposure to sPC, sZeta and sGamma differentially altered the mRNA levels of most inflammatory proteins and reduced those of antioxidant response markers in rat hippocampus. Furthermore, a decrease in the expression of mitochondrial biogenesis genes was induced by all serum samples, whereas a reduction in mitochondrial dynamics was only caused by sPC. Regarding the glial reactivity, S100B expression was modified by sPC and sZeta. These findings demonstrate that changes in the inflammatory response and a reduction of mitochondrial biogenesis and dynamics may contribute to the neurological damage observed in COVID-19 patients.

Expression changes of NaV channel subunits correlate with developmental maturation of electrophysiological characteristics of rat cerebellar Purkinje neurons

To investigate the variations in the expression of voltage-gated sodium (Nav) channel subunits during development of rat cerebellar Purkinje neurons and their correlation with maturation of electrophysiological characteristics of the neurons. We observed the changes in the expression levels of NaV1.1, 1.2, 1.3 and 1.6 during the development of Purkinje neurons using immunohistochemistry in neonatal (5-7 days after birth), juvenile (12-14 days), adolescent (21-24 days), and adult (42-60 days) SD rats. Using whole-cell patch-clamp technique, we recorded the spontaneous electrical activity of the neurons in ex vivo brain slices of rats of different ages to analyze the changes of electrophysiological characteristics of these neurons during development. The expression of NaV subunits in rat cerebellar Purkinje neurons showed significant variations during development. NaV1.1 subunit was highly expressed throughout the developmental stages and increased progressively with age (P < 0.05). NaV1.2 expression was not detected in the neurons in any of the developmental stages (P > 0.05). The expression level of NaV1.3 decreased with development and became undetectable after adolescence (P < 0.05). NaV1.6 expression was not detected during infancy, but increased with further development (P < 0.05). NaV1.1 and NaV1.3 were mainly expressed in the early stages of development. With the maturation of the rats, NaV1.3 expression disappeared and NaV1.6 expression increased in the neurons. NaV1.1 and NaV1.6 were mainly expressed after adolescence. The total NaV protein level increased gradually with development (P < 0.05) and tended to stabilize after adolescence. The spontaneous frequency and excitability of the Purkinje neurons increased gradually with development and reached the mature levels in adolescence. The developmental expression of NaV subunits was positively correlated with discharge frequency (r=0.9942, P < 0.05) and negatively correlated with the excitatory threshold of the neurons (r=0.9891, P < 0.05). The changes in the expression levels of NaV subunits are correlated with the maturation of high frequency electrophysiological properties of the neurons, suggesting thatmature NaV subunit expressions is the basis of maturation of electrophysiological characteristics of the neurons.

Deep isoflurane anesthesia is associated with alterations in ion homeostasis and specific Na+/K+-ATPase impairment in the rat brain.

Maintenance of ion homeostasis is essential for normal brain function. Inhalational anesthetics are known to act on various receptors but their effects on ion homeostatic systems, such as the Na+/K+-ATPase, remain largely unexplored. Based on reports demonstrating global network activity and wakefulness modulation by interstitial ions, we hypothesized that deep isoflurane anesthesia affects ion homeostasis and the key mechanism for clearing extracellular K+, the Na+/K+-ATPase. Using ion-selective microelectrodes, we assessed isoflurane-induced extracellular ion dynamics in cortical slices of male and female Wistar rats in the absence of synaptic activity, the presence of two-pore-domain K+ channel antagonists, during seizures, and spreading depolarizations. We measured specific isoflurane effects on Na+/K+-ATPase function using a coupled enzyme assay and studied the relevance of our findings in vivo and in silico. Isoflurane concentrations clinically relevant for burst suppression anesthesia increased baseline [K+]o (mean±SD: 3.0 ± 0.0 versus 3.9 ± 0.5mM, p<0.001, n=39) and lowered [Na+]o (153.4 ± 0.8 versus 145.2 ± 6.0mM, p<0.001, n=28). Similar changes in [K+]o, [Na+]o, and a substantial drop in [Ca2+]o (1.5 ± 0.0 versus 1.2 ± 0.1mM, p=0.001, n=16) during inhibition of synaptic activity and two-pore-domain K+ suggested a different underlying mechanism. Following seizure-like events and spreading depolarization, isoflurane greatly slowed [K+]o clearance (63.4 ± 18.2 versus 196.2 ± 82.4s, p<0.001, n=14). Na+/K+-ATPase activity was markedly reduced after isoflurane exposure (>25%), affecting specifically the α2/3 activity fraction. In vivo, isoflurane-induced burst suppression resulted in impaired [K+]o clearance and interstitial K+ accumulation. A computational biophysical model reproduced the observed effects on [K+]o and displayed intensified bursting when Na+/K+-ATPase activity was reduced by 35%. Finally, Na+/K+-ATPase inhibition with ouabain induced burst-like activity during light anesthesia in vivo. Our results demonstrate cortical ion homeostasis perturbation and specific Na+/K+-ATPase impairment during deep isoflurane anesthesia. Slowed K+ clearance and extracellular accumulation might modulate cortical excitability during burst suppression generation while prolonged Na+/K+-ATPase impairment could contribute to neuronal dysfunction after deep anesthesia.

The Formation of Morphologically Stable Lipid Nanocarriers for Glioma Therapy

Cerasomes are a promising modification of liposomes with covalent siloxane networks on the surface that provide outstanding morphological stability while maintaining all the useful traits of liposomes. Herein, thin film hydration and ethanol sol injection methods were utilized to produce cerasomes of various composition, which were then evaluated for the purpose of drug delivery. The most promising nanoparticles obtained by the thin film method were studied closely using MTT assay, flow cytometry and fluorescence microscopy on T98G glioblastoma cell line and modified with surfactants to achieve stability and the ability to bypass the blood-brain barrier. An antitumor agent, paclitaxel, was loaded into cerasomes, which increased its potency and demonstrated increased ability to induce apoptosis in T98G glioblastoma cell culture. Cerasomes loaded with fluorescent dye rhodamine B demonstrated significantly increased fluorescence in brain slices of Wistar rats compared to free rhodamine B. Thin film hydration with Tween 80 addition was established as a more reliable and versatile method for cerasome preparation. Cerasomes increased the antitumor action of paclitaxel toward T98G cancer cells by a factor of 36 and were able to deliver rhodamine B over the blood-brain barrier in rats.

γ-Aminobutyric Acid-Ergic Development Contributes to the Enhancement of Electroencephalogram Slow-Delta Oscillations Under Volatile Anesthesia in Neonatal Rats.

General anesthetics (eg, propofol and volatile anesthetics) enhance the slow-delta oscillations of the cortical electroencephalogram (EEG), which partly results from the enhancement of (γ-aminobutyric acid [GABA]) γ-aminobutyric acid-ergic (GABAergic) transmission. There is a GABAergic excitatory-inhibitory shift during postnatal development. Whether general anesthetics can enhance slow-delta oscillations in the immature brain has not yet been unequivocally determined. Perforated patch-clamp recording was used to confirm the reversal potential of GABAergic currents throughout GABAergic development in acute brain slices of neonatal rats. The power density of the electrocorticogram and the minimum alveolar concentrations (MAC) of isoflurane and/or sevoflurane were measured in P4-P21 rats. Then, the effects of bumetanide, an inhibitor of the Na+-K+-2Cl- cotransporter (NKCC1) and K+-Cl- cotransporter (KCC2) knockdown on the potency of volatile anesthetics and the power density of the EEG were determined in vivo. Reversal potential of GABAergic currents were gradually hyperpolarized from P4 to P21 in cortical pyramidal neurons. Bumetanide enhanced the hypnotic effects of volatile anesthetics at P5 (for MACLORR, isoflurane: 0.63% ± 0.07% vs 0.81% ± 0.05%, 95% confidence interval [CI], -0.257 to -0.103, P < .001; sevoflurane: 1.46% ± 0.12% vs 1.66% ± 0.09%, 95% CI, -0.319 to -0.081, P < .001); while knockdown of KCC2 weakened their hypnotic effects at P21 in rats (for MACLORR, isoflurane: 0.58% ± 0.05% to 0.77% ± 0.20%, 95% CI, 0.013-0.357, P = .003; sevoflurane: 1.17% ± 0.04% to 1.33% ± 0.04%, 95% CI, 0.078-0.244, P < .001). For cortical EEG, slow-delta oscillations were the predominant components of the EEG spectrum in neonatal rats. Isoflurane and/or sevoflurane suppressed the power density of slow-delta oscillations rather than enhancement of it until GABAergic maturity. Enhancement of slow-delta oscillations under volatile anesthetics was simulated by preinjection of bumetanide at P5 (isoflurane: slow-delta changed ratio from -0.31 ± 0.22 to 1.57 ± 1.15, 95% CI, 0.67-3.08, P = .007; sevoflurane: slow-delta changed ratio from -0.46 ± 0.25 to 0.95 ± 0.97, 95% CI, 0.38-2.45, P = .014); and suppressed by KCC2-siRNA at P21 (isoflurane: slow-delta changed ratio from 16.13 ± 5.69 to 3.98 ± 2.35, 95% CI, -18.50 to -5.80, P = .002; sevoflurane: slow-delta changed ratio from 0.13 ± 2.82 to 3.23 ± 2.49, 95% CI, 3.02-10.79, P = .003). Enhancement of cortical EEG slow-delta oscillations by volatile anesthetics may require mature GABAergic inhibitory transmission during neonatal development.

The Effects of Insulin on Spike Activity of the Suprachiasmatic Nucleus Neurones and Functional State of Afferent Inputs from the Arcuate Nucleus in Rats

In experiments on the sagittal hypothalamic slices of male Wistar rats, the effects of 15 nM insulin on the level of spike activity, parameters of spike information coding by the suprachiasmatic nucleus neurones, and functional state of afferent inputs to the neurones from the arcuate nucleus were studied. Application of insulin induced a decrease in the frequency of action potential generation and an increase in the entropy of interspike interval distribution in 33.3% of neurones recorded; in 12% of cells, the responses of opposite direction were found; in the remaining 54.7% neurones, spike activity did not change. The responses of the entropy of interspike interval distribution suggest the related changes in a degree of interspike interval irregularity induced by insulin. To characterise afferent inputs to the suprachiasmatic nucleus neurones from the arcuate nucleus, electrophysiological technique of the construction and analysis of the peristimulus time histogram (PSTH) was used. Statistically significant responses to the stimulation of the arcuate nucleus were recorded in 24 of 38 neurones of the suprachiasmatic nucleus. In 6 of the neurones, the responses were in the form of a short-latency (20 ms) excitation, in 1 neurone in the form of a long-latency excitation, in 6 neurones in the form of a short-latency inhibition; in 11 neurones complex two- or three-phase responses in the form of different compositions of excitation and inhibition were observed. Application of 15 nM insulin induced a qualitative transformation of the responses (disappearing of the initial responses or emergence of new responses) in 5 neurones initially responded to stimulation, and in 1 neurone initially not responded to stimulation of the arcuate nucleus. Statistically significant changes in the latency or duration of the responses in the presence of insulin were not found. The results of the study suggest the ability of insulin to influence the activity level and the spike code of a respectively numerous population of neurones in the suprachiasmatic nucleus circadian oscillator as well as modulate the functional state of afferent inputs to the circadian oscillator from hypothalamic arcuate nucleus playing an important role in the control of appetite and metabolism.

ДЕЙСТВИЕ ДВУХ НОВЫХ ПРОИЗВОДНЫХ НИКОТИНОВОЙ КИСЛОТЫ НА УРОВНЕ ГИППОКАМПА У КРЫС

Experiments on hippocampal slices in rats showed that new nicotinic acid derivatives LKhT 6-20 and LKhT 7-20 at 5 mM inhibited the СА1 orthodromic population responses by 43  4 and 69  5%, respectively, whereas preparation of comparison mexicor in equal concentration inhibited the responses by 52  4 %. Action of LKhT 7-20 exceeded LKhT6–20 in 1.6 times and mexicor, in 1.3 times. The refore, LKhT 6-20 and LKhT 7-20 inhibit synaptic transmission between Schaffer collaterals and pyramidal СА1 neurons. It can be inferred that hippocampus has a significant role in the central action of these new substances.

Effects of Phenolic-Rich Pinus densiflora Extract on Learning, Memory, and Hippocampal Long-Term Potentiation in Scopolamine-Induced Amnesic Rats.

Alzheimer's disease is the most common type of dementia with cognitive impairment. Various plant-derived phenolics are known to alleviate cognitive impairment in Alzheimer's disease by radical scavenging and strengthening synaptic plasticity activities. Here, we examined the cognition-improving effect of Pinus densiflora Sieb. et Zucc. bark extract (PBE). We identified and quantified phenolics in the PBE using a UHPLC-Orbitrap mass spectrometer. To evaluate the cognition-enhancing effects of PBE, scopolamine-induced amnesic Sprague-Dawley (SD) rats (5 weeks old) and ion channel antagonist-induced organotypic hippocampal slices of SD rats (7 days old) were used. Twenty-three phenolics were tentatively identified in PBE, 10 of which were quantified. Oral administration of PBE to the scopolamine-induced SD rats improved cognitive impairment in behavioral tests. PBE-fed SD rats showed significantly improved antioxidant indices (superoxide dismutase and catalase activities, and malondialdehyde content) and reduced acetylcholinesterase activity in hippocampal lysate compared with the scopolamine group. PBE increased the long-term potentiation (LTP) induction and rescued LTP from blockades by the muscarinic cholinergic receptor antagonist (scopolamine) and N-methyl-D-aspartate channel antagonist (2-amino-5-phosphonovaleric acid) in the organotypic hippocampal slices. These results suggest that polyphenol-rich PBE is applicable as a cognition-improving agent due to its antioxidant properties and enhancement of LTP induction.

Neonatal Isoflurane Exposure in Rats Impairs Short-Term Memory, Cell Viability, and Glutamate Uptake in Slices of the Frontal Cerebral Cortex, But Not the Hippocampus, in Adulthood.

Neonatal exposure to general anesthetics has been associated with neurotoxicity and morphologic changes in the developing brain. Isoflurane is a volatile anesthetic widely used in pediatric patients to induce general anesthesia, analgesia, and perioperative sedation. In the present study, we investigated the effects of a single neonatal isoflurane (3% in oxygen, 2h) exposure in rats at postnatal day (PND) 7, in short-term (24h - PND8) and long-term (adulthood) protocols. In PND8, ex vivo analysis of hippocampal and frontal cortex slices evaluated cell viability and susceptibility to in vitro glutamate challenge. In adult rats, behavioral parameters related to anxiety-like behavior, short-term memory, and locomotor activity (PND60-62) and ex vivo analysis of cell viability, membrane permeability, glutamate uptake, and susceptibility to in vitro glutamate challenge in hippocampal and cortical slices from PND65. A single isoflurane (3%, 2h) exposure at PND7 did not acutely alter cell viability in cortical and hippocampal slices of infant rats (PND8) per se and did not alter slice susceptibility to in vitro glutamate challenge. In rat's adulthood, behavioral analysis revealed that the neonatal isoflurane exposure did not alter anxiety-like behavior and locomotor activity (open field and rotarod tests). However, isoflurane exposure impaired short-term memory evaluated in the novel object recognition task. Ex vivo analysis of brain slices showed isoflurane neonatal exposure selectively decreased cell viability and glutamate uptake in cortical slices, but it did not alter hippocampal slice viability or glutamate uptake (PND65). Isoflurane exposure did not alter in vitro glutamate-induced neurotoxicity to slices, and isoflurane exposure caused no significant long-term damage to cell membranes in hippocampal or cortical slices. These findings indicate that a single neonatal isoflurane exposure did not promote acute damage; however, it reduced cortical, but not hippocampal, slice viability and glutamate uptake in the adulthood. Additionally, behavioral analysis showed neonatal isoflurane exposure induces short-term recognition memory impairment, consolidating that neonatal exposure to volatile anesthetics may lead to behavioral impairment in the adulthood, although it may damage brain regions differentially.