Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.     

THREE-DIMENSIONAL ULTRASTRUCTURE OF THE CRAYFISH NEUROMUSCULAR APPARATUS

The synapse-bearing nerve terminals of the opener muscle of the crayfish Procambarus were reconstructed using electron micrographs of regions which had been serially sectioned. The branching patterns of the terminals of excitatory and inhibitory axons and the locations and sizes of neuromuscular and axo-axonal synapses were studied. Excitatory and inhibitory synapses could be distinguished not only on the basis of differences in synaptic vesicles, but also by a difference in density of pre- and postsynaptic membranes. Synapses of both axons usually had one or more sharply localized presynaptic "dense bodies" around which synaptic vesicles appeared to cluster. Some synapses did not have the dense bodies. These structures may be involved in the physiological activity of the synapse. Excitatory axon terminals had more synapses, and a larger percentage of terminal surface area devoted to synaptic contacts, than inhibitory axon terminals. However, the largest synapses of the inhibitory axon exceeded in surface area those of the excitatory axon. Both axons had many side branches coming from the main terminal; often, the side branches were joined to the main terminal by narrow necks. A greater percentage of surface area was devoted to synapses in side branches than in the main terminal. Only a small fraction of total surface area was devoted to axo-axonal synapses, but these were often located at narrow necks or constrictions of the excitatory axon. This arrangement would result in effective blockage of spike invasion of regions of the terminal distal to the synapse, and would allow relatively few synapses to exert a powerful effect on transmitter release from the excitatory axon. A hypothesis to account for the development of the neuromuscular apparatus is presented, in which it is suggested that production of new synapses is more important than enlargement of old ones as a mechanism for allowing the axon to adjust transmitter output to the functional needs of the muscle.     

Innervation and vascular supply of the crayfish opener muscle: Scanning and transmission electron microscopy

Summary Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were employed to study the innervation and vascular supply of crayfish skeletal muscle.Blood vessels and nerve terminals identified by TEM were often closely associated. Synaptic regions of the nerve terminals were always located under sarcolemma and contained both dense-cored and agranular synaptic vesicles. Axo-axonal synapses of several different types were observed. Blood vessels consisted of several vessel cells or supporting cells enclosing a lumen, which was connected to the exterior by fine channels between the supporting cells.SEM of whole freeze-dried muscles revealed two types of ramifying structure, which often ran in parallel over the muscle surface. One, identified as nerve, was more cylindrical and had a smoother surface than the other, which was identified as blood vessel. Fine nerve branches disappeared under the sarcolemma, probably near synaptic regions, but synapses could not be seen. Blood vessels also had fine terminations which merged into the sarcolemma.Supported by grants from the National Research Council of Canada and The Muscular Dystrophy Association of Canada. The technical assistance of Mr. M. Uy is acknowledged. Dr. F. Lang held a Postdoctoral Fellowship from the Muscular Dystrophy Association of Canada. Acknowledgement is made for the use of the scanning electron microscope in the Royal Ontario Museum, established through a grant from N.R.C. to the Department of Zoology, University of Toronto for the development of a program in systematic and evolutionary Zoology.     

Circadian changes in salivary constituents and conductivity in women and men.

Circadian rhythms in salivary [glucose], [Na+], [K+] and conductivity were measured in 2 age groups of men (men A, 20-45 years and men B, 46-60 years) and 8 different states of fertility in women (normally menstruating, taking oral contraceptives, pregnant, lactational amenorrhea, lactational amenorrhea and taking oral contraceptives, lactating and menstruating, menopausal, and post-menopausal). Unstimulated whole saliva (2-3 ml) was collected every 3 h over a 48 h span. Analysis of Spearman Rank Correlations indicated significant circadian rhythms (significant positive coefficients) for all groups of [Na+] (mean = 0.577 +/- 0.040) and conductivity (mean = 0.410 +/- 0.050). There was no evidence of differences in prominence of rhythm across groups for [Na+] and conductivity. [K+] showed less evidence of rhythms and much greater variability between groups (mean correlation coefficient = 0.198 +/- 0.055). Rhythms in [glucose] (mean correlation coefficient = 0.409 +/- 0.051) were evident in all groups except men B (0.016), menopausal women (0.151) and post-menopausal women (0.310). Model analysis of the data showed no discernible rhythmic trend with age for either conductivity, [Na+] or [K+], where any differences were explainable by the group characteristics. The rhythm in [glucose] showed a significant weakening with age over all groups (F-ratio = 7.46**), and was different between men A and men B (F-ratio = 6.95**). It was concluded that circadian rhythms were present in whole unstimulated saliva for conductivity and [Na+] and that these rhythms were independent of reproductive state, whereas circadian rhythms in [K+] were dependent on reproductive state. Circadian rhythms for [glucose] were dependent on age. The loss of a rhythm in [glucose] with age indicates that glucose, Na+ and K+ are not linked in their entry into saliva. The influence of entry and reabsorption on the final concentrations of glucose, Na+ and K+ in saliva is discussed.     

The substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase. Analysis of myristic acid analogs containing oxygen, sulfur, double bonds, triple bonds, and/or an aromatic residue

We have explored the acyl-CoA substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by synthesizing 81 fatty acid analogs and surveying their activity in a coupled in vitro assay containing Pseudomonas acyl-CoA synthetase and Escherichia coli-derived yeast NMT. Single oxygen or sulfur substitution for C-3 through C-13 is well tolerated by both enzymes. Detailed kinetic analyses suggest that the acyl-CoA and peptide-binding sites of NMT are relatively insensitive to placement of single group 6B heteroatoms. By contrast, di-oxygen-substituted analogs were very poor substrates, producing dramatic reductions in the affinity of NMTs peptide-binding site for a synthetic octapeptide substrate derived from the NH2-terminal sequence of a known N-myristoylprotein, the gag poly-protein precursor of human immunodeficiency virus 1 (HIV-1). This observation provides an example of binding site cooperativity in NMT. Replacement of one oxygen with sulfur at either the 6, 9, or 12 position of dioxatetradecanoic acids results in a general increase in peptide catalytic efficiency (Vmax/Km). An analysis of five fatty acids from octanoic to dodecanoic having terminal phenyl groups indicated that the best substrate was 10-phenyldecanoic acid even though Corey-Pauling-Koltun molecular models indicate that it has a length equivalent to that of tridecanoic acid. Six analogs having an equivalent length of 13 carbon atoms were subsequently prepared in which the phenyl group was systematically moved one methylene group closer to carboxyl. Movement of the phenyl just one carbon closer to carboxyl (producing 9-(p-methylphenyl) nonanoic acid) decreases peptide catalytic efficiency (Vmax/Km) severalfold compared to 10-phenyldecanoic acid. 10-(4-Tolyl)decanoic acid has the same relative positions of phenyl and carboxyl as 10-phenyldecanoic acid even though a methyl group is present on the phenyl ring. It produces peptide Km and Vmax values that are the same as 10-phenyldecanoic acid. Substitution of either oxygen or sulfur for a methylene group fails to override the effects noted when the phenyl group position is altered in the C-14 equivalent fatty acid series. Several fatty acids of differing chain lengths with cyclohexyl-, 2-furyl, and 2-thienyl groups at their omega termnius had activity profiles that paralleled those of the comparable phenyl-substituted compounds. Myristic acid analogs with triple bonds (beginning at positions 2 through 13), cis-double bonds (positions 3 through 13) and trans-double bond isomers (E5, E6, and E7) were also tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane Potentials,Synaptic Responses,Neuronal Circuitry,Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell''s membrane potential and which we address in these experiments are: (1) Ion concentration of K⁺ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.