bluuebunny/crossref_metadata_2025 huggingface dataset:
Why Do IPO Firms Conduct Primary Seasoned Equity Offerings?
Authors: Harjoto Maretno , Garen John
Published: March 2003

This study examines the reason behind the IPO firm's decision to conduct a primary seasoned equity offering (SEO). First, we develop a two–period model of blockholder incentives starting from the IPO stage. The model suggests that the blockholder has an incentive to conduct an SEO after the IPO when the firm is experiencing growth that was not anticipated at the IPO stage. Using a sample of IPO firms during 1992 to 1997, we find that IPO firms with higher unanticipated positive growth are more likely to conduct an SEO during the four years after their IPOs. We find that the firm's unanticipated shock and growth positively affect the relative size of the firm's seasoned equity offering. We also find that the firm's risk measure reduces the probability of conducting an SEO and reduces the relative size of an SEO.
Probing the Will of God: Bonhoeffer and Discernment
Authors: Dahill Lisa E.
Published: March 2003

Like the Spiritual Exercises of St. Ignatius, the spiritual discernment of Dietrich Bonhoeffer seeks to bring one’s life into conformity (Gleichgestaltung) with Christ. Such conformation makes discipleship costly, but it also overflows with the fullness of life.
Toward a Theory of Entrepreneurial Cognition: Rethinking the People Side of Entrepreneurship Research
Authors: Mitchell Ronald K. , Busenitz Lowell , Lant Theresa , McDougall Patricia P. , Morse Eric A. , Smith J. Brock
Published: March 2003

The failure of past “entrepreneurial personality”—based research to clearly distinguish the unique contributions to the entrepreneurial process of entrepreneurs as people, has created a vacuum within the entrepreneurship literature that has been waiting to be filled. Recently, the application of ideas and concepts from cognitive science has gained currency within entrepreneurship research, as evidenced by the growing accumulation of successful studies framed in entrepreneurial cognition terms. In this article we reexamine “the people side of entrepreneurship” by summarizing the state of play within the entrepreneurial cognition research stream, and by integrating the five articles accepted for publication in this special issue into this ongoing narrative. We believe that the constructs, variables, and proposed relationships under development within the cognitive perspective offer research concepts and techniques that are well suited to the analysis of problems that require better explanations of the contributions to entrepreneurship that are distinctly human.
Alterations in Slow‐Twitch Muscle Phenotype in Transgenic Mice Overexpressing the Ca2+ Buffering Protein Parvalbumin
Authors: Chin Eva R. , Grange Robert W. , Viau Francois , Simard Alain R. , Humphries Caroline , Shelton John , Bassel‐Duby Rhonda , Williams R. Sanders , Michel Robin N.
Published: March 2003

The purpose of this study was to determine whether induced expression of the Ca2+ buffering protein parvalbumin (PV) in slow‐twitch fibres would lead to alterations in physiological, biochemical and molecular properties reflective of a fast fibre phenotype. Transgenic (TG) mice were generated that overexpressed PV in slow (type I) muscle fibres. In soleus muscle (SOL; 58 % type I fibres) total PV expression was 2‐ to 6‐fold higher in TG compared to wild‐type (WT) mice. Maximum twitch and tetanic tensions were similar in WT and TG but force at subtetanic frequencies (30 and 50 Hz) was reduced in TG SOL. Twitch time‐to‐peak tension and half‐relaxation time were significantly decreased in TG SOL (time‐to‐peak tension: 39.3 ± 2.6 vs. 55.1 ± 4.7 ms; half‐relaxation time: 42.1 ± 3.5 vs. 68.1 ± 9.6 ms, P < 0.05 for TG vs. WT, respectively; n= 8–10). There was a significant increase in expression of type IIa myosin heavy chain (MHC) and ryanodine receptor at the mRNA level in TG SOL but there were no differences in MHC expression at the protein level and thus no difference in fibre type. Whole muscle succinate dehydrogenase activity was reduced by 12 ± 0.4 % in TG SOL and single fibre glycerol‐3‐phosphate dehydrogenase activity was decreased in a subset of type IIa fibres. These differences were associated with a 64 % reduction in calcineurin activity in TG SOL. These data show that overexpression of PV, resulting in decreased calcineurin activity, can alter the functional and metabolic profile of muscle and influence the expression of key marker genes in a predominantly slow‐twitch muscle with minimal effects on the expression of muscle contractile proteins.
External Perturbation of the Trunk in Standing Humans Differentially Activates Components of the Medial Back Muscles
Authors: Moseley G. Lorimer , Hodges Paul W. , Gandevia S. C.
Published: March 2003

During voluntary arm movements, the medial back muscles are differentially active. It is not known whether differential activity also occurs when the trunk is perturbed unpredictably, when the earliest responses are initiated by short‐latency spinal mechanisms rather than voluntary commands. To assess this, in unpredictable and self‐initiated conditions, a weight was dropped into a bucket that was held by the standing subject (n= 7). EMG activity was recorded from the deep (Deep MF), superficial (Sup MF) and lateral (Lat MF) lumbar multifidus, the thoracic erector spinae (ES) and the biceps brachii. With unpredictable perturbations, EMG activity was first noted in the biceps brachii, then the thoracic ES, followed synchronously in the components of the multifidus. During self‐initiated perturbations, background EMG in the Deep MF increased two‐ to threefold, and the latency of the loading response decreased in six out of the seven subjects. In Sup MF and Lat MF, this increase in background EMG was not observed, and the latency of the loading response was increased. Short‐latency reflex mechanisms do not cause differential action of the medial back muscles when the trunk is loaded. However, during voluntary tasks the central nervous system exerts a ‘tuned response’, which involves discrete activity in the deep and superficial components of the medial lumbar muscles in a way that varies according to the biomechanical action of the muscle component.
Effect of atropine on the biomechanical properties of the oesophageal wall in humans
Authors: Takeda Torahiko , Kassab Ghassan , Liu Jianmin , Nabae Toshinaga , Mittal Ravinder K.
Published: March 2003

Recently, we reported a novel ultrasound technique to assess biomechanical properties of the oesophagus in human subjects. In the present study, we use the technique, in combination with atropine, to determine the active and passive biomechanical properties of the oesophagus in normal healthy humans. A manometric catheter equipped with a high‐compliance bag and a high‐frequency intraluminal ultrasonography probe was used to record pressure and oesophageal geometry. Oesophageal distensions with either isovolumic (5–20 ml water) or with isobaric (10–60 mmHg) technique were performed. Intra‐bag pressure and ultrasound images of the oesophagus were recorded simultaneously. Following injection of atropine (15 μg kg−1, i.v.), the oesophageal distensions were repeated. The oesophageal wall compliance, circumferential wall tension, stress, strain and elastic modulus were calculated. Atropine resulted in an increase in the oesophageal wall compliance during isobaric distension, but no change in compliance was observed during isovolumic distension. The stress–strain relationship was found to be linear during both types of distension, before as well as after atropine. The Young's modulus, which is the slope of a linear stress–strain relationship, was significantly higher after atropine in the isovolumic study but not in the isobaric study. The stress–strain relationship of the active component (muscle contraction) was different during isovolumic and isobaric distensions but the passive components were similar. The passive and active stress–strain relationships of the human oesophagus resemble those of other soft biological tissues. Furthermore, the method of oesophageal distension has significant influence on the active but not the passive biomechanical properties due to a strain‐rate effect.
Variation in Large‐Conductance, Calcium‐Activated Potassium Channels from Hair Cells Along the Chicken Basilar Papilla
Authors: Duncan R. K. , Fuchs P. A.
Published: March 2003

The mechanism for electrical tuning in non‐mammalian hair cells rests within the widely diverse kinetics of functionally distinct, large‐conductance potassium channels (BK), thought to result from alternative splicing of the pore‐forming α subunit and variable co‐expression with an accessory β subunit. Inside‐out patches from hair cells along the chicken basilar papilla revealed ‘tonotopic’ gradations in calcium sensitivity and deactivation kinetics. The resonant frequency for the hair cell from which the patch was taken was estimated from deactivation rates, and this frequency reasonably matched that predicted from the originating cell's tonotopic location. The rates of deactivation for native BK channels were much faster than rates reported for cloned chicken BK channels including both α and β subunits. This result was surprising since patches were pulled from hair cells in the apical half of the papilla where β subunits are most highly expressed. Heterogeneity in the properties of native chicken BK channels implies a high degree of molecular variation and hinders our ability to identify those molecular constituents.
Rod and cone contributions to the a‐wave of the electroretinogram of the macaque
Authors: Robson John G. , Saszik Shannon M. , Ahmed Jameel , Frishman Laura J.
Published: March 2003

The electroretinogram (ERG) of anaesthetised dark‐adapted macaque monkeys was recorded in response to ganzfeld stimulation and rod‐ and cone‐driven receptoral and postreceptoral components were separated and modelled. The test stimuli were brief (< 4.1 ms) flashes. The cone‐driven component was isolated by delivering the stimulus shortly after a rod‐saturating background had been extinguished. The rod‐driven component was derived by subtracting the cone‐driven component from the mixed rod–cone ERG. The initial part of the leading edge of the rod‐driven a‐wave scaled linearly with stimulus energy when energy was sufficiently low and, for times less than about 12 ms after the stimulus, it was well described by a linear model incorporating a distributed delay and three cascaded low‐pass filter elements. Addition of a simple static saturating non‐linearity with a characteristic intermediate between a hyperbolic and an exponential function was sufficient to extend application of the model to most of the leading edge of the saturated responses to high energy stimuli. It was not necessary to assume involvement of any other non‐linearity or that any significant low‐pass filter followed the non‐linear stage of the model. A negative inner‐retinal component contributed to the later part of the rod‐driven a‐wave. After suppressing this component by blocking ionotropic glutamate receptors, the entire a‐wave up to the time of the first zero‐crossing scaled with stimulus energy and was well described by summing the response of the rod model with that of a model describing the leading edge of the rod‐bipolar cell response. The negative inner‐retinal component essentially cancelled the early part of the rod‐bipolar cell component and, for stimuli of moderate energy, made it appear that the photoreceptor current was the only significant component of the leading edge of the a‐wave. The leading edge of the cone‐driven a‐wave included a slow phase that continued up to the peak, and was reduced in amplitude either by a rod‐suppressing background or by the glutamate analogue, cis‐piperidine‐2,3‐dicarboxylic acid (PDA). Thus the slow phase represents a postreceptoral component present in addition to a fast component of the a‐wave generated by the cones themselves. At high stimulus energies, it appeared less than 5 ms after the stimulus. The leading edge of the cone‐driven a‐wave was adequately modelled as the sum of the output of a cone photoreceptor model similar to that for rods and a postreceptoral signal obtained by a single integration of the cone output. In addition, the output of the static non‐linear stage in the cone model was subject to a low‐pass filter with a time constant of no more than 1 ms. In conclusion, postreceptoral components must be taken into account when interpreting the leading edge of the rod‐ and cone‐driven a‐waves of the dark‐adapted ERG.
Ketamine Increases Human Motor Cortex Excitability to Transcranial Magnetic Stimulation
Authors: Lazzaro V. Di , Oliviero A. , Profice P. , Pennisi M. A. , Pilato F. , Zito G. , Dileone M. , Nicoletti R. , Pasqualetti P. , Tonali P.A.
Published: March 2003

Subanaesthetic doses of the N‐methyl‐d‐aspartate (NMDA) antagonist ketamine have been shown to determine a dual modulating effect on glutamatergic transmission in experimental animals, blocking NMDA receptor activity and enhancing non‐NMDA transmission through an increase in the release of endogenous glutamate. Little is known about the effects of ketamine on the excitability of the human central nervous system. The effects of subanaesthetic, graded incremental doses of ketamine (0.01, 0.02 and 0.04 mg kg−1 min−1, i.v.) on the excitability of cortical networks of the human motor cortex were examined with a range of transcranial magnetic and electric stimulation protocols in seven normal subjects. Administration of ketamine at increasing doses produced a progressive reduction in the mean resting motor threshold (RMT) (F(3, 18) = 22.33, P < 0.001) and active motor threshold (AMT) (F(3, 18) = 12.17, P < 0.001). Before ketamine administration, mean RMT ±s.d. was 49 ± 3.3 % of maximum stimulator output and at the highest infusion level it was 42.6 ± 2.6 % (P < 0.001). Before ketamine administration, AMT ±s.d. was 38 ± 3.3 % of maximum stimulator output and at the highest infusion level it was 33 ± 4.4 % (P < 0.002). Ketamine also led to an increase in the amplitude of EMG responses evoked by magnetic stimulation at rest; this increase was a function of ketamine dosage (F(3, 18) = 5.29, P= 0.009). In contrast to responses evoked by magnetic stimulation, responses evoked by electric stimulation were not modified by ketamine. The differential effect of ketamine on responses evoked by magnetic and electric stimulation demonstrates that subanaesthetic doses of ketamine enhance the recruitment of excitatory cortical networks in motor cortex. Transcranial magnetic stimulation produces a high‐frequency repetitive discharge of pyramidal neurones and for this reason probably depends mostly on short‐lasting AMPA transmission. An increase in this transmission might facilitate the repetitive discharge of pyramidal cells after transcranial magnetic stimulation which, in turn, results in larger motor responses and lower thresholds. We suggest that the enhancement of human motor cortex excitability to transcranial magnetic stimulation is the effect of an increase in glutamatergic transmission at non‐NMDA receptors similar to that described in experimental studies.
Somatic Exocytosis of Serotonin Mediated by L‐Type Calcium Channels in Cultured Leech Neurones
Authors: Trueta Citlali , Méndez Bruno , De‐Miguel Francisco F.
Published: March 2003

We studied somatic exocytosis of serotonin and its mediation by L‐type calcium (Ca2+) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 mm potassium (K+), and was quantified by use of the fluorescent dye FM 1–43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1–43 fluorescent spots of 1.28 ± 0.01 μm in diameter, provided that Ca2+ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 mm K+. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 ± 5 spots per soma, 77.9 ± 13.9 spots per soma were produced at 10 Hz and 91.5 ± 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ. In the presence of the L‐type Ca2+ channel blocker nimodipine (10 μm), a 20 Hz train produced only 22.9 ± 6.4 spots per soma, representing a 75 % reduction compared to control cells (P < 0.05). Subsequent incubation with 10 mm caffeine to induce Ca2+ release from intracellular stores increased the number of spots to 73.22 ± 12.5. Blockers of N‐, P‐, Q‐ or invertebrate Ca2+ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.
Modulation of AMPA receptors by cAMP‐dependent protein kinase in PreBötzinger complex inspiratory neurons regulates respiratory rhythm in the rat
Authors: Shao Xuesi M. , Ge Qing , Feldman Jack L.
Published: March 2003

We hypothesize that phosphorylation of AMPA receptors or associated synaptic proteins modulates the excitability of respiratory neurons in the preBötzinger Complex (preBötC), affecting respiratory rhythm. Using neonatal rat medullary slices that spontaneously generate respiratory rhythm, we examined the role of the cAMP–PKA pathway (PKA:cAMP‐dependent protein kinase) in modulating glutamatergic synaptic transmission, the excitability of inspiratory neurons in the preBötC and respiratory rhythm. Microinjection of forskolin, an activator of adenylate cyclase, into the preBötC with or without the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX), decreased the period (increased the frequency) of respiratory‐related rhythmic motor output in the hypoglossal nerve (XIIn) to 84 % (without IBMX) and to 72% (with IBMX) of the pre‐injection baseline. In the presence of MK‐801, a non‐competitive NMDA receptor antagonist, microinjection of forskolin plus IBMX decreased the period to 66% of baseline levels. Microinjection of Rp‐adenosine 3′,5′‐cyclic monophosphothioate (Rp‐cAMPS), a PKA inhibitor, increased the period to 145% of baseline levels. Concurrent microinjection of Rp‐cAMPS and forskolin had no effect on the period. Bath application of 7β‐deacetyl‐7β‐[γ‐(morpholino)butyryl]‐forskolin hydrochloride (7Db‐forskolin, a water‐soluble derivative of forskolin): (1) decreased the period to 67% of baseline levels without affecting the amplitude of integrated XIIn inspiratory discharge, (2) induced a tonic inward current of 29 pA and enhanced inspiratory drive current (the amplitude increased to 183% and the integral increased to 184% of baseline) in voltage‐clamped (holding potential =−60 mV) preBötC inspiratory neurons and (3) increased the frequency to 195 % and amplitude to 118% of spontaneous excitatory postsynaptic currents (sEPSCs) during expiratory periods. Dideoxy‐forskolin did not have these effects. Intracellular perfusion with the catalytic subunit of PKA (cPKA) into preBötC inspiratory neurons progressively enhanced inspiratory drive currents and, in the presence of TTX, increased the inward currents induced by local ejection of AMPA; the latter currents were blocked by 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo[f]quinoxaline‐7‐sulphonamide (NBQX, an AMPA/kainate receptor antagonist). The effects of cPKA were blocked by co‐application of PKA inhibitor (6–22) amide (PKI). These results suggest that phosphorylation of postsynaptic AMPA receptors through the cAMP–PKA pathway modulates both tonic and phasic excitatory amino acid synaptic transmission and excitability of inspiratory neurons in the preBötC and, therefore, regulates respiratory rhythm. Moreover, the basal level of endogenous PKA activity appears to be a determinant of resting respiratory frequency.
Decreased Surface Tension of Upper Airway Mucosal Lining Liquid Increases Upper Airway Patency in Anaesthetised Rabbits
Authors: Kirkness Jason P. , Christenson Hugo K. , Garlick Sarah R. , Parikh Radha , Kairaitis Kristina , Wheatley John R. , Amis Terence C.
Published: March 2003

The obstructive sleep apnoea syndrome (OSA) is a disorder characterised by repetitive closure and re‐opening of the upper airway during sleep. Upper airway luminal patency is influenced by a number of factors including: intraluminal air pressure, upper airway dilator muscle activity, surrounding extraluminal tissue pressure, and also surface forces which can potentially act within the liquid layer lining the upper airway. The aim of the present study was to examine the role of upper airway mucosal lining liquid (UAL) surface tension (γ) in the control of upper airway patency. Upper airway opening (PO) and closing pressures (PC) were measured in 25 adult male, supine, tracheostomised, mechanically ventilated, anaesthetised (sodium pentabarbitone), New Zealand White rabbits before (control) and after instillation of 0.5 ml of either 0.9 % saline (n= 9) or an exogenous surfactant (n= 16; Exosurf Neonatal) into the pharyngeal airway. The γ of UAL (0.2 μl) was quantified using the ‘pull‐off’ force technique in which γ is measured as the force required to separate two curved silica discs bridged by the liquid sample. The γ of UAL decreased after instillation of surfactant from 54.1 ± 1.7 mN m−1 (control; mean ±s.e.m.) to 49.2 ± 2.1 mN m−1 (surfactant; P < 0.04). Compared with control, PO increased significantly (P < 0.04; paired t test, n= 9) from 6.2 ± 0.9 to 9.6 ± 1.2 cmH2O with saline, and decreased significantly (P < 0.05, n= 16) from 6.6 ± 0.4 to 5.5 ± 0.6 cmH2O with surfactant instillation. Findings tended to be similar for PC. Change in both PO and PC showed a strong positive correlation with the change in γ of UAL (both r > 0.70, P < 0.001). In conclusion, the patency of the upper airway in rabbits is partially influenced by the γ of UAL. These findings suggest a role for UAL surface properties in the pathophysiology of OSA.
Mast cell tryptase and proteinase‐activated receptor 2 induce hyperexcitability of guinea‐pig submucosal neurons
Authors: Reed David E. , Barajas‐Lopez Carlos , Cottrell Graeme , Velazquez‐Rocha Sara , Dery Olivier , Grady Eileen F. , Bunnett Nigel W. , Vanner Stephen J.
Published: March 2003

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long‐term hyperexcitability in submucosal neurons from the guinea‐pig ileum by activating proteinase‐activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT‐PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL‐NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min–hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR‐NH2) had no effect on neuronal membrane potential or long‐term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5‐HT, prostaglandins) also caused depolarization, and following washout of antigen, long‐term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long‐term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.
Activity of Brainstem Respiratory Neurones just before the Expiration‐Inspiration Transition in the Rat
Authors: Ezure Kazuhisa , Tanaka Ikuko , Saito Yoshiaki
Published: March 2003

Inspiratory activity of the hypoglossal nerve (XIIn) often precedes that of the phrenic nerve (PHRn). By manipulating artificial respiration, this preceding activity (pre‐I XIIn activity) can be lengthened or isolated prematurely (decoupled XIIn activity) without developing into overt PHRn‐associated inspiratory bursts. We hypothesized that these pre‐I and decoupled XIIn activities, collectively termed ‘XIIn‐w/o‐PHRn activity’, reflect certain internal states of the respiratory centre at the period just prior to the transition from the expiratory phase to the inspiratory phase. In decerebrate, neuromuscularly blocked and artificially ventilated rats, the firing properties of medullary respiratory neurones were examined during the period of the XIIn‐w/o‐PHRn activity. The majority of the inspiratory neurones examined could be classified into two types: one was active (XIIn‐type) and the other was inactive (PHRn‐type) during the XIIn‐w/o‐PHRn period. On the other hand, augmenting expiratory (E‐AUG) neurones of the Bötzinger complex (BOT) and the caudal ventral respiratory group (VRG) fired intensively during this period. Their firing stopped at the onset of the overt inspiratory bursts in the XIIn and PHRn, suggesting that BOT E‐AUG neurones inhibit PHRn‐type, but not XIIn‐type, inspiratory neurones. We hypothesize that XIIn‐type inspiratory activity facilitates the phase change from expiration to inspiration, through activation of certain inspiratory neurones that inhibit the firing of BOT E‐AUG neurones and generation of the overt inspiratory bursts in XIIn‐type and PHRn‐type inspiratory neurones.
Allosteric Regulation and Spatial Distribution of Kainate Receptors Bound to Ancillary Proteins
Authors: Bowie Derek , Garcia Elizabeth P. , Marshall John , Traynelis Stephen F. , Lange G. David
Published: March 2003

A diverse range of accessory proteins regulates the behaviour of most ligand‐ and voltage‐gated ion channels. For glutamate receptor 6 (GluR6) kainate receptors, two unrelated proteins, concanavalin‐A (Con‐A) and postsynaptic density protein 95 (PSD‐95), bind to extra‐ and intracellular domains, respectively, but are reported to exert similar effects on GluR6 desensitization behaviour. We have tested the hypothesis that distinct allosteric binding sites control GluR6 receptors via a common transduction pathway. Rapid agonist application to excised patches revealed that neither Con‐A nor PSD‐95 affect the onset of desensitization. The rate of desensitization elicited by 10 mm l‐glutamate was similar in control (τfast= 5.5 ± 0.4 ms), Con‐A‐treated patches (τfast= 6.1 ± 0.5 ms) and patches containing PSD‐95 and GluR6 receptors (τfast= 4.7 ± 0.6 ms). Likewise, the time course of recovery from GluR6 desensitization was similar in both control and Con‐A conditions, whereas PSD‐95 accelerated recovery almost twofold. Peak and steady‐state (SS) dose‐response relationships to glutamate were unchanged by lectin treatment (e.g. control, EC50(SS)= 31 ± 28 μmvs Con‐A, EC50(SS)= 45 ± 9 μm, n= 6), suggesting that Con‐A does not convert non‐conducting channels with high agonist affinity into an open conformation. Instead, we demonstrate that the effects of Con‐A on macroscopic responses reflect a shift in the relative contribution of different open states of the channel. In contrast, the effect of PSD‐95 on recovery behaviour suggests that the association between kainate receptors and cytoskeletal proteins regulates signalling at glutamatergic synapses. Our results show that Con‐A and PSD‐95 regulate kainate receptors via distinct allosteric mechanisms targeting selective molecular steps in the transduction pathway.
Local Suppression of Epileptiform Activity by Electrical Stimulation in Rat Hippocampus In Vitro
Authors: Lian Jun , Bikson Marom , Sciortino Christopher , Stacey William C. , Durand Dominique M.
Published: March 2003

High frequency electrical stimulation of deep brain structures (DBS) has been effective at controlling abnormal neuronal activity in Parkinson's patients and is now being applied for the treatment of pharmacologically intractable epilepsy. The mechanisms underlying the therapeutic effects of DBS are unknown. In particular, the effect of the electrical stimulation on neuronal firing remains poorly understood. Previous reports have showed that uniform electric fields with both AC (continuous sinusoidal) or DC waveforms could suppress epileptiform activity in vitro. In the present study, we tested the effects of monopolar electrode stimulation and low‐duty cycle AC stimulation protocols, which more closely approximate those used clinically, on three in vitro epilepsy models. Continuous sinusoidal stimulation, 50 % duty‐cycle sinusoidal stimulation, and low (1.68 %) duty‐cycle pulsed stimulation (120 μs, 140 Hz) could completely suppress spontaneous low‐Ca2+ epileptiform activity with average thresholds of 71.11 ± 26.16 μA, 93.33 ± 12.58 μA and 300 ± 100 μA, respectively. Continuous sinusoidal stimulation could also completely suppress picrotoxin‐ and high‐K+‐induced epileptiform activity with either uniform or localized fields. The suppression generated by the monopolar electrode was localized to a region surrounding the stimulation electrode. Potassium concentration and transmembrane potential recordings showed that AC stimulation was associated with an increase in extracellular potassium concentration and neuronal depolarization block; AC stimulation efficacy was not orientation‐selective. In contrast, DC stimulation blocked activity by membrane hyperpolarization and was orientation‐selective, but had a lower threshold for suppression.
Enhanced G protein‐dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+ channel‐mutant mouse, tottering
Authors: Zhou Yu Dong , Turner Timothy J. , Dunlap Kathleen
Published: March 2003

Tottering, a mouse model for absence epilepsy and cerebellar ataxia, carries a mutation in the gene encoding class A (P/Q‐type) Ca2+ channels, the dominant exocytotic Ca2+ channel at most synapses in the mammalian central nervous system. Comparing tottering to wild‐type mice, we have studied glutamatergic transmission between parallel fibres and Purkinje cells in cerebellar slices. Results from biochemical assays and electrical field recordings demonstrate that glutamate release from parallel fibre terminals of the tottering mouse is controlled largely by class B Ca2+ channels (N‐type), in contrast to the P/Q‐channels that dominate release from wild‐type terminals. Since N‐channels, in a variety of assays, are more effectively inhibited by G proteins than are P/Q‐channels, we tested whether synaptic transmission between parallel fibres and Purkinje cells in tottering mice was more susceptible to inhibitory modulation by G protein‐coupled receptors than in their wild‐type counterparts. GABAB receptors and α2‐adrenergic receptors (activated by bath application of transmitters) produced a three‐ to fivefold more potent inhibition of transmission in tottering than in wild‐type synapses. This increased modulation is likely to be important for cerebellar transmission in vivo, since heterosynaptic depression, produced by activating GABAergic interneurones, greatly prolonged GABAB receptor‐mediated presynaptic inhibition in tottering as compared to wild‐type slices. We propose that this enhanced modulation shifts the balance of synaptic input to Purkinje cells in favour of inhibition, reducing Purkinje cell output from the cerebellum, and may contribute to the aberrant motor phenotype that is characteristic of this mutant animal.
Sour Taste Stimuli Evoke Ca2+ and pH Responses in Mouse Taste Cells
Authors: Richter T. A. , Caicedo A. , Roper S. D.
Published: March 2003

Sour taste is elicited by acids. How taste cells transduce sour taste is controversial because acids (specifically protons) have diverse effects on cell membranes. Consequently, it is difficult to differentiate between events related to sour taste transduction per se and unrelated effects of protons. We have studied acid taste transduction in mouse taste buds using a lingual slice preparation where it is possible to measure changes in pH and [Ca2+]i simultaneously in taste cells. Focal application of citric acid or HCl to the apical tips of taste buds produced widespread acidification of the entire taste bud. Citric acid was effective at a pH of ∼4, but HCl only at a pH of ∼1.5. Despite acidification of the whole taste bud, only a select few taste cells exhibited Ca2+ responses. Acid‐evoked Ca2+ responses were dose dependent in a range consistent with them being sour‐taste responses. Cells exhibiting acid‐evoked Ca2+ responses also responded to KCl depolarization. Acid‐evoked Ca2+ responses were blocked by Ba2+ (2 mm) and Cd2+ (500 μm), suggesting that acid responses are generated by Ca2+ influx through depolarization‐gated Ca2+ channels. Removing extracellular Ca2+ reduced acid‐evoked Ca2+ responses, but depleting intracellular Ca2+ stores with thapsigargin had no effect, suggesting that acid taste responses are generated by an influx of extracellular Ca2+. Neither Cs+ (500 μm) nor amiloride (100 μm) affected acid‐evoked Ca2+ responses, suggesting that neither hyperpolarization‐activated cyclic nucleotide‐gated cation (pacemaker) channels nor epithelial Na+ channels, respectively, transduce sour taste. Collectively, the results indicate that acids, especially weak acids, acidify the taste bud and evoke depolarization‐induced Ca2+ entry into a select subset of taste cells. The primary transducer protein(s) for sour taste remain undiscovered.
The Wellcome Prize Lecture
Authors: Parekh Anant B.
Published: March 2003

In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca2+ concentration: an initial transient release of Ca2+ from intracellular stores is followed by a sustained phase of Ca2+ influx. This influx is generally store‐dependent and is required for controlling a host of Ca2+‐dependent processes ranging from exocytosis to cell growth and proliferation. In many cell types, store‐operated Ca2+ entry is manifest as a non‐voltage‐gated Ca2+ current called ICRAC (Ca2+ release‐activated Ca2+ current). Just how store emptying activates CRAC channels remains unclear, and some of our recent experiments that address this issue will be described. No less important from a physiological perspective is the weak Ca2+ buffer paradox: whereas macroscopic (whole cell) ICRAC can be measured routinely in the presence of strong intracellular Ca2+ buffer, the current is generally not detectable under physiological conditions of weak buffering following store emptying with the second messenger InsP3. In this review, I describe some of our experiments aimed at understanding just why InsP3 is ineffective under these conditions and which lead us to conclude that respiring mitochondria are essential for the activation of ICRAC in weak intracellular Ca2+ buffer. Mitochondrial Ca2+ uptake also increases the dynamic range over which InsP3 functions as the second messenger that controls Ca2+ influx. Finally, we find that Ca2+‐dependent slow inactivation of Ca2+ influx, a widespread but poorly understood phenomenon that helps shape the profile of an intracellular Ca2+ signal, is regulated by mitochondrial Ca2+ buffering. Thus, by enabling macroscopic store‐operated Ca2+ current to activate and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store‐operated Ca2+ influx. Store‐operated Ca2+ entry reflects therefore a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.
Role of the Transverse‐Axial Tubule System in Generating Calcium Sparks and Calcium Transients in Rat Atrial Myocytes
Authors: Kirk Malcolm M. , Izu Leighton T. , Chen‐Izu Ye , McCulle Stacey L. , Wier W. Gil , Balke C. William , Shorofsky Stephen R.
Published: March 2003

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse‐axial tubular system (TATS). To investigate the possible role of the TATS in excitation‐contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di‐8‐ANEPPS) simultaneously with Ca2+ transients and/or Ca2+ sparks (fluo‐4). In confocal transverse linescan images of field‐stimulated cells, whole‐cell Ca2+ transients had two morphologies: ‘U‐shaped’ transients and irregular or ‘W‐shaped’ transients with a varying number of points of origin of the Ca2+ transient. About half (54 %, n=289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 ± 2.8 μm) than cells without TATS (11.7 ± 2.0 μm) and were more common in the left atrium (n= 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca2+ sparks and sarcolemmal structures showed that cells without TATS had U‐shaped transients that started at the cell periphery, and cells with TATS had W‐shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) ‘spontaneous’ (non‐depolarized) Ca2+ sparks occurred within 1 μm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 μm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca2+ transient and for spontaneous Ca2+ sparks.
Arm Blood Flow and Oxygenation on the Transition from Arm to Combined Arm and Leg Exercise in Humans
Authors: Volianitis S. , Krustrup P. , Dawson E. , Secher N. H.
Published: March 2003

The cardiovascular response to exercise with several groups of skeletal muscle implies that work with the legs may reduce arm blood flow. This study followed arm blood flow (Q̇arm) and oxygenation on the transition from arm cranking (A) to combined arm and leg exercise (A+L). Seven healthy male subjects performed A at ∼80 % of maximum work rate (Wmax) and A at ∼80 % Wmax combined with L at ∼60 % Wmax. A transition trial to volitional exhaustion was performed where L was added after 2 min of A. The Q̇arm was determined by constant infusion thermodilution in the axillary vein and changes in biceps muscle oxygenation were measured with near‐infrared spectroscopy. During A+L Q̇arm was lowered by 0.38 ± 0.06 l min−1 (10.4 ± 3.3 %, P < 0.05) from 2.96 ± 1.54 l min−1 during A. Total (HbT) and oxygenated haemoglobin (HbO2) concentrations were also lower. During the transition from A to A+L Q̇arm decreased by 0.22 ± 0.03 l min−1 (7.9 ± 1.8 %, P < 0.05) within 9.6 ± 0.2 s, while HbT and HbO2 decreased similarly within 30 ± 2 s. At the same time mean arterial pressure and arm vascular conductance also decreased. The data demonstrate reduction in blood flow to active skeletal muscle during maximal whole body exercise to a degree that arm oxygen uptake and muscle tissue oxygenation are compromised.
Hydrogen‐Peroxide‐Induced Toxicity of Rat Striatal Neurones Involves Activation of a Non‐Selective Cation Channel
Authors: Smith M. A. , Herson P. S. , Lee K. , Pinnock R. D. , Ashford M. L. J.
Published: March 2003

Striatal neurones are particularly vulnerable to hypoxia/ischaemia‐induced damage, and free radicals are thought to be prime mediators of this neuronal destruction. It has been shown that hydrogen peroxide (H2O2), through the production of free radicals, induces rat insulinoma cell death by activation of a non‐selective cation channel, which leads to irreversible cell depolarization and unregulated Ca2+ entry into the cell. In the study presented here, we demonstrate that a subpopulation of striatal neurones (medium spiny neurones) is depolarized by H2O2 through the production of free radicals. Cell‐attached recordings from rat cultured striatal neurones demonstrate that exposure to H2O2 opens a large‐conductance channel that is characterized by extremely long open times (seconds). Inside‐out recordings show that cytoplasmically applied β‐nicotinamide adenine dinucleotide activates a channel with little voltage dependence, a linear current‐voltage relationship and a single‐channel conductance of between 70 and 90 pS. This channel is permeable to Na+, K+ and Ca2+ ions. Fura‐2 imaging from cultured striatal neurones reveals that H2O2 exposure induces a biphasic intracellular Ca2+ increase in a subpopulation of neurones, the second, later phase resulting in Ca2+ overload. This later component of the Ca2+ response is dependent on the presence of extracellular Ca2+ and is independent of synaptic activity or voltage‐gated Ca2+ channel opening. Consequently, this channel may be an important contributor to free radical‐induced selective striatal neurone destruction. These results are remarkably similar to those observed for insulinoma cells and suggest that this family of non‐selective cation channels has a widespread distribution in mammalian tissues.
Creatine Kinase Injection Restores Contractile Function in Creatine‐Kinase‐Deficient Mouse Skeletal Muscle Fibres
Authors: Dahlstedt Anders J. , Katz Abram , Tavi Pasi , Westerblad Håkan
Published: March 2003

Viable genetically engineered animals generally exhibit adaptations to the altered genotype, which may mask the role of the protein of interest. We now describe a novel method by which the direct effects of the altered genotype can be distinguished from secondary adaptive changes in isolated adult skeletal muscle cells. We studied contractile function and intracellular Ca2+ handling in single skeletal muscle fibres that are completely deficient of creatine kinase (CK; CK−/−) before and after microinjection of purified CK (injected together with the fluorescent Ca2+ indicator indo‐1). The mean total CK activity after CK injection was estimated to be ∼ 4 mm s−1, which is ∼ 5 % of the activity in wild‐type muscle fibres. After CK injection, CK−/− fibres approached the wild‐type phenotype in several aspects: (a) the free myoplasmic [Ca2+] ([Ca2+]i) increased and force showed little change during a period of high‐intensity stimulation (duty cycle, i.e. tetanic duration divided by tetanic interval = 0.67); (b) [Ca2+]i did not decline during a brief (350 ms) tetanus; (c) during low‐intensity fatiguing stimulation (duty cycle = 0.14), tetanic [Ca2+]i increased over the first 10 tetani, and thereafter it decreased; (d) tetanic [Ca2+]i and force did not display a transient reduction in the second tetanus of low‐intensity fatiguing stimulation. Conversely, tetanic force in the unfatigued state was lower in CK−/− than in wild‐type fibres, and this difference persisted after CK injection. Injection of inactivated CK had no obvious effect on any of the measured parameters. In conclusion, microinjection of CK into CK−/− fibres markedly restores many, but not all, aspects of the wild‐type phenotype.
Enhanced excitability of myenteric AH neurones in the inflamed guinea‐pig distal colon
Authors: Linden David R. , Sharkey Keith A. , Mawe Gary M.
Published: March 2003

The electrical and synaptic properties of myenteric neurones in normal and inflamed guinea‐pig distal colons were evaluated by intracellular microelectrode recording. Chronic inflammation was established 6 days following administration of trinitrobenzene sulfonic acid (TNBS). In S neurones, inflammation only altered synaptic inputs as the amplitude of fast excitatory postsynaptic potentials were significantly larger (31 ± 2 mV compared to 20 ± 1 mV) and they were more likely to receive slow excitatory synaptic input (85% compared to 55%). AH neurones displayed altered electrical properties in colitis compared to control tissues: they generated more action potentials during a maximal depolarising current pulse (7 ± 1 compared to 1.6 ± 0.2); they had a smaller afterhyperpolarisation (9 ± 2 mV s compared to 20 ± 2 mV s); and they were more likely to receive fast excitatory synaptic input (74% compared to 17%), possess spontaneous activity (46% compared to 3%), and generate anodal break action potentials (58% compared to 19%). Although the resting membrane potential, input resistance and action potential characteristics were unaltered in AH neurones from inflamed tissues, they exhibited an enhanced Cs+‐sensitive rectification of the current–voltage relationship. This suggests that the increase in excitability of AH neurones may involve a colitis‐induced augmentation of the hyperpolarisation‐activated cation current (Ih) in these cells. An increased excitability, selectively in AH neurones, suggests that the afferent limb of intrinsic motor reflexes is disrupted in the inflamed colon and this may contribute to dysmotility associated with inflammatory diseases.
Crucial Role of Sodium Channel Fast Inactivation in Muscle Fibre Inexcitability in a Rat Model of Critical Illness Myopathy
Authors: Rich Mark M. , Pinter Martin J.
Published: March 2003

Critical illness myopathy is an acquired disorder in which skeletal muscle becomes electrically inexcitable. We previously demonstrated that inactivation of Na+ channels contributes to inexcitability of affected fibres in an animal model of critical illness myopathy in which denervated rat skeletal muscle is treated with corticosteroids (steroid denervated; SD). Our previous work, however, did not address the relative importance of membrane depolarization versus a shift in the voltage dependence of fast inactivation in causing inexcitability. It also remained unknown whether changes in the voltage dependence of activation or slow inactivation play a role in inexcitability. In the current study we found that a hyperpolarizing shift in the voltage dependence of fast inactivation of Na+ channels is the principal factor underlying inexcitability in SD fibres. Although depolarization tends to decrease excitability, it is insufficient to account for inexcitability in SD fibres since many normal and denervated fibres retain normal excitability when depolarized to the same resting potentials as affected SD fibres. Changes in the voltage dependence of activation and slow inactivation of Na+ channels were also observed in SD fibres; however, the changes appear to increase rather than decrease excitability. These results highlight the importance of the change in fast inactivation in causing inexcitability of SD fibres.