Rosemary L Martin

Reduction by general anaesthetics of group Ia excitatory postsynaptic potentials and currents in the cat spinal cord.

1. The effects of thiopentone and halothane on excitatory synaptic transmission at group Ia afferent synapses on lumbosacral motoneurones were studied in the anaesthetized or decerebrate cat. 2. Thiopentone (10 mg kg‐1) infused on a background of light pentobarbitone anaesthesia caused a decrease in single‐fibre monosynaptic group Ia excitatory postsynaptic potentials (EPSPs) of between 0 and 24%. A step increase in inspired halothane concentration in the range 0.7‐0.9% produced a decrease in EPSP amplitude of between 0 and 31%. These effects were reversible when the anaesthetic level was reduced. 3. Fluctuation analysis of selected single‐fibre group Ia EPSPs revealed that these effects could be accounted for by a decrease in the probability of occurrence of EPSPs of larger amplitude, and an increase in the probability of occurrence of EPSPs of smaller amplitude. The mean separation between discrete amplitudes was not altered by either anaesthetic agent. 4. EPSPs whose time course indicated a somatic site of origin were voltage clamped to study the effect of the anaesthetics on the time course of the synaptic currents. Neither thiopentone nor halothane produced a consistent effect on the time constant of decay of the current, although they both depressed its peak amplitude. 5. The results are interpreted as indicating a presynaptic site of action of both anaesthetics at the concentrations studied: the probability of release of neurotransmitter is reduced, without any detectable change in the mean duration of the postsynaptic conductance increase. These findings are discussed in relation to the mechanisms of action of anaesthetics on exocytosis and presynaptic inhibition.

A pharmacological and biochemical investigation of the venom from the platypus (Ornithorhynchus anatinus)

In this study several activities of the venom of Ornithorhynchus anatinus have been investigated. Whole venom induced local oedema after subplantar injection and produced relaxation of the rat uterus in vitro. The relaxant activity was partially purified by gel permeation HPLC and subsequent analyses by SDS-PAGE revealed that this activity was associated with a 4200 mol. wt peptide. The N-terminal partial sequence of this peptide exhibited substantial identity with human and porcine C-type natriuretic peptide (CNP). Three other major proteins isolated from the venom had mol. wts of 140,000, 55,000 and 16,000. None was found to have any sequence homology with proteins listed in the SwissProt database. The 140,000 mol. wt protein exhibited hyaluronidase activity but the nature of the 55,000 and 16,000 mol. wt proteins remains to be determined. Platypus venom also exhibits protease activity, although the concentration of proteolytic enzymes was too low to be visualised by SDS-PAGE using Coomassie staining.

The natriuretic peptide (ovCNP-39) from platypus (Ornithorhynchus anatinus) venom relaxes the isolated rat uterus and promotes oedema and mast cell histamine release.

In this study we characterise the ability of a C-type natriuretic peptide from platypus (Ornithorhynchus anatinus) venom (ovCNP-39) to relax the rat uterus in vitro and we investigate the possibility that ovCNP-39 contributes to the acute effects of envenomation, which include oedema, pain and erythema. We have found that both ovCNP-39 and the endogenous C-type natriuretic peptide, CNP-22, produce oedema in the rat paw and release histamine from rat peritoneal mast cells. Two synthetic peptides, ovCNP-39(1-17) and ovCNP-39(18-39), corresponding to the N- and C-termini, respectively, are equipotent histamine releasers, suggesting that ovCNP-39 and other natriuretic peptides do not act through conventional natriuretic peptide receptors on mast cells.

Ionic basis of membrane potential changes induced by anoxia in rat dorsal vagal motoneurones.

1. The effects of anoxia on membrane properties of 119 dorsal vagal motoneurones (DVMs) were investigated in an in vitro slice preparation of the rat medulla. 2. Membrane potential was unaffected by anoxia in 11% of DVMs. An hyperpolarization accompanied by a decrease in input resistance occurred in 44% of DVMs; the remaining 45% depolarized with either an increase (60%) or decrease in input resistance (40%). TTX at a concentration of 0.3‐1 microM did not significantly affect these responses. 3. Anoxic artificial cerebrospinal fluid (ACSF) containing 20 mM‐TEA reversed the response of DVMs that hyperpolarized in standard ACSF to reveal a depolarization of 7.4 +/‐ 2.1 mV, and increased the anoxic depolarization from 5.0 +/‐ 0.7 to 8.7 +/‐ 1.4 mV. 4. Anoxic depolarization was converted to an hyperpolarization of 7.3 +/‐ 2.1 mV in ACSF containing 5 mM‐4‐aminopyridine (4‐AP) and 1 microM‐TTX. A residual depolarization of 4.5 +/‐ 3.5 mV was then observed in ACSF containing 5 mM‐4‐AP, 1 microM‐TTX and 20 mM‐TEA. Anoxic hyperpolarization was increased from 7.8 +/‐ 1.8 to 10.0 +/‐ 3.9 mV in 5 mM‐4‐AP and 1 microM‐TTX and converted to a depolarization of 5.3 +/‐ 4.5 mV in 5 mM‐4‐AP, 1 microM‐TTX and 20 mM‐TEA. 5. In anoxic ACSF containing TEA, the action potential width was increased from 0.92 +/‐ 0.04 to 8.1 +/‐ 1.1 ms in hyperpolarizing DVMs, and from 0.85 +/‐ 0.01 to 2.4 +/‐ 1.0 ms in depolarizing DVMs. The increase in width was prevented by 2‐3 mM‐Mn2+. 6. The long after‐hyperpolarization (AHP) of DVMs, which is contributed to by both an apamin‐sensitive IK(Ca) and an apamin, charybdotoxin and TEA insensitive IK(Ca) was decreased in duration from 2.59 +/‐ 0.14 to 1.94 +/‐ 0.12 s during anoxia. 7. It is concluded that anoxia enhances the delayed rectifier current (IK(DR)) and an inward current, probably ICa, but suppresses the A currents (IA). In DVMs that hyperpolarize during anoxia, the increase in IK(DR) outweighs the increase in ICa and the decrease in IA. In depolarizing DVMs the decrease in IA and increase in ICa outweight the increase in IK(DR). The change in input resistance is determined by the relative sizes of current enhancement or suppression.

Changes in Membrane Potential and the Intracellular Calcium Concentration During CSD and OGD in Layer V and Layer II/III Mouse Cortical Neurons

Cortical spreading depression (CSD) is an episode of electrical silence following intense neuronal activity that propagates across the cortex at ∼3-6 mm/min and is associated with transient neuronal depolarization. CSD is benign in normally perfused brain tissue, but there is evidence suggesting that repetitive CSD contributes to infarct growth following focal ischemia. Studies to date have assumed that the cellular responses to CSD are uniform across neuronal types because there are no data to the contrary. In this study, we investigated the effect of CSD on membrane potential and the intracellular calcium concentration ([Ca(2+)](i)) of mouse layer V and layer II/III pyramidal neurons in brain slices. To place the data in context, we made similar measurements during anoxic depolarization induced by oxygen and glucose deprivation (OGD). The [Ca(2+)](i) was quantified using the low-affinity ratiometric indicator Fura-4F. During both CSD- and OGD-induced depolarization, the membrane potential approached 0 mV in all neurons. In layer V pyramids OGD resulted in an increase in [Ca(2+)](i) to a maximum of 3.69 ± 0.73 (SD) μM (n = 12), significantly greater than the increase to 1.81 ± 0.70 μM in CSD (n = 34; P < 0.0001). Membrane potential and [Ca(2+)](i) returned to nearly basal levels following CSD but not OGD. Layer II/III neurons responded to CSD with a greater peak increase in [Ca(2+)](i) than layer V neurons (2.88 ± 0.6 μM; n = 9; P < 0.01). We conclude there is a laminar difference in the response of pyramidal neurons to CSD; possible explanations are discussed.

Block of rapid depolarization induced by in vitro energy depletion of rat dorsal vagal motoneurones

1 The ionic mechanisms contributing to the rapid depolarization (RD) induced by in vitro ischaemia have been studied in dorsal vagal motoneurones (DVMs) of brainstem slices. Compared with CA1 hippocampal neurones, RD of DVMs was slower, generally occurred from a more depolarized membrane potential and was accompanied by smaller increases in [K+]o. 2 RD was not induced by elevation of [K+]o to values measured around DVMs during in vitro ischaemia or by a combination of raised [K+]o and 2–5 μM ouabain. 3 Neither TTX (5–10 μM) nor TTX combined with bepridil (10–30 μM), a Na+‐Ca2+ exchange inhibitor, slowed RD. Block of voltage‐dependent Ca2+ channels with Cd2+ (0.2 mM) and Ni2+ (0.3 mM) led to an earlier onset of RD, possibly because [K+]o was higher than that measured during in vitro ischaemia in the absence of divalent ions. 4 When [Na+]o was reduced to 11.25–25 mM, RD did not occur, although a slow depolarization was observed. RD was slowed (i) by 10 mM Mg2+ and 0.5 mM Ca2+, (ii) by a combination of TTX (1.5–5 μM), 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 μM) and D‐2‐amino‐5‐phosphonovalerate (AP5, 50 μM) and (iii) by TTX (1.5–5 μM) and AP5 (50 μM). 5 Ni2+ at concentrations of 0.6 or 1.33 mM blocked RD whereas 0.6 mM Cd2+ did not. A combination of Cd2+ (0.2 mM), Ni2+ (0.3 mM), AP5 (50 μM) and bepridil (10 μM) was largely able to mimic the effects of high concentrations of Ni2+. 6 It is concluded that RD is due to Na+ entry, predominantly through N‐methyl‐D‐aspartate receptor ionophores, and to Ca2+ entry through voltage‐dependent Ca2+ channels. These results are consistent with known changes in the concentrations of extracellular ions when ischaemia‐induced rapid depolarization occurs.

Development of basic medical sciences in a new medical school with an integrated curriculum: The ANU experience

Background: The development of a basic medical science curriculum in a new medical school with a problem-based focus in Australia has been subject to a number of constraints. We describe the process and early evaluation. Aim: To describe the development of a basic medical science curriculum in an Australian medical school with a problem-based curriculum. Methods: We describe the process we used for curriculum development and the benefits and constraints that arose from pre-existing strong biomedical science on the Australian National University (ANU) campus. We outline methods we used to inform our curriculum content and report on accreditation and early internal evaluation. Results: Australian medical schools design their curriculum within a relatively restrictive framework put forward by a national accreditation system. The curriculum achieved accreditation from the external accrediting agency, but early student evaluation has been mixed. Conclusion: Although our internal faculty evaluation and external review by the accrediting agency has supported the view that this aspect of the curriculum has performed reasonably well, student feedback is mixed and further evaluation is needed and adjustments probably warranted.

Experimental neuronal protection in cerebral ischaemia part I: experimental models and pathophysiological responses.

Development of neuroprotective strategies for the treatment of cerebral ischaemia depends upon knowledge of the pathophysiological responses which occur during the period of ischaemia and during reperfusion. Both in vitro and in vivo studies have been used to accumulate this base of knowledge; a brief examination of the results from the two approaches reveal that they are in general agreement and often provide complementary information. Ischaemia in the absence of reperfusion can lead to neuronal death from protein degradation and DNA breakup. Resupply of blood permits increased free radical attack but energy supply can still be limited as the cerebral circulation reacts to release of cicosanoids, and DNA repair utilizes ATP.

Experimental neuronal protection in cerebral ischaemia Part II: Potential neuroprotective drugs

The search for agents that could protect neurons from ischaemia-induced death has largely been based upon knowledge of the pathophysiological events that cause neuronal damage. Thus, the main classes of drugs that have been tested in animal experiments include ion channel blockers, those targeting neurotransmitter receptors, calpain inhibitors, inhibitors of enzymes in the arachidonic acid cascade, antagonists of platelet-activating factor, antioxidants and antagonists of inflammatory cytokines. The therapeutic potential of neurotrophins, growth factors and gangliosides has also been explored. As this review indicates, nearly all of these drugs have demonstrable neuroprotective capabilities. Broad spectrum neuroprotective drugs appear to have more potential than specifically targeted drugs, a finding which is in keeping with the multifactorial nature of ischaemia-induced neuronal death.