A study of the electrical basis for the inhibition and excitation in autonomically innervated smooth muscle

The aim of this thesis was to investigate the electrical and mechanical responses to inhibitory non-adrenergic noncholinergic (NANC) nerve stimulation in the bovine retractor penis muscle (BRP) and compare them with those to an inhibitory extract made from this muscle. The extract may contain the NANC inhibitory transmitter of the BRP and possibly of other smooth muscles. Because of species differences in the electrical response to NANC nerves in the rat and rabbit anococcygeus the effects of the extract on these tissues was also investigated. Prior to the investigation of the extract, both the excitatory and inhibitory responses to field stimulation in the BRP, and the effects of passive membrane potential displacement were studied using conventional intra- or extracellular (sucrose gap) recording techniques. The majority of cells in the BRP were electrically quiescent independent of the resting tone. The most frequent (in approximately 25% of preparations) form of spontaneous activity, oscillations in membrane potential and tone, may represent a pacemaker activity. The BRP had cable properties; the time constant and space constant indicated a high membrane resistance. In the absence of tone, field stimulation of the BRP evoked excitatory junction potentials (ejps) in every cell impaled and contractions, graded with the strength, frequency and number of pulses; spikes were not observed. Guanethidine (1-3 x 10-5M) abolished the ejps and contractions, confirming their adrenergic origin. Noradrenaline added exogenously depolarised and contracted the muscle. These effects were blocked by the a-adrenoceptor antagonists, phentolamine and prazosin. However, phentolamine (2.5x 10-6M) inhibited the contraction without reducing the ejp significantly. These effects may be independent of adrenoceptor blockade or the ejp may be mediated by a substance other than noradrenaline (e.g. ATP) released from adrenergic nerves. Prazosin (1.4 x lO-6M) failed to block either the ejp or contraction, indicating the possible existence of two types of adrenoceptor in the BRP; one activated by neuronally-released and the other by exogenously-added noradrenaline. ATP, a contaminant in the extract, also depolarised and contracted the BRP. Physostigmine reduced whilst atropine enhanced the ejps and contractions without similarly affecting the response to exogenous noradrenaline. This confirmed the presence of a cholinergic inhibitory innervation acting on the excitatory adrenergic fibres (Klinge and Sjostrand, 1977). TEA (1 x lO-4M) enhanced the ejp and contraction. Higher concentrations (0.5 to 10 x 10-3M) depolarised, increased the tone and evoked electrical and mechanical oscillations but no spikes. The depolarisation and contraction to exogenous noradrenaline were not enhanced, indicating that TEA acts on the adrenergic nerves. Some post-synaptic effect to block K+ channels also seems likely. The relationship between ejp amplitude and membrane potential in the double sucrose gap was linear and indicated a reversal potential more positive than -30mV. Electrotonic pulse amplitude decreased during the ejp, indicating an increased membrane conductance. Ejps and contractions were reduced following the replacement of the NaCl of the Krebs solution with sodium glutamate. This may be due to the effects of glutamate itself (e.g. Ca2+ chelation) rather than reduction in the membrane Cl- gradient. Tone usually developed spontaneously and was accompanied by membrane depolarisation (from -53 to -45mV) which may open voltage-dependent channels, causing Ca2+ entry and/or its release from intracellular binding sites. Field stimulation produced inhibitory potentials (ijps) and relaxations graded with the strength and number of pulses but showing little frequency dependence. Rebound depolarisation and contraction often followed the ijp and relaxation. Tetrodotoxin (3 x IO-6M), but not adrenergic or cholinergic antagonists, abolished the ijp and relaxation, confirming their non-adrenergic non-cholinergic neurogenic nature. The extract, prepared and acid-activated as described by Gillespie, Hunter and Martin (1981), hyperpolarised and relaxed the BRP, as did sodium nitroprusside and adenosine triphosphate (ATP). Unlike the activated extract or sodium nitroprusside, desensitisation to ATP occurred rapidly and without any change in the inhibitory electrical or mechanical responses to field stimulation. The ijp and relaxation in the BRP were insensitive to apamin but abolished by oxyhaemoglobin (4-8 x 10-6M), as were the responses to extract and sodium nitroprusside. In TEA (10-2M), field stimulation evoked relaxations with no accompanying electrical change. The ijp may be unconnected with or additional to another mechanism producing relaxation. The relationship between membrane potential and ijp in the BRP was non-linear. Ijp amplitude was initially increased during membrane potential displacement from -45mV to approximately -60mV. Thereafter (-60 to -l03mV) the ijp was reduced. Ijps were abolished at -27 and -103mV; reversal was not observed. The hyperpolarisation to extract was also enhanced during passive displacement of the membrane potential to more negative values (-57mV). Membrane resistance increased during the ijp. The extract produced inconsistent changes in membrane resistance, possibly because of the presence of more than one active component. K+ withdrawal failed to enhance the ijp or hyperpolarisation to extract and 20mM K+ did not abolish the the ijp at membrane potentials exceeding EK (-49mV). Thus, the ijp or hyperpolarisation to extract are unlikely to be mediated by an increased K+ conductance. Reducing the Cl- abolished the hyperpolarisation to field stimulation and extract. This occurred more quickly than the anticipated reduction in the Cl- gradient and may be due to Ca2+ chelation by the anion substitute (glutamate or benzenesulphonate) or blockade of the resting conductance which is normally inactivated by the transmitter. Ouabain (1-5x 10-5M), which reduces both the Na+ and Cl- gradients, abolished the ijp, implicating either of these ions as the ionic species involved. In the rat and rabbit anococcygeus, field stimulation and extract each reduced guanethidine-induced tone. This was unaccompanied in the majority of cells in the rat by any significant electrical response. In the remaining cells, inhibition of the membrane potential oscillations occurred. The rabbit anococcygeus differed in that inhibition of the electrical oscillations was observed in every cell exhibiting this behaviour. However, the majority of cells in the rabbit were electrically quiescent and showed only small hyperpolarisations to field stimulation and no electrical response to extract. Apamin (1 x 10-7M) failed to block the electrical and mechanical response to field stimulation in the rabbit but did inhibit transiently that to extract. The latter effect may be due to the initial excitatory effects of apamin. The similarities between the electrical effects of the extract and those of inhibitory nerve stimulation in the BRP, rat and rabbit anococcygeus muscles are generally consistent with their being mediated by the same active component. Moreover, the ijp in the BRP shows properties which have not been reported in other non-adrenergic noncholinergically innervated smooth muscles.

LHC main dipole magnet circuits: sustaining near-nominal beam energies

Crossing the Franco-Swiss border, the Large Hadron Collider (LHC), designed to collide 7 TeV proton beams, is the world's largest and most powerful particle accelerator the operation of which was originally intended to commence in 2008. Unfortunately, due to an interconnect discontinuity in one of the main dipole circuit's 13 kA superconducting busbars, a catastrophic quench event occurred during initial magnet training, causing significant physical system damage. Furthermore, investigation into the cause found that such discontinuities were not only present in the circuit in question, but throughout the entire LHC. This prevented further magnet training and ultimately resulted in the maximum sustainable beam energy being limited to approximately half that of the design nominal, 3.5-4 TeV, for the first three years of operation (Run 1, 2009-2012) and a major consolidation campaign being scheduled for the first long shutdown (LS 1, 2012-2014). Throughout Run 1, a series of studies attempted to predict the amount of post-installation training quenches still required to qualify each circuit to nominal-energy current levels. With predictions in excess of 80 quenches (each having a recovery time of 8-12+ hours) just to achieve 6.5 TeV and close to 1000 quenches for 7 TeV, it was decided that for Run 2, all systems be at least qualified for 6.5 TeV operation. However, even with all interconnect discontinuities scheduled to be repaired during LS 1, numerous other concerns regarding circuit stability arose. In particular, observations of an erratic behaviour of magnet bypass diodes and the degradation of other potentially weak busbar sections, as well as observations of seemingly random millisecond spikes in beam losses, known as unidentified falling object (UFO) events, which, if persist at 6.5 TeV, may eventually deposit sufficient energy to quench adjacent magnets. In light of the above, the thesis hypothesis states that, even with the observed issues, the LHC main dipole circuits can safely support and sustain near-nominal proton beam energies of at least 6.5 TeV. Research into minimising the risk of magnet training led to the development and implementation of a new qualification method, capable of providing conclusive evidence that all aspects of all circuits, other than the magnets and their internal joints, can safely withstand a quench event at near-nominal current levels, allowing for magnet training to be carried out both systematically and without risk. This method has become known as the Copper Stabiliser Continuity Measurement (CSCM). Results were a success, with all circuits eventually being subject to a full current decay from 6.5 TeV equivalent current levels, with no measurable damage occurring. Research into UFO events led to the development of a numerical model capable of simulating typical UFO events, reproducing entire Run 1 measured event data sets and extrapolating to 6.5 TeV, predicting the likelihood of UFO-induced magnet quenches. Results provided interesting insights into the involved phenomena as well as confirming the possibility of UFO-induced magnet quenches. The model was also capable of predicting that such events, if left unaccounted for, are likely to be commonplace or not, resulting in significant long-term issues for 6.5+ TeV operation. Addressing the thesis hypothesis, the following written works detail the development and results of all CSCM qualification tests and subsequent magnet training as well as the development and simulation results of both 4 TeV and 6.5 TeV UFO event modelling. The thesis concludes, post-LS 1, with the LHC successfully sustaining 6.5 TeV proton beams, but with UFO events, as predicted, resulting in otherwise uninitiated magnet quenches and being at the forefront of system availability issues.