Noise-induced precursors of tonic-to-bursting transitions in hypothalamic neurons and in a conductance-based model.

The dynamics of neurons is characterized by a variety of different spiking patterns in response to external stimuli. One of the most important transitions in neuronal response patterns is the transition from tonic firing to burst discharges, i.e., when the neuronal activity changes from single spikes to the grouping of spikes. An increased number of interspike-interval sequences of specific temporal correlations was detected in anticipation of temperature induced tonic-to-bursting transitions in both, experimental impulse recordings from hypothalamic brain slices and numerical simulations of a stochastic model. Analysis of the modelling data elucidates that the appearance of such patterns can be related to particular system dynamics in the vicinity of the period-doubling bifurcation. It leads to a nonlinear response on de- and hyperpolarizing perturbations introduced by noise. This explains why such particular patterns can be found as reliable precursors of the neurons' transition to burst discharges.

Dynamics of heart rate and sleep stages in normals and patients with sleep apnea.

Sleep is not just the absence of wakefulness but a regulated process with an important restorative function. Based on electroencephalographic recordings and characteristic patterns and waveforms we can distinguish wakefulness and five sleep stages grouped into light sleep, deep sleep, and rapid-eye-movement (REM) sleep. In order to explore the functions of sleep and sleep stages, we investigated the dynamics of sleep stages over the night and of heart-rate variability during the different sleep stages. Sleep stages and intermediate wake states have different distributions of their duration and this allows us to create a model for the temporal sequence of sleep stages and wake states. Heart rate is easily accessed with a high precision by the recording and analysis of the electrocardiogram (ECG). Heart-rate regulation is part of the autonomous nervous system and sympathetic tone is strongly influenced by the sleep stages.

Local and systemic autonomic nervous effects on cell migration to the spleen.

This work is based on the hypothesis that sympathetic nerves regulate the uptake of circulating cells by the spleen by affecting splenic blood flow and that the quantity of cells sequestered depends on whether changes in noradrenergic transmission occur at local or systemic levels. Fluorescently labeled lymphoid cells were injected into rats, and organ blood flow was measured by the microsphere method. Increased retention of cells in the spleen paralleled by increased blood flow was detected after local denervation of this organ or administration of bacterial endotoxin. A comparable enhanced splenic blood flow was observed after general sympathectomy. However, the redistribution of blood perfusion during general vasodilatation resulted in deviation of leukocyte flow from the spleen, thus resulting in reduced uptake of cells by this organ. These results indicate that, although the uptake of cells by the spleen depends on arterial blood supply, enhanced perfusion does not always result in increased cell sequestration because general vasodilatation reduces cell uptake by this organ and even overrides stimulatory effects of endotoxin.

Fever induction by localized subcutaneous inflammation in guinea pigs: the role of cytokines and prostaglandins.

In guinea pigs, dose-dependent febrile responses can be induced by injection of a high (100 micro g/kg) or low (10 micro g/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. In this fever model, LPS does not enter the systemic circulation from the site of localized tissue inflammation in considerable amounts but causes a local induction of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6), which can be measured in lavage fluid collected from the chamber area. Only in response to the high LPS dose, small traces of TNF are measurable in blood plasma. A moderate increase of circulating IL-6 occurs in response to administration of both LPS doses. To investigate the putative roles of TNF and prostaglandins in this fever model, a neutralizing TNF binding protein (TNF-bp) or a nonselective inhibitor of cyclooxygenases (diclofenac) was injected along with the high or low dose of LPS into the subcutaneous chamber. In control groups, both doses of LPS were administered into the chamber along with the respective vehicles for the applied drugs. The fever response to the high LPS dose remained unimpaired by treatment with TNF-bp despite an effective neutralization of bioactive TNF in the inflamed tissue area. In response to the low LPS dose, there was an accelerated defervescence under the influence of TNF-bp. Blockade of prostaglandin formation with diclofenac completely abolished fever in response to both LPS doses. In conclusion, prostaglandins seem to be essential components for the manifestation of fever in this model.

Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat endothelium.

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.

Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine.

Low-molecular-weight polyethylenimine (LMW-PEI) was synthesized by the acid-catalyzed, ring-opening polymerization of aziridine and compared with commercially available high-molecular-weight PEI (HMW-PEI) of 25 kDa. Molecular weights were determined by size-exclusion chromatography in combination with multi-angle laser light scattering. The weight average molecular weight (M(w)) of synthesized LMW-PEI was determined as 5.4+/-0.5 kDa, whereas commercial HMW-PEI showed a M(w) of 48+/-2 kDa. DNA polyplexes of LMW-PEI and HMW-PEI were characterized with regard to DNA condensation (ethidium bromide fluorescence quenching), size (photon correlation spectroscopy) and surface charge (laser Doppler anemometry). Compared with HMW-PEI, DNA condensation of LMW-PEI was slightly impaired at lower N/P ratios. Complexes with plasmid DNA at a N/P ratio of 6.7 showed significantly increased hydrodynamic diameters (590+/-140 vs. 160+/-10 nm), while the zeta-potential measurements were similar (23+/-2 vs. 30+/-3 mV). The cytotoxicity of LMW-PEI in L929 fibroblasts was reduced by more than one order of magnitude compared with HMW-PEI, as shown by MTT assay. LMW-PEI exhibited increased transfection efficiency in six different cell lines. Reporter gene expression was found to be increased by a factor of 2.1-110. The pharmacokinetics and biodistribution of 125I-PEI in mice were similar for both molecular weights with an AUC of ca. 330+/-100% ID/ml min. Approximately half of the injected dose accumulated in the liver. LMW-PEI proved to be an efficient gene delivery system in a broad range of cell lines. Due to differences in polyplex structure, as well as its relatively low cytotoxicity, which makes the application of high N/P ratios possible, LMW-PEI appears to possess advantageous qualities with regard to transfection efficiency over PEI of higher molecular weight.

Oscillations, resonances and noise: basis of flexible neuronal pattern generation.

Modulation of neuronal impulse pattern is examined by means of a simplified Hodgkin-Huxley type computer model which refers to experimental recordings of cold receptor discharges. This model essentially consists of two potentially oscillating subsystems: a spike generator and a subthreshold oscillator. With addition of noise the model successfully mimics the major types of experimentally recorded impulse patterns and thereby elucidate different resonance behaviors. (1) There is a range of rhythmic spiking or bursting where the spike generator is strongly coupled to the subthreshold oscillator. (2) There is a pacemaker activity of more complex interactions where the spike generator has overtaken part of the control. (3) There is a situation where the two subsystems are decoupled and only resonate with the help of noise.

A mathematical model of homeostatic regulation of sleep-wake cycles by hypocretin/orexin.

We introduce a physiology-based mathematical model of sleep-wake cycles, suggesting a novel mechanism of homeostatic regulation of sleep. In this model, the homeostatic process is determined by the neuropeptide hypocretin/ orexin, which is a cotransmitter of the lateral hypothalamus. Hypocretin/ orexin neurons are silent during sleep and active during wakefulness. Firing of these neurons is sustained by reciprocal excitatory synaptic connections with local glutamate interneurons. This feedback loop has been simulated with a minimal but physiologically plausible model. It includes 2 simplified Hodgkin-Huxley type neurons that are connected via glutamate synapses, one of which additionally contains hypocretin/orexin as the functionally relevant cotransmitter. During the active state (wakefulness), the synaptic efficacy of hypocretin/orexin declines as a result of the ongoing firing. It recovers during the silent (sleep) state. We demonstrate that these homeostatic changes can account for typical alterations of sleep-wake transitions, for example, introduced by napping, sleep deprivation, or alarm clock. In combination with a circadian input, the model mimics the transitions between silent and firing states in agreement with sleep-wake cycles. These simulation results support the concept of state-dependent alterations of hypocretin/orexin effects as an important homeostatic process in sleep-wake regulation, although additional mechanisms can be involved.

Neural synchronization at tonic-to-bursting transitions.

We studied the synchronous behavior of two electrically-coupled model neurons as a function of the coupling strength when the individual neurons are tuned to different activity patterns that ranged from tonic firing via chaotic activity to burst discharges. We observe asynchronous and various synchronous states such as out-of-phase, in-phase and almost in-phase chaotic synchronization. The highest variety of synchronous states occurs at the transition from tonic firing to chaos where the highest coupling strength is also needed for in-phase synchronization which is, essentially, facilitated towards the bursting range. This demonstrates that tuning of the neuron's internal dynamics can have significant impact on the synchronous states especially at the physiologically relevant tonic-to-bursting transitions.

Crosslinked nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery: in vitro characterization and in vivo studies in mice.

Crosslinked poly(ethylene imine) (PEI) polyplexes for intracellular DNA release were generated using a low molecular weight crosslinking reagent, Dithiobis(succinimidyl propionate) (DSP). Disulfide bonds of the crosslinked polyplexes were susceptible to intracellular redox conditions and DNA release was observed using an ethidium bromide exclusion assay and dynamic light scattering. Transfection experiments were performed to elucidate the effect of extra- and intracellular redox conditions. Pharmacokinetics and organ accumulation of uncrosslinked and crosslinked polyplexes were compared and gene expression patterns were measured in mice 24 h after intravenous injection. Crosslinked PEI and plasmid DNA formed stable polyplexes in a size range of 100-300 nm, with zeta potentials between +16.4 and +26.1 mV. DNA release occurred after cleavage of the disulfide bonds. Cell culture experiments under reducing conditions as well as with glutathione loaded cells confirmed the proposed intracellular activation. A significant influence of the intracellular glutathione status on the transfection efficiency was observed. Pharmacokinetic profiles of crosslinked PEI/DNA polyplexes in mice after intravenous administration showed higher blood levels for crosslinked polyplexes. These polyplexes accumulated mainly in the liver and the lungs. In vivo transfection data revealed significantly reduced (unwanted) lung transfection while liver transfection predominated. These studies suggest that crosslinked polyplexes are more stable in circulation and retain their transfection efficiency after intravenous administration.

The sympathetic control of blood supply is different in the spleen and lymph nodes.

OBJECTIVES: The noradrenergic innervation of lymphoid organs controls several immune cell functions and local blood perfusion. Considering that cell and antigen uptake depend on the blood supply to lymphoid organs, the hypothesis was tested that feedback signals from activated immune cells control sympathetic vasomotor activity. METHODS: We determined the blood flow in spleen and mesenteric lymph nodes (mLN) of Wistar Kyoto rats during immune stimulation with endotoxin (LPS; 10 microg/kg) and following disruption of the noradrenergic transmission. RESULTS: Our data indicate that (a) the splenic noradrenaline content, which reflects the density of the sympathetic innervation, is 5 times higher in the spleen than in other peripheral organs and the spleen receives stronger tonic sympathetic input than mLN; (b) immune stimulation with LPS causes a 4-fold increase in the IL-1beta production in the spleen, but only 2-fold in mLN; (c) IL-1beta causes an inhibition of the sympathetic vasoconstrictor tonus in the spleen, but has no significant effect on the noradrenergic vascular tonus in mLN, and (d) in mLN, the local hyperemia induced by LPS is attenuated by the degranulation of vesicular stores of histamine and serotonin, indicating that these monoamines participate in the vasodilator effect of LPS in mLN. CONCLUSIONS: The present experiments, taken together with our previous studies, indicate that the control of blood supply to the spleen and mLN involves different mechanisms. While blood perfusion in the spleen depends on the inhibition of the noradrenergic vasoconstriction by endogenously produced IL-1beta, other vasoactive mediators such as serotonin and histamine play a role in the control of mLN perfusion.

Calcitonin receptor-like receptor is expressed on gastrointestinal immune cells.

BACKGROUND/AIMS: Pharmacological and morphological studies suggest that the gut mucosal immune system and local neuropeptide-containing neurones interact. We aimed to determine whether gut immune cells are targets for calcitonin gene-related peptide (CGRP), which has potent immune regulatory properties. METHODS: Using density gradient centrifugation, rat lamina propria mononuclear cells (LP-MNCs) and intra-epithelial lymphocytes (IELs) were isolated. RT-PCR was employed for the detection of mRNA of rat calcitonin receptor-like receptor (CRLR), which is considered to represent the pharmacologically defined CGRP receptor-1 subtype, as well as mRNA of the receptor activity-modifying proteins, which are essential for CRLR function and determine ligand specificity. A radioreceptor assay was employed for the detection of specific CGRP binding sites. RESULTS: RT-PCR and DNA sequencing showed that LP-MNCs and IELs express CRLR. Incubation of isolated LP-MNCs with radiolabelled alphaCGRP revealed the existence of specific binding sites for CGRP. CONCLUSION: These novel data indicate that mucosal immune cells of the rat gut are a target for CGRP and provide significant evidence that CGRP functions as an immune regulator in the gut mucosa.

Interactions of temperature and angiotensin II in paraventricular neurons of rats in vitro.

We recorded extracellular impulse activity of hypothalamic paraventricular neurons ( n=75) in rat brain slices during application of angiotensin II (ANG II, 10(-9)-10(-6) M) and/or temperature changes (32-42 degrees C). ANG II, with a threshold concentration of 10(-8) M, increased the firing rate in more than 80% of the neurons with strongest excitations occurring in bursting neurons. Increasing the temperature also raised the discharge rate in the majority of the neurons, often together with enhanced burst discharges. When ANG II was applied during ongoing sinusoidal temperature changes, its effects were more pronounced at elevated temperatures. These electrophysiological data illustrate that stimulus-encoding properties at the neuronal level can contribute to the interactions between osmoregulatory and thermoregulatory mechanisms including mutual sensitization when different stimuli (here: ANG II and temperature changes) are applied simultaneously.

The structure of PEG-modified poly(ethylene imines) influences biodistribution and pharmacokinetics of their complexes with NF-kappaB decoy in mice.

PURPOSE: To study the relationship between structure of poly(ethylene imine-co-ethylene glycol), PEI-PEG, copolymers and physicochemical properties as well as in vivo behavior of their complexes with NF-kappaB decoy. METHODS: A variety of copolymers of PEG grafted onto PEI as well as PEI grafted onto PEG were synthesized and their complexes with a double stranded 20mer oligonucleotide were examined regarding size, surface charge, biodistribution and pharmacokinetics. RESULTS: Polyplexes of copolymers were smaller compared to polyplexes formed by non-PEGylated PEI 25 kDa (58 - 334 nm vs. 437 nm for a nitrogen/phosphate ratio of 3.5 and 85 - 308 nm vs. 408 nm for N/P 6.0) and showed reduced zeta potential (-2.5 - 6.4 mV vs. 14.5 mV for N/P 6.0). IV injection into mice revealed liver (35-76% of injected dose), kidney (3 - 22%) and spleen (2 - 16%) to be the main target organs for all injected complexes. Complexes formed by copolymers with few PEG blocks of higher molecular weight (5 kDa and 20 kDa) grafted onto PEI 25 kDa did not show different blood levels from PEI 25 kDa. In contrast, a copolymer with more short PEG blocks (550 Da) grafted onto PEI showed elevated blood levels with an increase in AUC of 62 %. CONCLUSIONS: A sufficiently high density of PEG molecules is necessary to effectively prevent opsonization and thereby rapid clearance from blood stream.