Comparison of electrical transients and corrosion responses of pulsed MP35N and 316LVM electrodes

Functional neuromuscular stimulation (FNS) is often limited by electrode malfunctions such as corrosion and breakage, particularly for intramuscular and epimysial type electrodes. As a result, the electrochemical charge injection characteristics and corrosion responses of single strand 316LVM stainless steel and MP35N nickel-cobalt alloy electrodes were evaluatedin vitro. For charge balance, capacitor coupled monophasic protocols with varying charge injections were employed. Electrodes were evaluated with either positive-first or negative-first pulses, 60 Hz, 100 μsec pulse duration, and stimulation periods from 100 to 240 hours. Charge injection densities ranged from 20 to 80 μC/cm². For both anodic-first and cathodic-first pulsing, the potential transients for the MP35N electrodes were more extreme than for the 316LVM electrodes over the test period, and increased corrosion was apparent on the MP35N electrodes from both optical and scanning electron microscopy. Therefore, 316LVM, but not MP35N, may be suitable for FNS applications with charge injection densities less than 40 μC/cm².     

Effects of neuromuscular electrical stimulation parameters on specific tension

This study examined the effects of altering surface neuromuscular electrical stimulation (SNMES) parameters on the specific tension of the quadriceps femoris muscle. Seven able-bodied subjects had magnetic resonance images taken of both thighs prior to and immediately after four SNMES protocols to determine the activated muscle cross-sectional area (CSA). The four protocols were: (1) research (RES, 100 Hz, 450 μs, and amplitude set to evoke 75% of maximal voluntary isometric torque, MVIT); (2) pulse duration (PD, 100 Hz, 150 μs, same current as in RES); (3) frequency (FREQ, 25 Hz, 450 μs, and same current as in RES); (4) amplitude (AMP, 100 Hz, 450 μs, and current set to evoke the average of the initial torques of PD and FREQ, 45 ± 9% of MVIT). Reducing the amplitude of the current from 75 to 45% of MVIT did not alter specific tension, 25 ± 8 N/cm², suggesting that the amplitude probably affects torque and the area of activated muscle proportionally. Shortening the pulse duration from 450 to 150 μs caused specific tension to drop from 25 ± 6 to 20 ± 6 N/cm² (P < 0.05), indicating that pulse duration increased torque and the activated CSA disproportionally. Alternatively, reducing the frequency from 100 to 25 Hz decreased specific tension from 25 ± 6 to 17 ± 4 N/cm² (P < 0.05), suggesting that the frequency increased torque without affecting the activated CSA. Clinicians who administer SNMES should be aware of the magnitude of adaptations to a given amplitude, pulse duration, and frequency.     

60 Hz ventricular fibrillation thresholds for large-surface-area electrodes

The paper describes a series of animal experiments in which large-surface-area disk electrodes were used to study the current density required for ventricular fibrillation. The electrical currents were introduced to the heart both by applying the electrodes directly to the heart and by applying the electrodes to the surface of the chest near the heart. The electrode areas studied ranged from 1000 to 30 000 mm². The results show that, for large-area electrodes, fibrillation thresholds are determined by current density. The thresholds approach a constant value of 3·5 μA mm⁻²     

Spatial variation of aortic wall oxygen diffusion coefficient from transient polarographic measurements

Polarographic current transients following a voltage step (turn-on transient) were measured with bare cathodes (25 μm diameter) and shallowly recessed oxygen microelectrodes (<5μm diameter). Except for the initial part of the current transient, the experimental measurements were in excellent agreement with simple models in the literature, which predict an inverse relationship with . Turn-on transients were measured in aqueous solutions with known physical properties, and in aortic wall tissue from three different species (n=6 rabbits, n=3 dogs, and n=1 miniature pig). Oxygen diffusion coefficients (D) were determinedin vitro by comparing time constants measured by the same microelectrode in saline and in strips of aortic wall tissue at 37°C. On the inner side (endothelium and intima) of the aorta, D averaged (±S.E.) 7.0 (±0.8)×10⁻⁶ cm²/s in 6 rabbits, 6.4 (±1.0)×10⁻⁶ cm²/s in 3 dogs, and was 4.6×10⁻⁶ cm²/s in the pig. On the adventitial side, D was 9.5×10⁻⁶ cm²/s in 1 rabbit, 11.4 (±1.2)×10⁻⁶ cm²/s in 3 dogs, and 8.1×10⁻⁶ cm²/s in the pig. For every aortic strip on which D was measured from both sides, D for the inner wall was always lower, overall by a little more than one third (p<0.001). The lower D on the endothelial side may limit oxygen transport to the vascular wall and play a role in atherogenesis.     

Late sodium current contributes to diastolic cell Ca<Superscript>2+</Superscript> accumulation in chronic heart failure

We elucidate the role of late Na⁺ current (I_NaL) for diastolic intracellular Ca²⁺ (DCa) accumulation in chronic heart failure (HF). HF was induced in 19 dogs by multiple coronary artery microembolizations; 6 normal dogs served as control. Ca²⁺ transients were recorded in field-paced (0.25 or 1.5 Hz) fluo-4-loaded ventricular myocytes (VM). I_NaL and action potentials were recorded by patch-clamp. Failing VM, but not normal VM, exhibited (1) prolonged action potentials and Ca²⁺ transients at 0.25 Hz, (2) substantial DCa accumulation at 1.5 Hz, and (3) spontaneous Ca²⁺ releases, which occurred after 1.5 Hz stimulation trains in ~31% cases. Selective I_NaL blocker ranolazine (10 μM) or the prototypical Na⁺ channel blocker tetrodotoxin (2 μM) reversibly improved function of failing VM. The DCa accumulation and the beneficial effect of I_NaL blockade were reproduced in silico using an excitation-contraction coupling model. We conclude that I_NaL contributes to diastolic Ca²⁺ accumulation and spontaneous Ca²⁺ release in HF.     

Imbalanced biphasic electrical stimulation: Muscle tissue damage

The effects of imbalanced biphasic stimulation were studied on cat skeletal muscle to determine if greater charge densities can be safely used than with balanced or monophasic stimulation. The results of the study indicate that imbalanced biphasic stimulation can be tolerated safely by tissue at or below a net dc current density of 35 μA/mm² and not safely tolerated at or above a net dc current of 50 μA/mm². Monophasic stimulation has been shown to be safe at or below net dc current levels of 10 μA/mm² and in these studies we found it was not safe at or above net dc current levels of 20 μA/mm². Stimuli were applied to muscles via coiled wire intramuscular electrodes using a regulated current source. Since the safe average current density was higher for imbalanced biphasic stimulation than for monophasic stimulation, this suggests that: (a) pH change is not the primary reaction causing tissue damage and (b) the damaging electrochemical process that takes place during a cathodic stimulation pulse can be reversed by an anodic pulse having substantially less charge than its companion cathodic pulse. We conclude that greater cathodic charge densities can be safely employed with imbalanced biphasic stimulation than with either monophasic stimulation or balanced charge biphasic stimulation. Supported by the Veterans Administration, Grant No. V541P-3141 and the National Institutes of Health-National Institute of Neurological and Communicative Disorders and Stroke, Neural Prosthesis Program, Contract No. NOI-NS-4-2362.     

Short pulses of acetylcholine stimulation induce cytosolic Ca2+ signals that are excluded from the nuclear region in pancreatic acinar cells

 We have investigated the spreading of cytosolic Ca²⁺ signals generated by acetylcholine stimulation (using either microionophoresis or pressure application) of isolated pancreatic acinar cells (or small cell clusters) using confocal microscopy of Ca²⁺-sensitive fluorescence (fura red). We have been particularly interested in the effects of short vigorous pulses of acetylcholine (ACh) stimulation since, in the pancreas, ACh secreted from nerve endings is quickly eliminated by the action of ACh esterase. We focused on three regions: the secretory pole (secretory granule area), the nucleus and the basal area outside the nucleus. The nuclei were visualized by using the specific nuclear stain Hoechst 33342. With ionophoretic application, a long-lasting stimulation with ACh (10 s and longer) induces large Ca²⁺ transients of similar amplitude in all the three selected regions of the cell. Short applications (about 3 s) of ACh result in a Ca²⁺ rise in the secretory pole, whereas no changes in cytoplasmic Ca²⁺ were detected in the basal, nonnuclear region or in the nucleus. We found that at the peak of such localised Ca²⁺ responses, evoked either by ACh ionophoresis or pressure application, significant Ca²⁺ concentration gradients (up to 400 nM/μm) can be established along the line connecting the secretory pole with the nucleus. In some experiments slightly longer applications (about 5 s) of ACh produce Ca²⁺ transients in both the secretory region and in the basal, nonnuclear regions of the cells, whereas the nuclear [Ca²⁺] remained largely unaffected. Estimation of the ACh concentration in the vicinity of the cell under investigation indicated that values of about 1 μM were attained in the pressure application experiments. These results show directly that the nucleus of pancreatic acinar cells can be effectively protected from relatively large Ca²⁺ transients generated in the secretory pole of pancreatic acinar cells by short pulses of near-maximal ACh concentrations. This indicates that calcium-dependent secretion (both fluid and digestive enzymes) can occur without changes of the intranuclear [Ca²⁺] and consequently without activation of numerous calcium dependent nuclear processes. Received: 6 May 1996 / Accepted: 5 July 1996     

Comparison of neural damage induced by electrical stimulation with faradaic and capacitor electrodes

Arrays of platinum (faradaic) and anodized, sintered tantalum pentoxide (capacitor) electrodes were implanted bilaterally in the subdural space of the parietal cortex of the cat. Two weeks after implantation both types of electrodes were pulsed for seven hours with identical waveforms consisting of controlled-current, chargebalanced, symmetric, anodic-first pulse pairs, 400 μsec/phase and a charge density of 80–100 μC/cm² (microcoulombs per square cm) at 50 pps (pulses per second). One group of animals was sacrificed immediately following stimulation and a second smaller group one week after stimulation. Tissues beneath both types of pulsed electrodes were damaged, but the difference in damage for the two electrode types was not statistically significant. Tissue beneath unpulsed electrodes was normal. At the ultrastructural level, in animals killed immediately after stimulation, shrunken and hyperchromic neurons were intermixed with neurons showing early intracellular edema. Glial cells appeared essentially normal. In animals killed one week after stimulation most of the damaged neurons had recovered, but the presence of shrunken, vacuolated and degenerating neurons showed that some of the cells were damaged irreversibly. It is concluded that most of the neural damage from stimulations of the brain surface at the level used in this study derives from processes associated with passage of the stimulus current through tissue, such as neuronal hyperactivity rather than electrochemical reactions associated with current injection across the electrode-tissue interface, since such reactions occur only with the faradaic electrodes.     

Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline

 We evaluated the relationship between intracellular calcium concentration ([Ca²⁺]_i) and vasoconstriction during the presence of exogenous noradrenaline (NA) and sympathetic nerve stimulation. An imaging technique was used to determine calcium/tension relationships in isolated rat mesenteric resistance arteries that had been mounted for recording of isometric tension development and loaded with Fura-2/AM. Experiments were performed after depletion of vasodilator neuropeptides and in the continuous presence of 1 μM propranolol, 3 μM indomethacin, and 30 μM nitro-l-arginine. NA (10 μM) was shown first to induce a further increase in tension, but not [Ca²⁺]_i, during the contraction induced by 125 mM K⁺. Subsequently, calcium/tension relationships were determined during stimulation with graded increases in extracellular [K⁺] (5.9–125 mM K⁺), cumulative administration of NA (0.2–10 μM) and electrical field stimulation of perivascular nerves (EFS, 1–16 Hz). A basal calcium/tension relationship without the calcium-sensitizing property of NA was constructed using a cumulative concentration/response curve of 5.9–125 mM K⁺ in arteries after prior exposure to the irreversible α-adrenoceptor antagonist phenoxybenzamine (POB). K⁺ series before and during α-blockade were also studied using the combination of the α₁-antagonist prazosin and α₂-antagonist yohimbine yielding comparable results as with POB. Calcium/tension curves obtained in the presence of NA, K⁺ and during EFS all were shifted to the left compared with the basal condition and all showed a similar slope indicating that neurogenically released NA is equally capable of inducing calcium sensitization in smooth muscle of mesenteric resistance arteries as exogenously applied NA. In the presence of exogenous and endogenous NA we not only observed an elevated contractile response for a given increase in [Ca²⁺]_i, but also an attenuated rise in [Ca²⁺]_i for a given intensity of stimulation. This suggests that the agonist-induced calcium-sensitization is accompanied by a reduction of the rise in [Ca²⁺]_i. Received: 24 October 1997 / Received after revision: 26 January 1998 / Accepted: 26 February 1998     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

Non-invasive imaging of optical parameters of biological tissues

The normalised back-scattered intensity (NBI) profiles at various locations on the forearms of ten human subjects were obtained by moving the multi-probe of a laser reflectometer. The statistical analysis of the NBI data showed that the variation in the NBI was significantly higher at the ulnar region compared with that at other regions. For determination of the scattering (μ _s ) and absorption (μ _a ) coefficients and the anisotropy parameter g at each location on the forearm, these profiles were matched with the NBI profiles simulated by a Monte Carlo procedure (χ _0.99 ² ). For the reconstruction of images of variation of these parameters, the averaged values ofμ _a ,μ _s and g at all locations on the forearms of the subjects were determined. The absorption coefficient had a minimum (1.92 cm⁻¹) and maximum (2.21 cm⁻¹) at the wrist and the lateral region of the forearm, respectively. The scattering coefficient had a maximum (194 cm⁻¹) at the medial side and near the elbow, and a minimum (186 cm⁻¹) at the lateral side of the forearm. Similar changes in the anisotropy parameter were also observed. By interpolation of the data of each parameter on a 100×100 image matrix and after median filtering, colour-coded images of the variation in the optical parameters were constructed. These images could be useful for diagnostic and therapeutic applications of lasers.     

Electrolyte transport across the rabbit caecum in vitro

Electrolyte transport across rabbit caecal epithelium was investigated in vitro using conventional shortcircuiting and radioisotope techniques. In standard saline the caecum exhibited a relatively high short-circuit current (I _sc=4.4 μEq · cm⁻² · h⁻¹) and conductance (6.43 mS · cm⁻²). Both sodium and chloride were absorbed (J _net ^Na =6.40 andJ _net ^Cl =3.40 μEq · cm⁻² · h⁻¹) and potassium was secreted (J _net ^K =−0.5 μEq · cm⁻² · h⁻¹). Removal of Na⁺ abolishedI _sc andJ _net ^Cl whereas removal of Cl⁻ reducedJ _net ^Na to 2.92 μEq · cm⁻² · h⁻¹ but did not alterI _sc. In HCO ₃ ⁻ free salines containing 10⁻⁴ M acetazolamideJ _net ^Cl was abolished andJ _net ^Na andI _sc were reduced to 2.3 and 2.5 μEq · cm⁻² · h⁻¹ respectively. A positive residual ion flux (∼ 1 μEq · cm⁻² · h⁻¹) was detected in standard and Cl⁻-free salines but not in Na⁺-free or HCO ₃ ⁻ buffers. Mucosal amiloride (10⁻³ M) decreased net Na⁺ and Cl⁻ absorption but did not decreaseI _sc. Mucosal DIDS (10⁻⁴ M) decreasedJ _net ^Cl while mucosal bumetanide (10⁻⁴ M) did not affect any of the measured parameters. Finally, addition of theophylline (8 mM) stimulated Cl⁻ secretion and increasedI _sc. It is concluded that net sodium absorption by caecal epithelia occurs by both electrogenic and electroneutral mechanisms whereas net chloride absorption occurs only by an electroneutral process. Coupling of the absorptive fluxes of Na⁺ and Cl⁻ may result from Na⁺/H⁺ and Cl⁻/HCO ₃ ⁻ antiport systems in this tissue. Finally, it is proposed that up to half of theI _sc is due to a Na⁺-dependent secretion of bicarbonate ion.     

Trisk 32 regulates IP(3) receptors in rat skeletal myoblasts

To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation–contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6.G8 myoblasts. Co-localization of Trisk 32 and IP₃ receptors (IP₃R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP₃-mediated Ca²⁺ release were assessed by measuring changes in [Ca²⁺]_i following the stimulation by bradykinin or vasopressin. The amplitude of the Ca²⁺ transients evoked by 20 μM bradykinin was significantly higher in Trisk 32-overexpressing (p < 0.01; 426 ± 84 nM, n = 27) as compared to control cells (76 ± 12 nM, n = 23). The difference remained significant (p < 0.02; 217 ± 41 nM, n = 21, and 97 ± 29 nM, n = 31, respectively) in the absence of extracellular Ca²⁺. Similar observations were made when 0.1 μM vasopressin was used to initiate Ca²⁺ release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca²⁺ entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin- or vasopressin-evoked Ca²⁺ transients; rather, they were due to the enhanced activity of IP₃R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca²⁺ release via IP₃R.     

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres

 The Ca²⁺ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca²⁺ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake decreased from the control values of 0.384 ± 0.030 μmol (mg fibre protein)^–1 and 19.8 ± 1.9 nmol min^–1 (mg fibre protein)^–1, respectively, to 0.210 ± 0.016 μmol (mg fibre protein)^–1 and 13.5 ± 0.9 nmol min^–1 (mg fibre protein)^–1; the calculated amount of Ca²⁺ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 μmol (mg fibre protein)^–1. In the 7-day denervated muscle, the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake increased to 0.517 ± 0.06 μmol (mg fibre protein)^–1 and 21.6 ± 2.3 nmol min^–1 (mg fibre protein)^–1, respectively; the calculated amount of Ca²⁺ released increased to 0.217 μmol (mg fibre protein)^–1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca²⁺ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation. Received: 15 October 1996 / Received after revision: 28 March 1997 / Accepted: 8 April 1997     

Structure-property relationships in the optimization of polysilicon thin films for electrical recording/stimulation of single neurons

We had earlier demonstrated the use of polysilicon microelectrodes for recording electrical activity from single neurons in vivo. Good machinability and compatibility with CMOS processing further make polysilicon an attractive interface material between biological environments on one hand and MEMS technology and digital circuits on the other hand. In this study, we focus on optimizing the polysilicon thin films for (a) electrical recording and (b) stimulation of single neurons by minimizing its electrochemical impedance spectra and maximizing its charge storage/injection capacity respectively. The structure-property relationships in ion-implanted (phosphorus) LPCVD polysilicon thin films under different annealing and doping conditions were carefully assessed during this optimization process. A 2D model of the polysilicon thin film consisting of 4 grains and 3 grain boundaries was constructed and the effect of grain size and grain boundaries on dc resistivity was simulated using device simulator ATLAS. Optimal processing conditions and doping concentrations resulted in a 10-fold decrease in electrochemical impedance from 1.1 kΩ to 0.1 kΩ at 1 kHz (area of polysilicon interface = 4.8 mm²). Subsequent characterizations showed that evolution of secondary grains within the polysilicon thin films at optimal doping and annealing conditions (10²¹/cm³ of phosphorus and annealed at 1200°C) was responsible for decreasing the impedance. Cyclic voltammetry studies demonstrated that charge storage properties of low doped (10¹⁵/cm³) thin films was 111.4 μC/cm² in phosphate buffered saline which compares well with platinum wires (∼50 μC/cm²) and the double-layered capacitance (C _dl ) could be sustained between –1 to 1 V before breakdown and hydrolysis. We conclude that polysilicon can be optimized for recording and stimulating single neurons and can be a valuable interface material between neurons and CMOS or MEMS devices.     

Experimental Study of Relationship between Biological Hazards of Low-Dose Radiofrequency Exposure and Energy Flow Density in <Emphasis Type="Italic">Spirostomum Ambiguum</Emphasis> Infusoria Exposed at a Mobile Connection Frequency (1 GHz)

Radiofrequency exposure at the mobile connection frequency (1 GHz) at different energy flow densities, 5 μW/cm² (2-fold below the maximum permissible level) and 50 μW/cm² (5-fold surpassing this level), caused a reduction of motor activity in unicellular hydrobionts Spirostomum ambiguum Ehrbg infusoria. In all cases, the effect was similar by the intensity and developed in a jump-wise manner after exposure of a certain duration, after which did not increase with prolongation of the exposure. The duration of radiofrequency exposure safe for the object varied signifi cantly: 8–9 h and 10 min at 5 and 50 μW, respectively. These innovation data on harmful biological effects of very low radiofrequency exposure (5 μW/cm²), the threshold form of biological reaction, presence of “safe” periods of exposure, and the data demonstrating a clear-cut relationship between these periods and energy flow density are interesting from theoretical viewpoint and in connection with the problem of evaluating permissible levels of radiofrequency exposure of biological objects.     

A near infrared spectroscopy study investigating oxygen utilisation in hydrocephalic rats

Determination of hydrocephalus and its severity is important for optimal management of the condition. We have used near infrared spectroscopy (NIRS) to assess changes in concentrations of oxygenated (O₂Hb), deoxygenated (HHb), total haemoglobin (tHb) and cytochrome c oxidase (Caa₃) in normal and hydrocephalic Texas (HTx) rats in response to a 5 min head down tilt and a sodium pentobarbitone (NaPB) challenge. The former was used to test vascular responses and the latter to test metabolic responses. The haemoglobin oxygenation index (HbD) was derived which provides information regarding oxygen utilisation ([HbD]=[O₂Hb]−[HHb]). With the tilt challenge, a significant (P=0.001) difference was observed in [HbD] between normal (n=24) and hydrocephalic (n=14) rats (–3.50 (−6.00 to 0.00) μM cm⁻¹ and 7.50 (0.75 to 14.25) μM cm⁻¹, respectively). In another experiment we tested the response of ten rats to NaPB administration and observed a significant difference (P=0.008) in [Caa₃] between normal (n=5) and hydrocephalic (n=5) rats (−6.60 (−7.55 to −5.50) μM cm⁻¹ and −2.20 (−5.60 to −1.05) μM cm⁻¹, respectively). Coronal sections of these ten rat brains were analysed and significant (P<0.05) relationships were found between some of the NIRS parameters and cortical thickness or lateral ventricle area measurements. Our studies demonstrate that a significant difference in cerebral oxygenation and haemodynamics can be observed between normal and hydrocephalic HTx rats using NIRS.     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

 The effects of the sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca²⁺ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca²⁺] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca²⁺] of 100 nM, introduction of 20 μM CPA induced a slow release of Ca²⁺ from the SR. The following one to two caffeine-induced Ca²⁺ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca²⁺ transients decreased progressively and were barely detectable 6–7 min after introduction of CPA. However, increasing the bathing [Ca²⁺] or increasing the Ca²⁺ loading period resulted in a partial recovery of the caffeine-induced Ca²⁺ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 μM CPA. The slow Ca²⁺ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca²⁺ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca²⁺ transient reflects a balance between efflux via the SR Ca²⁺ channel and reuptake by the Ca pump. Ca²⁺ release upon addition of CPA may result from inhibition of SR Ca²⁺ uptake, which reveals a tonic Ca²⁺ efflux that is independent of the Ca²⁺ release channels. Received: 26 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998