System In Slices Research Articles

Improved seizure liability detection by combining rat hippocampal brain slice electrophysiology with in vivo behavior observation following intracerebroventricular drug administration

An adverse effect of drug candidates, seizure is a serious issue in drug development. Improving evaluation systems for seizure liability is crucial for selecting good candidates. Firstly, in vitro electrophysiological measurement by a multielectrode array system in rat hippocampal brain slices was employed to confirm an increase in electrically evoked population spike (PS) area, the occurrence of multiple population spikes (MPSs), and thereby the seizure liability of five positive control chemicals: picrotoxin, 4-aminopyridine, pentylenetetrazole, penicillin G, and chlorpromazine. Aspirin, a negative control, did not affect PS area or generate MPSs. Furthermore, baclofen, an anticonvulsant drug, decreased PS area and inhibited the increase in PS area or occurrence of MPSs induced by picrotoxin. A comparative study of seizure liability among carbapenem antibiotics revealed that tienam > carbenin > omegacin and finibax. Despite leading to a strong decrease in PS area, physostigmine, cisplatin, and paroxetine still produced MPSs. Therefore, the increase in PS area or the occurrence of the MPS are considered significant evaluation parameters for seizure liability.In contrast, the in vitro electrophysiological measurement could not detect the seizure liability of diphenhydramine or fluvoxamine. A follow-up study of in vivo mouse behavioral change induced by intracerebroventricular administration of these drugs clearly detected convulsions. The in vitro electrophysiological study using hippocampal brain slices combined with in vivo behavior observation study of drug candidates administered by intracerebroventricular injection can implement to assess the seizure liability of even small amounts, especially in the early stages of drug development.

Somatostatin contributes to long-term potentiation at excitatory synapses onto hippocampal somatostatinergic interneurons

Somatostatin-expressing interneurons (SOM-INs) are a major subpopulation of GABAergic cells in CA1 hippocampus that receive excitation from pyramidal cells (PCs), and, in turn, provide feedback inhibition onto PC dendrites. Excitatory synapses onto SOM-INs show a Hebbian long-term potentiation (LTP) mediated by type 1a metabotropic glutamate receptors (mGluR1a) that is implicated in hippocampus-dependent learning. The neuropeptide somatostatin (SST) is also critical for hippocampal long-term synaptic plasticity, as well as learning and memory. SST effects on hippocampal PCs are well documented, but its actions on inhibitory interneurons remain largely undetermined. In the present work, we investigate the involvement of SST in long-term potentiation of CA1 SOM-IN excitatory synapses using pharmacological approaches targeting the somatostatinergic system and whole cell recordings in slices from transgenic mice expressing eYFP in SOM-INs. We report that application of exogenous SST14 induces long-term potentiation of excitatory postsynaptic potentials in SOM-INs via somatostatin type 1–5 receptors (SST1-5Rs) but does not affect synapses of PC or parvalbumin-expressing interneurons. Hebbian LTP in SOM-INs was prevented by inhibition of SSTRs and by depletion of SST by cysteamine treatment, suggesting a critical role of endogenous SST in LTP. LTP of SOM-IN excitatory synapses induced by SST14 was independent of NMDAR and mGluR1a, activity-dependent, and prevented by blocking GABAA receptor function. Our results indicate that endogenous SST may contribute to Hebbian LTP at excitatory synapses of SOM-INs by controlling GABAA inhibition, uncovering a novel role for SST in regulating long-term synaptic plasticity in somatostatinergic cells that may be important for hippocampus-dependent memory processes.

Electrophysiological field potential identification of an intact GABAergic system in mouse cortical slices

In brain slice experiments there’s currently no validated electrophysiological method for differentiating viability between GABAergic and glutamatergic cell populations. Here we investigated the neurophysiology of high frequency field potential activity — and its utility for probing the functional state of the GABAergic system in brain slices.Field potentials were recorded from mouse cortical slices exposed to 50 mM potassium (“elevated-K”) and the induced high frequency (>20 Hz) response characterized pharmacologically. The elevated-K responses were also related to the high frequency activity imbedded in no-magnesium seizure-like events (SLE) from the same slices.The elevated-K response, comprising a transient burst of high frequency activity, was strongly GABAA-dependent. The size of the high frequency response was reduced by 71% (p = 0.001) by picrotoxin, but not significantly attenuated by either APV or CNQX. High frequency activity embedded in no-magnesium SLEs correlated with the elevated-K response. The success rate for generating an elevated-K response — and high frequency SLE activity — declined rapidly with increasing time since slicing.These findings support the hypothesis that in cortical slices, a functioning synaptic GABAergic system is evidenced by a strong high frequency component to no-magnesium SLE activity — and that the integrity of the GABAergic system degrades quicker than the excitatory glutamatergic system in this preparation.

Native T1 mapping in diffuse myocardial diseases using 3-Tesla MRI: An institutional experience.

Aims:Newer cardiac magnetic resonance techniques like native T1 mapping are being used increasingly as an adjunct to diagnose myocardial diseases with fibrosis. However, its full clinical utility has not been tested extensively, especially in the Indian population. The purpose of this study was to find native T1 values in healthy individuals without cardiac disease in our 3-Tesla MRI system and examine whether native myocardial T1 values can be used to differentiate between normal and diffuse myocardial disease groups.Subjects and Methods:After approval from the institutional ethics committee, native T1 mapping was performed in 12 healthy individuals without cardiac disease who served as controls and in 26 patients with diffuse myocardial diseases (acute myocarditis (n = 5), hypertrophic cardiomyopathy (HCM) (n = 8), nonischemic dilated cardiomyopathy (DCM) (n = 7), restrictive cardiomyopathy (RCM) due to amyloidosis (n = 6)) in a 3-Tesla MRI system in short axis slices and four-chamber view using a modified Look-Locker inversion recovery sequence. The mean native T1 values and standard deviations were calculated for control and disease groups and compared. The ability of native myocardial T1 mapping to differentiate between normal and diffuse myocardial disease groups was assessed. One-way ANOVA with Tukey's Post-Hoc test was used to find significant difference in the multivariate analysis and Chi-Square test was used to find the significance in categorical data.Results:The native T1 values for the healthy group in our 3-Tesla MRI system was 1186.47 ± 45.67 ms. The mean T1 values of the groups acute myocarditis (1418.68 ± 8.62 ms), HCM (1355.86 ± 44.67 ms), nonischemic DCM (1341.31 ± 41.48 ms), and RCM due to amyloidosis (1370.37 ± 90.14 ms) were significantly higher (P = 0.0005) than that of the healthy control group.Conclusion:Native myocardial T1 mapping is a promising tool for differentiating between healthy and diffuse myocardial disease groups.

СИСТЕМА БИОЛОГИЧЕСКОЙ ОБРАТНОЙ СВЯЗИ ДЛЯ ПОДАВЛЕНИЯ ЭПИЛЕПТИЧЕСКОЙ АКТИВНОСТИ В ОПТОГЕНЕТИЧЕСКОМ ЭКСПЕРИМЕНТЕ, "Российский физиологический журнал им. И.М. Сеченова"

One of the promising options for treating pharmacoresistant forms of epilepsy is low-frequency stimulation of the brain. However, to avoid the side effects, it is required to minimize the stimulation of the brain. This can be achieved if stimulation occurs only at the time of increased probability of the ictal discharge and if it has a specific action on only one type of cells. A way to meet these conditions is to use a closed-loop system between the nervous tissue and a computer in the optogenetic experiment. We have developed a closed-loop system in the optogenetic experiment for effective and harmless suppression of epileptic activity and implemented it in slices of the rat hippocampus and the entorhinal cortex as well as in the primary hippocampal culture. First, we tested the closed-loop system in slices in 4-aminopyridine model of epilepsy. Epileptic activity was detected automatically via monitoring of spiking activity of a representative neuron in the entorhinal cortex. Since a neuron is firing only in response to synaptic input in the used epilepsy model, the spike threshold is a valid indicator of the beginning of epileptic discharge. The software realizing the closed-loop system starts the low-frequency stimulation in response to detection of an epileptic event. Next, we tested the developed system in an optogenetic experiment in culture after expression of channelrhodopsins. These results indicate the potential effectiveness of this approach for the suppression of ictal events

Resin glycosides evoke the Gaba release by sodium- and/or calcium-dependent mechanism

Ipomoea tyrianthina Lindley (syn. I. orizabensis Pelletan, Lebed. ex Steud., Convolvulaceae) is known as a purgative, but it has been also used in Mexican traditional medicine in the treatment of seizures and pain for their anticonvulsive, hypnotic and sedative properties. Some glycolipids isolated from this plant have shown significant effects on Central Nervous System, modifying inhibitory or excitatory processes. The mechanism for such activity it is not clear; studies with these metabolites have suggested that a pore-forming mechanism is involved in their activity. Therefore, the present work explores a possible not pore-forming mechanism related to the effect of four resin glycosides, Scammonin 1 (S-1), tyrianthin C (T-C), tyrianthin A (T-A) and tyrianthinic acid VI (TA-VI), isolated from Ipomoea tyrianthina root on GABAergic transmission system in cerebral cortex slices of mouse brain in an in vitro model. The results obtained show that all glycolipids tested evoked endogenous GABA release and increased its concentration within the incubation medium compared with controls; T-C demonstrated a dose-dependent effect. Sodium absence and guvacine presence did not affect significantly the activity of S-1 and T-C in contrast to T-A and TA-VI. S-1 and T-C effects were calcium-dependent, where GABA concentrations were considerably reduced. These results suggest that the increase of endogenous γ-aminobutyric acid (GABA) released evoked by these glycolipids is possibly done through a Na+- and/or Ca2+-dependent mechanisms, discarding a pore-forming mechanism.

Integration of autopatching with automated pipette and cell detection in vitro.

Patch clamp is the main technique for measuring electrical properties of individual cells. Since its discovery in 1976 by Neher and Sakmann, patch clamp has been instrumental in broadening our understanding of the fundamental properties of ion channels and synapses in neurons. The conventional patch-clamp method requires manual, precise positioning of a glass micropipette against the cell membrane of a visually identified target neuron. Subsequently, a tight "gigaseal" connection between the pipette and the cell membrane is established, and suction is applied to establish the whole cell patch configuration to perform electrophysiological recordings. This procedure is repeated manually for each individual cell, making it labor intensive and time consuming. In this article we describe the development of a new automatic patch-clamp system for brain slices, which integrates all steps of the patch-clamp process: image acquisition through a microscope, computer vision-based identification of a patch pipette and fluorescently labeled neurons, micromanipulator control, and automated patching. We validated our system in brain slices from wild-type and transgenic mice expressing channelrhodopsin 2 under the Thy1 promoter (line 18) or injected with a herpes simplex virus-expressing archaerhodopsin, ArchT. Our computer vision-based algorithm makes the fluorescent cell detection and targeting user independent. Compared with manual patching, our system is superior in both success rate and average trial duration. It provides more reliable trial-to-trial control of the patching process and improves reproducibility of experiments.

ELECTRICAL CHARACTERISTICS OF RAA AND LAA IN 48 H AF CANINE WITH MEA CHIP TECHNIQUE

ObjectivesTo explore electrical characteristics of RAA and LAA in 48 h AF canine with MEA chip technique.MethodsTwelve adult healthy mongrel canine were randomly divided into two groups:pacing group and control...

Triple mammalian/yeast/bacterial shuttle vectors for single and combined Lentivirus- and Sindbis virus-mediated infections of neurons

Today, a large variety of viral vectors is available for ectopic gene expression in mammalian cell cultures or in vivo. Among them, infection with Sindbis virus- or Lentivirus-derived constructs is often used to address biological questions or for applications in neuronal therapies. However, cloning of genes of interest is time consuming, since it relies on restriction and ligation, frequently of PCR-generated DNA fragments with suitable restriction sites introduced by the primers employed. We here take advantage of the unusually high capacity for homologous recombination in Saccharomyces cerevisiae to circumvent this problem, and introduce a new set of triple shuttle vectors, which can be shuffled between E. coli, yeast, and mammalian cells. The system allows the introduction of genes of interest largely independent of the target site in the vectors. It also allows the removal of the yeast selection marker by Cre-recombinase directed recombination in E. coli, if vector size limits transfection efficiency in the mammalian cells. We demonstrate the expression of genes encoding fluorescent proteins (EGFP and mCherry) both separately and in combination, using two different viral systems in mammalian cell lines, primary neurons and organotypic slices.

Impaired hippocampal glucoregulation in the cannabinoid CB1 receptor knockout mice as revealed by an optimized in vitro experimental approach

Several techniques exist to study the rate of glucose uptake and metabolism in the brain but most of them are not sufficiently robust to permit extensive pharmacological analysis. Here we optimized an in vitro measurement of the simultaneous accumulation of the metabolizable and non-metabolizable 3H and 14C d-glucose analogues; permitting convenient large-scale studies on glucose uptake and metabolism in brain slices. Next, we performed an extensive pharmacological characterization on the putative glucoregulator role of the endocannabinoid system in the hippocampal slices of the rat, and the wild-type and the CB1 cannabinoid receptor (CB1R) knockout mice.We observed that 3H-3-O-methylglucose is a poor substrate to measure glucose uptake in the hippocampus. 3H-2-deoxyglucose is a better substrate but its uptake is still lower than that of 14C-U-d-glucose, from which the slices constantly metabolize and dissipate 14C atoms. Thus, uptake and the metabolism values are not to be used as standalones but as differences between a control and a treatment.The CB1R knockout mice exhibited ∼10% less glucose uptake and glucose carbon atom dissipation in comparison with the wild-type mice. This may represent congenital defects as acute treatments of the rat and mouse slices with cannabinoid agonists, antagonists and inhibitors of endocannabinoid uptake/metabolism failed to induce robust changes in either the uptake or the metabolism of glucose.In summary, we report here an optimized technique ideal to complement other metabolic approaches of high spatiotemporal resolution. This technique allowed us concluding that CB1Rs are at least indirectly involved in hippocampal glucoregulation.

Comparison of approaches for microscopic imaging of skin lymphatic vessels

Assessment of skin lymphatic vessels is of great significance in understanding their roles in many pathological conditions. Our aim was to identify the optimal approach for investigation of cutaneous lymphatic system. We performed comparative studies on skin lymphatic vessels using immunohistochemistry of tissue sections, computer graphic reconstruction method together with immunohistochemically stained serial sections and whole mount fluorescence in human lower limb. Lymphatic vessels were identified with podoplanin antibody. The relative merits and drawbacks of each method in evaluation of structure, spatial organization, and distribution of cutaneous lymphatic vessels were described. Immunohistology of tissue sections enabled the investigation of the structure and distribution of the whole cutaneous lymphatic system in two-dimensional slices, whereas three-dimensional morphology of only the most superficial lymph capillary network immediately under the epidermis could be evaluated with the whole mount technique. Meanwhile, only little segmentation of skin lymphatic vessel from five immunohistochemically stained serial sections was reconstructed and evaluated due to expense and special skills required using computer graphic three-dimensional reconstruction. Furthermore, a great number of artifacts and special skills required in its processes leaded to less accurate structure of skin lymphatic vessels. Our findings demonstrated that the use of either of the proposed techniques alone could not allow a comprehensive analysis of the skin lymphatic system due to their relative drawbacks. Combination of immunohistology of tissue sections and three-dimensional whole-mount preparations appears to be the best candidate for comprehensive evaluation of skin lymphatic system.

Effect of prednisone on transforming growth factor‐β1, connective tissue growth factor, nuclear factor‐κBp65 and tumor necrosis factor‐α expression in a murine model of hepatic sinusoidal obstruction syndrome induced by Gynura segetum

One major cause of hepatic sinusoidal obstruction syndrome (HSOS) is the consumption of products containing pyrrolizidine alkaloids (PA). As the use of herbal preparations has increased in China, so has the number of reports of HSOS induced by ingesting PA-containing herbs. The aim of the present study was to investigate the mechanisms by which prednisone and the related factors, transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF), prevent liver fibrosis and the pathogenesis of HSOS. A murine model of HSOS was created by oral gavage with Gynura segetum with or without prednisone for 30 days. Histological changes in liver tissue were evaluated by a scoring system in tissue slices subjected to hematoxylin-eosin and Masson trichrome staining. Hepatic expression of TGF-β1 and CTGF mRNA and protein was detected by immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. RT-PCR was also used to detect tumor necrosis factor (TNF)-α and nuclear factor (NF)-κBp65 mRNA expression. Activation of NF-κBp65 was detected by immunohistochemistry. Intervention with prednisone diminished the symptoms of HSOS in mice treated with G. segetum. Prednisone treatment significantly inhibited expression of TGF-β1 and CTGF mRNA and protein (P < 0.05), and inhibited expression of TNF-α and NF-κBp65 mRNA (P < 0.05) in the liver tissue of HSOS mice. Prednisone suppresses the development of liver fibrosis in HSOS mice by inhibiting TGF-β1, CTGF, TNF-α and NF-κBp65 expression.

Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination

Wide-field temporal focusing is a novel technique that provides optical sectioning for imaging without the need for beam scanning. However, illuminating over large areas greatly reduces the photon density which limits the technique applicability to small regions, precluding functional imaging of cellular networks. Here we present a strategy that combines beam shaping and temporal focusing of amplified pulses (>1 µJ/pulse) for fast imaging of cells from the central nervous system in acute slices. Multiphoton video-rate imaging over total areas as wide as 4800 µm2 with an optical sectioning under 10 µm at 800 nm is achieved with our setup, leading to imaging of calcium dynamics of multiple cells simultaneously in thick tissue.

Up-regulation of cytochrome P450 and phase II enzyme systems in rat precision-cut rat lung slices by the intact glucosinolates, glucoraphanin and glucoerucin

It is believed that the chemopreventive activity of cruciferous vegetables in the lung and other tissues is exclusively the result of exposure to degradation products of glucosinolates, such as the isothiocyanates, and that the parent glucosinolates make no contribution. In the present study, evidence is presented for the first time that, in rat lung, the intact glucosinolates, glucoraphanin and glucoerucin, can modulate carcinogen-metabolising enzyme systems. The glucosinolates were isolated from cruciferous vegetables and incubated (1–25μM) with precision-cut rat lung slices for 24h. Both glucosinolates, at concentrations as low as 1μM, up-regulated the O-deethylation of ethoxyresorufin and the apoprotein levels of CYP1A1 and CYP1B1; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished the rise in ethoxyresorufin O-deethylase activity. In contrast, neither glucosinolate, at the concentrations studied, influenced quinone reductase activity in the lung slices, but addition of myrosinase to the glucosinolate incubations led to a marked rise in activity. Glutathione S-transferase activity, monitored using 1-chloro-2,4-dinitrobenzene as the accepting substrate, was elevated in lung slices exposed to glucoraphanin. GSTα protein levels were increased by glucoraphanin and, to a much lesser extent, glucoerucin. It may be concluded that intact glucosinolates can modulate the activity of pulmonary carcinogen-metabolising enzyme systems, and can thus contribute to the documented chemopreventive activity of cruciferous vegetables in the lung.

The multi-herbal medicine Gongjin-dan enhances memory and learning tasks via NGF regulation

Nerve growth factor (NGF) decreases degeneration of cholinergic neurons, improves memory loss, and increases long-term potentiation and learning tasks. Therefore, NGF mimetics or NGF inducers may be important targets for the treatment of various neurodegenerative disorders. Traditionally, Gongjin-dan (GJD) has been used clinically for the treatment of central nervous system disorders. In this study, we examined the effects of GJD on NGF mimetic activity in PC12 cells and the induction of NGF secretion in primary astrocytes. Moreover, we also measured neuron survival by MAP-2 staining in an immobilization stress rat model and induction of long-term potentiation by the MEA system in rat hippocampus slices treated with dexamethasone. The behavioral syndrome by novel object test was also performed in mice. GJD increased neurite outgrowth in PC12 cells and NGF secretion in primary astrocytes. Also, it reduced neuronal cell death and increased long-term potentiation in the rat hippocampus. Moreover, the number of entries, the time spent and the distance moved in the center area of the test region by the mice was increased by oral administration of GJD in comparison with the distance moved over the total area. These data suggest that administration of GJD may improve memory and learning tasks via NGF regulation, and that it may have a potential for multiple function neuroprotection via NGF regulation.

Lipid rafts regulate 2‐arachidonoylglycerol metabolism and physiological activity in the striatum

Several G protein-associated receptors and synaptic proteins function within lipid rafts, which are subdomains of the plasma membranes that contain high concentrations of cholesterol. In this study we addressed the possible role of lipid rafts in the control of endocannabinoid system in striatal slices. Disruption of lipid rafts following cholesterol depletion with methyl-beta-cyclodestrin (MCD) failed to affect synthesis and degradation of anandamide, while it caused a marked increase in the synthesis and concentration of 2-arachidonoylglycerol (2-AG), as well as in the binding activity of cannabinoid CB1 receptors. Surprisingly, endogenous 2-AG-mediated control of GABA transmission was not potentiated by MCD treatment and, in contrast, neither basal nor 3,5-Dihydroxyphenylglycine-stimulated 2-AG altered GABA synapses in cholesterol-depleted slices. Synaptic response to the cannabinoid CB1 receptor agonist HU210 was however intact in MCD-treated slices, indicating that reduced sensitivity of cannabinoid CB1 receptors does not explain why endogenous 2-AG is ineffective in inhibiting striatal GABA transmission after cholesterol depletion. Confocal microscopy analysis suggested that disruption of raft integrity by MCD might uncouple metabotropic glutamate 5-CB1 receptor interaction by altering the correct localization of both receptors in striatal neuron elements. In conclusion, our data indicate that disruption of raft integrity causes a complex alteration of the endocannabinoid signalling in the striatum.

Spatiotemporal Profiles of Field Potentials in Mouse Superior Colliculus Analyzed by Multichannel Recording

The onset and vector of orienting behaviors, such as saccades, are controlled by commands that descend from a population of neurons in deep layers of the superior colliculus (dSC). In this study, to characterize the role of the collicular local circuitry that generates the spatiotemporal pattern of command activity in the dSC neuronal population, responses evoked by single-pulse electrical stimulation in superficial layers of the superior colliculus (sSC) were analyzed by a 64-channel field potential recording system (planar electrode, 8 x 8 pattern; 150 microm interelectrode spacing) in slices obtained from 16- to 22-d-old mice. A negative field potential with short latency and short duration spatially restricted to the recording sites in sSC was evoked adjacent to the stimulation site. After bath application of 10 mum bicuculline, the same stimulus induced a large negative field response with long duration that spread from sSC to dSC. The dSC potential initially showed a positive response, presumably because of reversal of the negative potential that originated in sSC, and then a long negative response that spread horizontally as far as 1 mm. These responses disappeared after application of an NMDA receptor antagonist, 2-amino-5-phosphonovelarate, indicating that NMDA receptors have an important role in the generation of these responses. Simultaneous whole-cell patch-clamp recordings showed that the long-lasting negative field potentials corresponded to the depolarization accompanying burst spike activity of SC neurons. The present study revealed an extensive excitatory network in the dSC that may contribute to the generation of activity by a large population of neurons that discharge before a saccade.

Stability of cytochromes P450 and phase II conjugation systems in precision-cut rat lung slices cultured up to 72 h

The objective of the present study was to evaluate the stability of cytochrome P450 enzymes and of the conjugation enzyme systems epoxide hydrolase, glucuronosyl transferase, sulphotransferase and glutathione S-transferase in precision-cut rat lung slices incubated in RPMI media for different time periods up to 72 h. Moreover, the effect of culturing of lung slices on total glutathione levels and glutathione reductase was also investigated. Monitoring of cytochrome P450 activity was achieved using established diagnostic probes, but when activity in the lung was low the maintenance of the various enzymes in culture was determined immunologically using Western blotting. The dealkylation of pentoxyresorufin declined markedly during the first 4 h of incubation but in the case of ethoxyresorufin loss of activity was more gradual and less severe. Western blot analysis revealed that the rate of decrease in cytochrome P450 apoprotein levels was isoform-specific with CYP2E1 being the most stable and CYP3A the least stable. Generally, phase II activities, especially cytosolic sulphotransferase, were relatively more stable throughout the incubation period compared with cytochromes P450. Finally, glutathione reductase activity and total glutathione levels were maintained throughout the 72 h incubation. The present studies indicate that xenobiotic-metabolising enzymes in precision-cut rat lung slices decline in culture, but the rate of loss differs and depends on the nature of the enzyme.

Lack of interaction between orexinergic and α 2-adrenergic neuronal systems in rat cerebrocortical slices

Orexinergic and norepinephrinergic α 2-adrenoceptor expressing neurons contribute to the regulation of the sleep–wakefulness cycle. In the present study, we have examined a possible interaction between orexinergic and α 2-adrenergic systems in orexin-A (100 nM)- and K + (25 mM)-evoked norepinephrine release from slices of rat cerebrocortex. In this tissue norepinephrinergic neurons are predominantly innervated via the locus coeruleus. Clonidine concentration-dependently inhibited K +-evoked norepinephrine release with pIC 50 ( I max) of 6.44 ± 0.38 (48.8 ± 6.9%). A selective orexin-1 receptor antagonist, SB-334867 was ineffective. SB-334867 concentration-dependently inhibited orexin A-evoked norepinephrine release with pIC 50 ( I max) of 6.05 ± 0.14 (86.4 ± 5.4%); clonidine (α 2-agonist) was ineffective. In contrast, yohimbine reversed the inhibitory effects of clonidine (1 μM) on K +-evoked norepinephrine release with pIC 50 ( I max) of 6.50 ± 0.34 (77.6 ± 10.9%); orexin A was ineffective. The present data suggest a lack of interaction between orexinergic and α 2-adrenergic neurons in rat cerebral cortex.

Detection and classification of neurotoxins using a novel short-term plasticity quantification method

A tissue-based biosensor is described for screening chemical compounds that rapidly affect the nervous system. The proposed sensor is an extension of a previous work on cultured hippocampal slices [Biosens. Bioelectron. 16 (2001) 491]. The detection of the chemical compounds is based on a novel quantification method of short-term plasticity (STP) of the CA1 system in acute hippocampal slices, using random electrical impulse sequences as inputs and population spike (PS) amplitudes as outputs. STP is quantified by the first and the second order kernels using a variant of the Volterra modeling approach. This approach is more specific and time-efficient than the conventional paired pulse and fixed frequency train methods [J. Neurosci. Methods 2 (2002) 111]. Describing the functional state of the biosensor, the kernels changed accordingly as chemical compounds were added. The second order kernel was decomposed into nine Laguerre functions. The corresponding Laguerre coefficients along with the first order kernel were used as features for classification purposes. The biosensor was tested using picrotoxin (100 μM), trimethylopropane phosphate (10 μM), tetraethylammonium (4 mM), valproate (5 mM), carbachol (5 mM), DAP5 (25 μM), CNQX (3 μM), and DNQX (0.15, 1.5, 3, 5 and 10 μM). Each chemical compound gave a different feature profile corresponding to its pharmacological class. The first order kernel and the Laguerre coefficients formed the input to an artificial neural network (ANN) comprised of a single layer of perceptrons. The ANN was able to classify each tested compound into its respective class.