Oil droplet formation on pellicle covered tooth surfaces studied with environmental scanning electron microscopy.

Lipophilic components are known to modulate the process of bioadhesion on the tooth surface. However, the presence of lipid droplets at the acquired pellicle under oral conditions has not been demonstrated, yet. The purpose of the present study was to establish a method for direct visualisation of lipids on the surface of hydrated, pellicle covered tooth samples by environmental scanning electron microscopy (ESEM), and to use this technique for studying the effects of rinsing with edible oils on the acquired pellicle under in vivo conditions. In situ pellicle formation was performed by 3 min exposure of enamel and dentin specimens in the oral cavity of volunteers. Subsequently, the volunteers rinsed in vivo with safflower oil or linseed oil for 30 s, and the specimens were further carried intraorally for periods from 0 min up to several hours. After intraoral exposure the specimens were treated by osmium tetroxide vapour, and were subsequently analysed by ESEM. This technique was capable to directly visualise the presence of lipid droplets at the pellicle's surface under hydrated conditions. ESEM analyses revealed that surface bound nano- and micro-sized lipid droplets were present at the acquired pellicle's surface even several hours after rinsing with edible oils indicating that these droplets had tightly adhered to the pellicle surface. Pellicle modification by edible oil rinsing as demonstrated in the present study might have the potential to be beneficial as an adjunct in dental prophylaxis.

Silicon nitride windows for electron microscopy of whole cells.

Silicon microchips with thin, electron transparent silicon nitride windows provide a sample support that accommodates both light-, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation steps. The windows are robust enough that cellular samples can be cultured directly onto them, with no addition of a supporting film, and there is no need to embed or section the sample, as is typically required in electron microscopy. By combining two microchips, a microfluidic chamber can be constructed for the imaging of samples in liquid in the electron microscope. We provide microchip design specifications, a fabrication outline, instructions on how to prepare the microchips for biological samples, and examples of images obtained using different light and electron microscopy modalities. The use of these microchips is particularly advantageous for correlative light and electron microscopy.

Electron microscopy of whole cells in liquid with nanometer resolution.

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 micros were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 +/- 1 microm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods.

Prodynorphin and kappa opioid receptor mRNA expression in the cingulate and prefrontal cortices of subjects diagnosed with schizophrenia or affective disorders.

The present study examined the prodynorphin and kappa opioid receptor mRNA expression levels in the anterior cingulate and dorsolateral prefrontal cortices of subjects diagnosed with schizophrenia, bipolar disorder, or major depression as compared with normal controls without a psychiatric diagnosis. Multivariate analyses failed to reveal any differences in the mRNA expression levels between the four diagnostic groups, though a group trend (non-significant) was evident for the expression of the kappa opioid receptor and prodynorphin mRNAs in the prefrontal cortex. The mRNA expression levels were not associated with lifetime history of antipsychotic treatment or with suicide as a cause of death. The results, however, suggested an influence of certain drugs of abuse on the prodynorphin cortical mRNA expression. Prodynorphin mRNA expression levels were found to be elevated in individuals with a history of marihuana or stimulant use, but not alcohol. Overall, our data do not provide strong evidence for impaired prodynorphin or kappa opioid receptor mRNA levels in the dorsolateral prefrontal or cingulate cortices of schizophrenic, bipolar disorder, or major depressed subjects.

Associative long-term depression in the hippocampus is dependent on postsynaptic N-type Ca2+ channels.

Long-term depression (LTD) is a form of synaptic plasticity that can be induced either by low-frequency stimulation of presynaptic fibers or in an associative manner by asynchronous pairing of presynaptic and postsynaptic activity. We investigated the induction mechanisms of associative LTD in CA1 pyramidal neurons of the hippocampus using whole-cell patch-clamp recordings and Ca(2+) imaging in acute brain slices. Asynchronous pairing of postsynaptic action potentials with EPSPs evoked with a delay of 20 msec induced a robust, long-lasting depression of the EPSP amplitude to 43%. Unlike LTD induced by low-frequency stimulation, associative LTD was resistant to the application of d-AP-5, indicating that it is independent of NMDA receptors. In contrast, associative LTD was inhibited by (S)-alpha-methyl-4-carboxyphenyl-glycine, indicating the involvement of metabotropic glutamate receptors. Furthermore, associative LTD is dependent on the activation of voltage-gated Ca(2+) channels by postsynaptic action potentials. Both nifedipine, an L-type Ca(2+) channel antagonist, and omega-conotoxin GVIA, a selective N-type channel blocker, abolished the induction of associative LTD. 8-hydroxy-2-dipropylaminotetralin (OH-DPAT), a 5-HT(1A) receptor agonist, inhibited postsynaptic Ca(2+) influx through N-type Ca(2+) channels, without affecting presynaptic transmitter release. OH-DPAT also inhibited the induction of associative LTD, suggesting that the involvement of N-type channels makes synaptic plasticity accessible to modulation by neurotransmitters. Thus, the modulation of N-type Ca(2+) channels provides a gain control for synaptic depression in hippocampal pyramidal neurons.

Expression of mu, kappa, and delta opioid receptor messenger RNA in the human CNS: a 33P in situ hybridization study.

The existence of at least three opioid receptor types, referred to as mu, kappa, and delta, is well established. Complementary DNAs corresponding to the pharmacologically defined mu, kappa, and delta opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using 33P-labelled RNA probes. In the present study we report the regional and cellular expression of mu, kappa, and delta opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the mu and kappa opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the delta opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of mu, kappa, and delta opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between mu opioid receptor receptor binding and mu opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous expression pattern in many regions. However, in the human brain, kappa opioid receptor messenger RNA expression was more widely distributed than in rodents. The differential and region specific expression of opioid receptors may help to identify targets for receptor specific compounds in neuronal circuits involved in a variety of physiological functions including pain perception, neuroendocrine regulation, motor control and reward.

Opioid receptor-mediated control of acetylcholine release in human neocortex tissue.

The effects of various opioid receptor agonists and antagonists on evoked acetylcholine release were studied in slices of human neocortex prelabelled with [3H]-choline, superfused and depolarized electrically (2 min, 3 Hz, 2 ms, 24 mA) or by K+ (20 mM). The delta-opioid receptor agonist DPDPE and the kappa-opioid receptor agonist U50488 reduced the evoked [3H]-overflow (acetylcholine release) in a concentration-dependent fashion; the delta-opioid receptor antagonist naltrindole and the kappa-opioid receptor antagonist norbinaltorphimine, respectively, antagonized these effects. Application of the mu-opioid receptor agonist DAGO also resulted in an inhibition of acetylcholine release; however, both delta- and kappa-opioid receptor antagonists were able to block this effect. The mu-opioid receptor agonists morphine and (+)-nortilidine had no effect. These results indicate that acetylcholine release in human neocortex is inhibited through delta- and kappa-opioid receptors, but not through mu-opioid receptors. Acetylcholine release was significantly increased by the delta-opioid receptor antagonist naltrindole in the presence of a mixture of peptidase inhibitors providing evidence for a delta-opioid receptor-mediated inhibition of acetylcholine release by endogenous enkephalin. K(+)-evoked acetylcholine release in the presence of TTX was inhibited by U50488, but not by DPDPE, suggesting the presence of kappa-opioid receptors on cholinergic terminals and the localization of delta-receptors on cortical interneurons. Therefore, the potent effect of DPDPE on acetylcholine release is likely to be indirect, by modulation of intrinsic cortical neurons. These interneurons probably do not use GABA as neurotransmitter since both GABAA and GABAB receptor agonists (muscimol and baclofen, respectively) were without effect on acetylcholine release.