The Royal Society

By using the patch–clamp technique the effect of 2-decenoic acid (DA) on Ca ²⁺ -activated potassium (K ⁺ ) channels in the membrane of smooth muscle cells from the human aorta was studied. In the presence of 0.5 μM Ca ²⁺ and 2 mM Mg ²⁺ on the cytoplasmic side of the membrane, a more than tenfold elevation in the probability of the channels being open ( p _o ) was observed under the effect of DA. With divalent cation concentrations of less than 1 nM DA caused a more than twofold elevation in p _o . In the DA-treated membranes Mg ²⁺ ions, which normally fail to activate the channels, brought about a nearly threefold increase in the channel activity when applied to the inner membrane surface. Channel sensitivity to the activating effect of cytoplasmic Ca ²⁺ ions did not increase with the application of DA. Single-channel conductance was unchanged by DA exposure. We suggest that DA alters the Ca ²⁺ -binding mechanism of the channel, increasing its sensitivity to Mg ²⁺ ions, presumably owing to membrane fluidization.

The new antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), which blocks responses to kainate and quisqualate, has been used in conjunction with D-2-amino-5-phosphonovalerate (APV), which blocks selectively responses to N -methyl-D-aspartate (NMDA), to determine the role of excitatory amino acid receptors in synaptic transmission. An excitatory postsynaptic potential (EPSP) – inhibitory postsynaptic potential (IPSP) sequence was evoked in CA1 neurons by stimulation of the Schaffer collateral–commissural pathway in rat hippocampal slices. CNQX (10 μm) substantially reduced the EPSP without having any effect on input resistance or membrane potential. The IPSP was also reduced provided that the stimulating electrode was place approximately 1 mm from the recording electrode. The EPSP that remained in the presence of CNQX had characteristics of an NMDA receptor-mediated potential; it had a slow timecourse, summated at high frequencies, was blocked reversibly by APV, increased greatly in size in Mg ²⁺ -free medium. and showed an anomalous voltage dependence in Mg ²⁺ -containing medium. In the presence of CNQX, an APV-sensitive polysynaptic GABAergic IPSP could be evoked, indicating that NMDA receptors can mediate suprathreshold EPSPS in inhibitory interneurons. It is suggested that either NMDA or non-NMDA receptors can, under different circumstances, mediate the synaptic excitation of pyramidal neurons and inhibitory interneurons in area CA1 of the hippocampus.

By means of electron microscopy applied to wild material prepared as dry whole mounts, descriptions are given of external morphology, including lorica construction, of four new species of collared flagellates, namely: Pleurasiga caudata, Salpingoeca longicaudata, Parvicorbicula serrulata and Diaphanoeca aperta . Reasons are given for the choice of generic names although future changes in some generic boundaries are expected. Aspects of the ecology and geographical distributions are discussed in a preliminary way.

On the basis of wild material processed into dry who mounts immediately following collection in two arctic localities (Hudson Bay and under sea ice in North Alaska), new insight has been obtained into lorica structure and development in Diaphanoeca grandis (Choanoflagellata) by means of scanning electron microscopy supplementing transmission electron microscopy and light microscopy. The more important new findings include demonstration of tectiform replication and of the two-layered nature of the lorica wall, the outer layer being limited throughout to longitudinal costae only. Details of transverse costae and of the fibrillar or membranous components involved in suspension of the protoplast are also described and illustrated. Outstanding problems, notably those involved in nutrient uptake, are discussed and the nature of the observations most needed to resolve them indicated. A revised description of the species replaces a summary.

The chemical evidence for the enzymic activity of lysozyme will be discussed in detail by other speakers at this meeting, but in order to describe our crystallographic studies of the interactions between the enzyme and its substrates it is necessary to summarize briefly what was known about them at the beginning of our work. Simultaneously with his discovery of lysozyme Fleming (1922) discovered a Gram-positive species of bacteria, Micrococcus lysodeikticus , which is particularly susceptible to the action of the enzyme. It was not until much later, however, that Salton (1952) demonstrated that the substrate is located entirely within the bacterial cell wall and it is only very recently that its chemical constitution has been established. Valuable early experiments (for example, by Meyer, Palmer, Thomson & Khorazo 1936; Meyer, Hahnel & Steinberg 1946; and by Epstein & Chain 1940) showed that lysozyme releases N -acetyl-amino sugars from M. lysodeikticus , but the first indication of the type of linkage attacked by lysozyme came when Berger & Weiser (1957) showed that lysozyme also degrades chitin, the linear polymer of N -acetylghicosamine.

It was previously shown that the addition of cytochrome c to a heart-muscle preparation greatly increases its power of catalysing the oxidation of such substances as p -phenylene diamine, hydroquinone, cysteine and ascorbic acid. It was demonstrated that the oxidations of these substances, which can be used for the detection and estimation of intracellular oxidase, are catalysed not directly by the oxidase but through the co-operation of cytochrome. The only direct function of the oxidase, so far ascertained, is the oxidation of reduced cytochrome, and the enzyme can therefore be considered as cytochrome oxidase (Keilin and Hartree 1938 a ). Several properties which have been previously ascribed to it do not complete cytochrome-oxidase system. The object of this paper is the study of the mechanism of oxidation and reduction of cytochrome in order to determine the properties and the nature of cytochrome oxidase.

Retinoic acid (vitamin A acid), the carboxylic acid corresponding to the primary alcohol retinol (vitamin A), has previously been thought to fulfil all the functions of vitamin A except in vision, since rats fed a diet deficient in retinol but supplemented with retinoic acid grow well, outwardly appearing healthy, yet become blind. This paper reports that female rats on such a diet had normal oestrous cycles and became pregnant when mated, but always resorbed the foetuses and no litters were born. The first abnormalities detected were necrosis and slight polymorph infiltration around the periphery of the placental disk about the sixteenth day of pregnancy. Supplementation with retinol as late as the tenth day resulted in the birth of a healthy litter. Retinoic acid therefore maintained the early but not the later stages of gestation. When very small amounts of retinol were given during pregnancy, dead or weak young were born; on higher supplements of the vitamin, litters were weaned successfully. By this means young rats were produced with negligible stores of retinol. Male rats fed retinoic acid but not retinol had small and often oedematous testes. The germinal epithelium sloughed off and in some tubules the lumen was obliterated, but in others the lumen remained, and in these some spermatocytes and spermatogonia were held tenaciously. The seminal vesicles were smaller than in controls given retinol. In rats born with negligible stores of retinol—see above—and maintained on retinoic acid, the testes remained infantile; spermatids were never formed. Feeding retinol restored spermatogenesis in degenerate testes and promoted the normal development of testes that had remained infantile; it also ensured the growth of the seminal vesicles. Retinoic acid did not therefore serve in reproduction, although it replaced the true vitamin in maintaining life, growth and general health. Besides the latter so-called systemic function, vitamin A must have a discrete and specific role in reproduction, viz. that performed by retinol but not by retinoic acid. From among the many previously reported features of disordered reproduction in vitamin A-deficient animals, it was possible to distinguish which had arisen from a failure of this specifically ‘reproductive’ role and which from a ‘systemic’ deficiency. The inactivity of retinoic acid in reproduction demonstrates that in rats vitamin A has not two, as previously thought, but three dissociable modes of action: (1) systemic; (2) in vision; and (3) in reproduction.

Computer-controlled operant-conditioning training procedures were used to raise (up-learning) or lower (down-learning) the mean frequency of discharge of the anterior adductor coxa motoneurone of the locust Schistocerca gregaria . Intracellular recordings were made from the soma of the motoneurone during training. The neurone appeared capable of spontaneous discharge in the absence of synaptic input since its mean pacemaker rate was measured after blocking synaptic inputs by infusing high Mg ²⁺ /zero Ca ²⁺ saline into the neuropile associated with the neurone. Rates were determined before and after the training procedure was applied. It was found that a stable increase in the mean frequency of the pacemaker occurred during up-learning and a decrease during down-learning. The pacemaker shift accounted for a little over half the overall learning change. The remainder was attributed to changes in the activities of interneurones that directly, or indirectly, affect the motoneurone pacemaker. Conventional synaptic potentials that could have accounted for the remainder were not conspicuous in the soma recordings.

Potentials in the tectum of large (12─20 cm) goldfish, evoked by stimulation of the optic nerve, were recorded extracellularly with double-barrelled electrodes (d. c., saline and a. c., Woods metal─Pt). Four fibre groups (E, M ₁ , M ₂ , M ₃ ) were recorded at latencies of approximately 2, 3, 5 and 8 ms after stimulation (conduction velocities of approximately 7, 5, 3 and 2 m/s). The same four groups were recorded from the optic nerve when the tectum was stimulated. The fastest fibre group (E) did not give rise to a postsynaptic wave. Fibre groups M ₁ , M ₂ and M ₃ gave rise to postsynaptic potentials which, following computation of their second spatial derivatives with depth, were found to have current sinks at depths of approximately 100─50 μm, 150─200 μm and 250-350 μm respectively. Thus the fastest conducting retinotectal fibres make their synapses most superficially, the opposite of the arrangement in the frog tectum. These postsynaptic waves fatigued at repetitive stimulus rates of 20─50 per second, and in twin pulses at interstimulus intervals of 10─15 ms; and they were reversibly blocked by topical application of pentobarbitol. The fibre potentials, however, were virtually undecremented under these conditions. To compare these electrophysiological findings with the anatomy, the cobalt procedure was used to visualize the profiles of the optic fibres in the various tectal laminae. A thick dense projection filled the superficial grey and white (s. g. w.) layer, and there was a thin satellite band just superficial to it. In addition, there were two deeper bands of sparse innervation, in the middle of the central grey zone (c. g.) and in the deep white (d. w.) layer. These bands were associated with the field potential sinks through lesions made with recording electrodes. The two deep bands correspond to the M ₃ fibre group. The dense s. g. w. innervation contains both the M ₁ and M ₂ fibre groups, the M ₁ just superficial to the M ₂ . The fastest fibre group, E, which had no postsynaptic wave associated with it, persisted at least six weeks after retinal removal, and probably represents efferent cells with fibres projecting back through the optic nerve to the retina. Filled cell profiles could not be positively identified with the cobalt technique, but could be seen with the HRP technique, when the optic afferents were first allowed to degenerate. The filled cells were the pyramidals of the s. g. w. layer.

Interplexiform cells are a class of retinal neuron that extends processes widely in both plexiform layers. In goldfish they contain dopamine and readily take up certain biogenic amines. Two of these amines, 6-hydroxyopamine (6-HDA) and 5, 6-dihydroxytryptamine (5,6-DHT), induce fine structural changes in the neurons that accumulate them, allowing the processes of the cells to be recognized by electron microscopy. Typically, the synaptic vesicles within the processes show electron-dense cores. The terminal cytoplasm may also show increased density, as may the cellular and cytoplasmic membranes, presumably an indication of degenerative changes induced by the drugs. 5, 6-DHT gives more readily observable changes than 6-HDA but labels both dopaminergic and indoleamine-accumulating neurons. The terminals of the indoleamine-accumulating terminals were therefore removed by intraocular injections of 5, 7-dihydroxytryptamine (5, 7-DHT) prior to the labelling with 5, 6-DHT. This procedure allowed an analysis of the dopaminergic terminals without interference by the terminals of the indoleamine-accumulating cells. The dopaminergic neurons were found to make synapses of the conventional type. In the outer plexiform layer they contacted both external horizontal cells and bipolar cell dendrites, but not hotoreceptor terminals or intermediate (rod) horizontal cells. No synapses onto the dopaminergic processes were found in the outer plexiform layer despite an extensive search. In the inner plexiform layer the dopaminergic processes were observed to be both pre- and postsynaptic to amacrine cells and their processes. No synaptic contacts between dopaminergic processes and bipolar cell terminals or ganglion cell dendrites were seen. We conclude that the dopaminergic interplexiform cells provide a centri­fugal pathway for information flow in the retina from inner to outer plexiform layer.