Automatic determination of EMG-contaminated components and validation of independent component analysis using EEG during pharmacologic paralysis.

OBJECTIVE: Validate independent component analysis (ICA) for removal of EMG contamination from EEG, and demonstrate a heuristic, based on the gradient of EEG spectra (slope of graph of log EEG power vs log frequency, 7-70 Hz) from paralysed awake humans, to automatically identify and remove components that are predominantly EMG. METHODS: We studied the gradient of EMG-free EEG spectra to quantitatively inform the choice of threshold. Then, pre-existing EEG from 3 disparate experimental groups was examined before and after applying the heuristic to validate that the heuristic preserved neurogenic activity (Berger effect, auditory odd ball, visual and auditory steady state responses). RESULTS: (1) ICA-based EMG removal diminished EMG contamination up to approximately 50 Hz, (2) residual EMG contamination using automatic selection was similar to manual selection, and (3) task-induced cortical activity remained, was enhanced, or was revealed using the ICA-based methodology. CONCLUSION: This study further validates ICA as a powerful technique for separating and removing myogenic signals from EEG. Automatic processing based on spectral gradients to exclude EMG-containing components is a conceptually simple and valid technique. SIGNIFICANCE: This study strengthens ICA as a technique to remove EMG contamination from EEG whilst preserving neurogenic activity to 50 Hz.

Surface Laplacian of scalp electrical signals and independent component analysis resolve EMG contamination of electroencephalogram.

The serious impact of electromyogram (EMG) contamination of electroencephalogram (EEG) is well recognised. The objective of this research is to demonstrate that combining independent component analysis with the surface Laplacian can eliminate EMG contamination of the EEG, and to validate that this processing does not degrade expected neurogenic signals. The method involves sequential application of ICA, using a manual procedure to identify and discard EMG components, followed by the surface Laplacian. The extent of decontamination is quantified by comparing processed EEG with EMG-free data that was recorded during pharmacologically induced neuromuscular paralysis. The combination of the ICA procedure and the surface Laplacian, with a flexible spherical spline, results in a strong suppression of EMG contamination at all scalp sites and frequencies. Furthermore, the ICA and surface Laplacian procedure does not impair the detection of well-known, cerebral responses; alpha activity with eyes-closed; ERP components (N1, P2) in response to an auditory oddball task; and steady state responses to photic and auditory stimulation. Finally, more flexible spherical splines increase the suppression of EMG by the surface Laplacian. We postulate this is due to ICA enabling the removal of local muscle sources of EMG contamination and the Laplacian transform being insensitive to distant (postural) muscle EMG contamination.

Reduction of medical error at the point-of-care using electronic clinical information delivery.

There is accumulating evidence that point-of-care delivery of clinical information such as evidence-based medicine, practice guidelines and drug information can streamline clinical practice and reduce preventable errors. In Australia, hospital-based physicians have -generally been slow to fully use these resources to enhance their clinical practice. Here we provide an introduction to the practical application of several hand-held and electronic information systems available to Australian physicians.

Muscarinic cholinoceptor-stimulated synthesis and degradation of inositol 1,4,5-trisphosphate in the rat cerebellar granule cell.

A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [3H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 mM) caused an immediate increase in level of Ins(1,4,5)P3 (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP5 and InsP6 was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P3 metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P3 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidyl-inositol 4-phosphate cannot be excluded.

Myo-inositol 1,3,4,5-tetrakisphosphate can independently mobilise intracellular calcium, via the inositol 1,4,5-trisphosphate receptor: studies with myo-inositol 1,4,5-trisphosphate-3-phosphorothioate and myo-inositol hexakisphosphate.

Myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] acts as a full agonist for Ca2+ release in saponin-permeabilised SH-SY5Y neuroblastoma cells. Studies were conducted in the presence of myo-inositol hexakisphosphate (InsP6, 10 microM), to inhibit the Ins(1,3,4,5)P(4)-3-phosphatase catalysed back conversion of Ins(1,3,4,5)P4 to Ins(1,4,5)P3. HPLC analysis confirmed that Ins(1,3,4,5)P4 releases the entire content of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores, independent of 3-phosphatase activity. Further we utilised racemic myo-inositol 1,4,5-trisphosphate-3-phosphorothioate [DL-Ins(1,3,4,5)P(4)-3S], a novel intrinsically Ins(1,3,4,5)P(4)-3-phosphatase resistant Ins(1,3,4,5)P4 analogue. DL-Ins(1,3,4,5)P(4)-3S specifically displaced [3H]Ins(1,4,5)P3 from bovine adrenal cortex Ins(1,4,5)P3 binding sites (IC50 = 889 nM, compared to Ins(1,4,5)P3, IC50 = 4.4 nM and Ins(1,3,4,5)P4, IC50 = 152 nM). DL-Ins(1,3,4,5)P(4)-3S was a full agonist for Ca2+ release (EC50 = 4.7 microM), being 90- and 2-fold less potent than Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (with InsP6), respectively. DL-Ins(1,3,4,5)P(4)-3S will be an important tool for identification of potentially exclusive Ins(1,3,4,5)P4 second messenger functions, since its resistance to 3-phosphatase action precludes the inconvenient artefact of steady state Ins(1,4,5)P3 generation.

Muscarinic receptors, phosphoinositide metabolism and intracellular calcium in neuronal cells.

1. We have utilised SH-SY5Y human neuroblastoma cells and primary cultures of rat neonatal cerebellar granule cells, both expressing M3 muscarinic receptors, to examine agonist driven polyphosphoinositide hydrolysis and alterations in intracellular calcium. 2. Stimulation of SH-SY5Y cells leads to a biphasic increase in intracellular calcium, the initial peak being due to the release of calcium from an intracellular store and the second maintained phase being due to calcium entry across the plasma membrane. The channel involved does not appear to be voltage sensitive, to involve a pertussis toxin sensitive G protein, or be opened by inositol polyphosphates. 3. Muscarinic receptor stimulation also leads to increased inositol polyphosphate formation in SH-SY5Y cells. Ins(1,4,5)P3 mass formation was biphasic in profile whereas Ins(1,3,4,5)P4 mass formation was slower and monophasic in profile. These data are consistent with substantial activity of 5-phosphatase (dephosphorylating Ins(1,4,5)P3 to Ins(1,4)P2) and 3-kinase (phosphorylating Ins(1,4,5)P3 to Ins(1,3,4,5)P4) in SH-SY5Y cells. 4. In order to better understand the role of Ins(1,4,5)P3 and its metabolites in calcium homeostasis we have examined the ability of a variety of natural and synthetic analogues to release intracellular sequestered calcium. The Ins(1,4,5)P3 calcium mobilizing receptor displays a remarkable degree of stereo- and positional selectivity with the most potent agonist to date being Ins(1,4,5)P3 (EC50 = 0.09 microM). 5. As an alternative to the continuous SH-SY5Y neuroblastoma (tumour derived) cell line we have used the primary cultured cerebellar granule cell. These cells also display a biphasic increase in Ins(1,4,5)P3 mass and a subsequent release of intracellular stored calcium. In our hands carbachol appears to increase calcium influx, a response which is only visible in the absence of magnesium.

Inositol 1,4,5-triphosphate-stimulated calcium release from permeabilized cerebellar granule cells.

1. Muscarinic cholinoceptor stimulation of phosphoinositide hydrolysis in rat cultured cerebellar granule cells results in a rapid, transient accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which has been implicated in the release of non-mitochondrial intracellular Ca2+ stores. In the present study, the release of Ca2+ from intracellular stores and the Ins(1,4,5)P3 receptor responsible for this process have been investigated. 2. Monolayers of saponin-permeabilized granule cells accumulate 45Ca2+ in an ATP-dependent manner and the sequestered 45Ca2+ can be concentration-dependently released by Ins(1,4,5)P3 by a stereospecific and heparin-sensitive mechanism. The EC50 for Ins(1,4,5)P3-stimulated 45Ca2+ release was 80 +/- 3 nM. 3. Radioligand binding studies performed on a crude granule cell membrane fraction indicated the presence of an apparently homogeneous population of stereo-specific Ins(1,4,5)P3 receptors (KD 54.7 +/- 2.0 nM; Bmax 1.37 +/- 0.29 pmol mg-1 protein). 4. This study provides evidence for Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in primary cultures of cerebellar granule cells and suggest that these cells provide an excellent model neuronal system in which to study the relative functional roles of Ca2+ release from intracellular stores and Ca(2+)-entry in neuronal Ca2+ homeostasis.

M3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells.

Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.

Different mechanisms of Ca2+ entry induced by depolarization and muscarinic receptor stimulation in SH-SY5Y human neuroblastoma cells.

Depolarization by elevated K+ and stimulation of muscarinic M3 receptors evoke rises in [Ca2+]i in Fura 2-loaded SH-SY5Y human neuroblastoma cells. The response to K+ (30 and 60 mM) could be inhibited by the dihydropyridine L-channel antagonist +PN 200-110 and totally suppressed by Ni2+, the N-channel blocker omega-conotoxin reduced the response to 60 mM K+. Carbachol-stimulated increase in [Ca2+]i was blocked by atropine and Ni2+ but was totally resistant to the L- and N-channel blockers. This study reveals the presence of L- and N-type voltage-sensitive Ca2+ channels on undifferentiated SH-SY5Y cells that are opened by K+ depolarization but not by muscarinic stimulation.

Effects of potassium channel blockade on endogenous glutamate release from cerebellar slices.

The effects of potassium channel blockade on the spontaneous release of endogenous glutamate from rat cerebellar slices was studied. Tetrapentylammonium (TPeA), 4-aminopyridine and quinine all increased the spontaneous release of glutamate. The effect of TPeA and 4-AP was potentiated in the absence of Ca2+ from the perfusing fluid. In normal artificial cerebrospinal fluid (ACSF) the Ca2+ channel antagonist, verapamil, mimicked the effects of TPeA seen in Ca2+-free ACSF. The increased release of glutamate produced by TPeA under Ca2+-free conditions was inhibited by the sodium channel blocker, tetrodotoxin, and by Ruthenium red, which inhibits mobilization of mitochondrial Ca2+. The results suggest that external Ca2+ is not required in the TPeA-induced release of glutamate. It is proposed that the prolongation of depolarization seen with potassium channel blocking drugs enables sufficient sodium to enter the neurone and release Ca2+ from intraneuronal stores in order to facilitate transmitter release.

The effects of vibration frequency and direction on the location of areas of discomfort caused by whole-body vibration.

Although much research has been devoted to the determination of equivalent comfort contours for human response to whole-body vibration little consideration has been given to the source of the feelings that give rise to such comfort contours. This paper shows that for vertical vibration there is a distinct difference in the locations of discomfort on the body at different frequencies and that the locations are not much affected by the vibration level. For horizontal motions, feelings of discomfort predominated in the lower abdomen and buttocks irrespective of vibration frequency or direction. A semantic scaling technique indicates the maximum sensitivity to vertical vibration acceleration in the 4 to 16 Hz range, but for both fore-and aft and lateral vibration there is a decrease in sensitivity with increasing frequency above 2Hz.