PROCEEDINGS OF THE PHYSIOLOGICAL SOCIETY GLASGOW MEETING
16-17 September 1977 DEMONSTRATIONS The construction of implantable strain-gauge transducers By V. RAYNER. Rowett Re8earch Institute, Buck8burn, Aberdeen AB2 9SB Bass & Wiley (1972) described the construction of implantable transducers by bonding strain gauges to beryllium-copper strips. These transducers have been used to study stomach motility in the dog (Walker, Stewart & Bass, 1974). But satisfactory transducers can be made by embedding strain gauges in Araldite.
5N _-**|l I ~~~~~0 I min time trace I _~ I| 1 .. J| A1
Fed 575 g
Fig. 1. Contractions (T) and electrical activity (E) of the stomach of a pig. The transducer was aligned to record from the circular muscle layer 5 cm from the pylorus. The bipolar nichrome wire electrodes were nearby. Before feeding, contractions occurred in bursts. On feeding, continuous rhythmic contractions occurred at the frequency of the electrical control activity.
Each etched foil strain gauge (Phillips 9833K/03FE) was embedded on the upper surface of an Araldite block (MY753 + HY956 hardener) with the two terminals bent so that they were deeply embedded in the block.
2P PROCEEDINGS OF THE 2P The block was cut down to 20 x 6 x 4 mm. The terminals were exposed from the underside of the block. Two wires (Radiospares 7/0.2 mm) were soldered to one terminal and one to the other. Strips of silicone rubber sheet (Silastic 501-3) were fixed in grooves cut at either end with stainlesssteel pins. These pins and the wire-terminal junctions were covered with Araldite. The completed gauge was given a final coating of silicone rubber (Silastic Medical Adhesive type A). Gauges were wired as one arm of a balanced Wheatstone bridge using the three-wire system to remove drift caused by temperature changes. The bridge excitation potential was 1 6 V. A bending moment applied to the transducer caused a change in resistance. The subsequent change in potential across the bridge circuit was shown on a DC pen recorder. To calibrate, weights (2-500 g) were hung from one end of the transducer. There was a linear relationship between the force applied and the pen deflexion. Gauges have been used for the study of stomach motility in the pig (Rayner, 1977). The silicone rubber sheet at either end of the gauge was sewn to the serosa of the stomach. Contractions occurred at the frequency of the electrical control activity recorded by nichrome wire electrodes. At each contraction action potentials were also recorded from the electrodes
Gauge life was 1-3 months. They usually failed because body fluids leaking into the gauge corroded the soldered joints. REFERENCES BAs, P. & Wm-By, J. N. (1972). J. apple. Phyaiol. 32, 567-570. RAYNER, V. (1977). J. Phyaiol. 273, 73-74P. WALxER, G. D., STEWART, J. J. & BAss, P. (1974). Ann. Surg. 174, 853-858.
Relative potency of prostacyclin, prostaglandin E1 and D2 as inhibitors of platelet aggregation in several species By S. MONCADA, J. R. VANE and B. J. R. WHITTLE. Wellcome Re8earch Laboratories, Langley Court, Beckenham, Kent BR3 3BS. Prostacyclin (prostaglandin I'2 PGI2) is the most potent inhibitor of human platelet aggregation in vitro so far described (Moncada, Gryglewski, Bunting & Vane, 1976), and like prostaglandin D2 (PGD2) and PGE1 it elevates platelet cyclic AMP levels (Tateson, Moncada & Vane, 1977). We have now investigated whether the effects of these prostaglandins are mediated through activation of a common platelet receptor. Blood was freshly collected into trisodium citrate (3-15 %, w/v; 0-1 vol.) and centrifuged (200 g for 15 min). Inhibition of platelet aggregation was
3P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 determined in an aggregometer by incubating aliquots (0.5 mI.) of the platelet-rich plasma (PRP) with the prostaglandin for 1 min prior to the addition of sufficient adenosine diphosphate (ADP, 0-5-2 /Sm) to cause 'irreversible' aggregation. Prostacyclin was the most potent inhibitor of platelet aggregation in the PRP of all species tested (Table 1). PGD2 was a weak inhibitor of platelet aggregation in rabbit or rat PRP whilst both PGE1 and prostacyclin were highly active. Thus, the sensitivity ofthe PRP from the different species to PGE1 followed more closely the sensitivity to prostacyclin than to PGD2. We have also found that low doses of the compound N-0164 (100-200 utm), which abolish PGD2-induced inhibition of human platelet aggregation without any significant effect on PGE1 inhibition (MacIntyre, 1977), did not antagonize the inhibition by prostacyclin. Higher concentrations of N-0164 (1-2 mM) did, however, antagonize the inhibition with PGE1 or prostacyclin. TABLr 1. Inhibition of ADP-induced platelet aggregation in different species by prostaglandins. The results show the concentration of PGI2 required to cause complete inhibition of aggregation and the relative potencies of PGE1 and PGD, (means of 5-10 determinations) Relative potency ConcentraA tion of PGI, PGE1 (nm) Species PGI2 PGD2 26-4 1 0-14 0.5 Sheep 0-4 Horse 25.2 1 0.11 0-02 0.05 1 Human 3-6 > 0-0001 Rat 0-09 1 7*5 1 0-002 Rabbit 6-9 0-06
These results suggest that prostacyclin and PGE1 act on the same or similar sites on platelets, which differ from those for PGD2 and extend the proposal that PGE1 and PGD2 act at separate receptors on a common adenylate cyclase (Mills & Macfarlane, 1974). Interestingly, in species in which PGD2 has little anti-aggregating activity, the conversion by plasma protein of the endoperoxide PGH2 to PGD2 is also low (Hamberg & Fredholm, 1976), whereas in those species in which PGD2 is highly active (e.g. sheep) there is a greater conversion by the plasma protein. It is, therefore, possible that in some species, PGD2 formed by conversion of endoperoxides released by platelets may play an additional role to the more potent prostacyclin which is generated by the vascular wall (Moncada et al. 1976), as an endogenous anti-aggregating agent.
PROCEEDINGS OF THE REFERENCES
HffA uRGa M. & FREDHOLM, B. B. (1976). Biochim. biophys. Ads 431, 189-193. MACINTYR, D. E. (1977). Br. J. Pharmac. 60, 293-294P. MLLs, D. C. B. & MAcFARLANE, D. E. (1974). Thromboei8 Re8. 5, 401-412. MONCADA, S., GRYGLEWsKI, R., BUNTNG, S. & Wm, J. R. (1976). Nature, Lond. 263, 663-665. TATESON, J. E., MONCADA, S. & VANE, J. R. (1977). Pro8taglandinm 13, 389-397.
Technique for studying interactions between ahemeral light cycles, egglay and calcium metabolism in Japanese quail BY C. G. DACKE. Department of Phy8iology, Mari8chal College, Univer8ity of Aberdeen, Aberdeen AB9 lAS Ahemeral light cycles (i.e. other than 24 hr) have been used extensively for studying the regulation of ovulation cycles in chickens (Gallu8 domedticus). In a recent study Morris (1974) demonstrated significant effects of long (27-30 hr) cycles on egg production, egg weight and eggshell thickness in the chicken. Japanese quail (Coturnix coturnix japonica), although physiologically similar to chickens, have the advantage of small size (about 120 g) and can be conveniently housed in small boxes in which it is simpler to control environmental factors such as temperature and light cycle, than in large rooms. Boxes of a construction suitable for studying the influence of ahemeral light cycles on the ovulation cycle and related parameters in Japanese quail were demonstrated. These are adapted from an original design by B. K. Follett (personal communication) and are of simple construction. The boxes are made of plywood, with internal volume 0-25 ms and can accommodate a total of sixteen adult quail in four separate plastic cages.
Lighting is provided by two 21 in., 13 W fluorescent tubes. Adequate air circulation is maintained by a noise-dampened fan. Light cycles are regulated by two Planet CV timers (Electronic Remote Control Co. Ltd.) linked in tandem to give cycle lengths ranging from a few minutes to 48 hr, with any ratio of light (L) to dark (D) periods. It is found that egglaying birds removed from a stock colony to these boxes require a period of at least 3 weeks before settling back into a regular pattern of egglay on a normal 24 hr cycle. Preliminary experimental data were shown which demonstrate that on moving from a normal (14 hr L, 10 hr D) to an ahemeral (I6j hr L, IIhr I D) cycle-length, Japanese quail displayed a transient period of depression of egg production, returning to normal within about 4 weeks. This suggests
PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 5P that the ovulation pattern undergoes a period of entrainment when the light cycle is changed. Eggshell thickness showed an immediate and significant increase of about 7 % (as measured by eggshell weight per unit area, see Dacke, 1976), which was maintained for several months, until the birds were killed. These results indicate that the technique may be of value in studying interactions between cycle-length and calcium metabolism in the Japanese quail. REFERENCES DACKE, C. G. (1976). J. Endocr. 71, 239-243. MoRm~s, T. R. (1974). Poult. Sci. 52, 423-445.
Measurement of leucocyte locomotion BY J. K. KERR, V. A. Moss, J. A. ROBERTS and H. K. L. SmiiSON. Institute of Phy8iology, The University, Glawgow G12 8QQ A system has been developed to measure the movement, in response to chemical stimuli, of leucocytes isolated from human blood and peritoneal macrophages harvested from the guinea-pig. The distance moved by the leucocytes or macrophages through a micropore filter, whose pores are smaller than the cells being tested, is measured using a technique based on the method of Boyden (1962). The original method was simplified by Wilkinson (1974), who used a chamber made from the sawn-off barrel of a 1 ml. syringe with a disk of micropore filter attached to the end with an inert glue. An incubation unit has been designed with circulating water to control accurately the temperature of incubation of the 200-350 filter units that can be tested with the leucocytes isolated from a 20 ml. sample of blood. The movement of the leucocytes is measured by focusing a microscope through the thickness of each stained filter. The filters are clarified in xylene to permit the cells within the filter to be seen clearly. At least five readings of cell movement must be made on each filter to obtain a sufficiently accurate estimate of cell movement. To facilitate this a potentiometer is coupled to the microscope fine-focus control so that the distance can be displayed as a voltage. The subtraction of the reading for the top surface of the filter from that of the limit of cell movement is carried out electrically using a capacitor to store the first reading. Further expansion of the system is made possible by using a computer to measure and record the data. The computer program is controlled remotely at the microscope by the addition of three push-button controls which read the distance, end the readings on the current filter, and correct for errors. Combinations of these buttons enable other options to be
APPROCEEDINGS OF THE up accessed. The computer also checks the incoming data and warns the operator of errors. In this system the electrical subtraction of voltages is essential to permit the A/D converter to measure accurately small differences. This system has been used to distinguish chemotaxis from chemokinesis by measuring several dose-response relationships simultaneously. It has also been employed to measure differences in leucocyte response between apparently healthy subjects. Using a modified incubation system, to produce a stable temperature gradient, the effect of temperature on the response of leucocytes has been measured. We gatefully acknowledge the financial support given by the Boyd Medical Research Trust and the Scottish Homoeopathic Research and Education Trust. REFERENCES BoYDmw, S. V. (1962). J. axp. Med. 115, 453. WnxrsoN, P. C. (1974). Chemotaxie and Inflammation, p. 168. Edinburgh and London: Churchill Livingstone.
Treadmill apparatus for the electrophysiological analysis of locomotion in crayfish By W. J. P. BARNES and J. A. KIDD. Department of Zoology, University of Glasgow, Glasgow G12 8QQ This demonstration illustrated a treadmill arrangement in which the experimental animal, the crayfish Astaus leptodactylus, is able to walk normally while being sufficiently restrained to allow recordings to be made of nerve and muscle activity. The treadmill consists of a large wheel, 38 cm in diameter, which is completely submerged in water. It is free to turn under the animal as it walks and thus differs from the power-driven treadmills commonly used by workers on cat locomotion and from those used by Macmillan (1975) and Ayers & Davis (1977) in their studies of locomotion in lobsters. The main advantage of this design is that the kinetic energy and hydrodynamic drag of the treadmill when it is turning at the average walking speed of a crayfish are approximately equivalent to the kinetic energy and hydrodynamic drag of a freely walking crayfish. The animal is held over the treadmill by a lever, which is counterbalanced so that the animal is at its normal weight in water. The coupling between animal and lever is flexible, allowing the crayfish a few degrees of movement about all three axes in addition to the movement in the vertical plane permitted by the lever assembly. The experimental animals
7P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 walk readily on the treadmill for long periods of time, using gaits which do not differ significantly from those of free-walking crayfish. So far this apparatus has been used in a study of proprioceptive influences upon motor output during walking (Barnes, 1977), but since an animal whose carapace has been clamped firmly above the treadmill still walks readily, it should be possible to develop this preparation to allow intracellular recordings from the central nervous system to be made from walking animals. Supported by the Alexander von Humboldt Stiftung and the Science Research Council. The initial version of this apparatus was constructed at the University of Konstanz, West Germany. REFERENCES AymEs, J. L. & DAVIs, W. J. (1977). J. cell. comp. Physiol. 115, 1-27. BARNES, W. J. P. (1977). J. Phyeiol. 273, 54-55P. MACMILAN, D. L. (1975). Phil. Trans. R. Soc. B 270, 1-59.
The effect of cooling on neuromuscular transmission in the guineapig vas deferens By A. G. H. BTAKELEY and T. C. CtUNOMmNE. Department of P1armoacology, University of Glasgow, Glasgow G12 8QQ Excitatory junction potentials (EJP) were intracellularly recorded in vitro from the guinea-pig vas deferens. EJP's were evoked by stimulation of the hypogastric nerve, 0 5 cm from the muscle, with trains of ten pulses at 0-91 Hz (pulse width 0-1-2-0 msec). Cell penetrations were accepted if the following criteria were satisfied: (1) the cell penetration was abrupt, (2) the resting membrane potential (RMP) sealed to a more negative value, (3) spontaneous EJP's were recorded, (4) following submaximal hypogastric nerve stimulation the RMP returned to the same or more negative value. At 35 'C the mean RMP was 64-0 + 0 4 mV (n = 120). The amplitude of the first EJP was 5*1 + 0-2 mV (n = 120) and the time to decay to half amplitude 241 + 12 msec (n = 30). The amplitude of the EJP's showed facilitation and reached a plateau value of 17-5 + 0 4 mV (n = 95) by the 8th pulse of the train. At room temperature (20-22 'C) the fraction of cells failing to fulfil the criteria for successful penetration increased. The mean RMP was 60-9 + 0 5 mV (n = 29) but the amplitude of the first EJP was larger, 15-9 + 0-5 mV (n = 29). The time of decay to half amplitude was prolonged, 621 + 18 msec (n = 21). The effects were reversible by rewarming. At room temperature summation of EJP's always occurred with stimulation at 0-91 Hz, and action potentials occurred after two or three stimuli. i8
8P PROCEEDINGS OF THE Mechanical responses of the guinea-pig vas deferens are also potentiated by cooling (Della Bella, Gandini & Preti, 1965; Ambache & Zar, 1971). Kuriyama (1964) found that cooling the vas from 36 to 20 0C decreased the mean RMP and amplitude of the EJP. We have found that at room temperature many more cells failed to seal (criterion 2) after penetration by the micro-electrode. These cells with lower RMPs produce EJPs which are often smaller than control values obtained at 35 00. This effect was demonstrated. REFERENCES
AmBACHE, N. & ZAZ, M. A. (1971). J. Phy-ol. 216, 359-389. DELIA ByTIA, D., GANDHI, A. & PR=TI, M. (1965). J. Pharm. Pharmacol. 17,
KuRIYAmA, H. (1964). J. Phyaiol. 170, 561-570.
Moving vector display for the study of balance and of the reactions to perturbation BY T. D. M. ROBERTS and G. STENHOUSE. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ It has been argued elsewhere (Roberts, 1975, 1977a) that the otolith apparatus is concerned, not with gravity itself, but with the local stress gradients associated with the forces that support the skull and normally prevent it falling under the action of gravity. Accordingly, so long as the skull is not accelerating relative to the trunk, measurement of the changing external forces on the trunk will show what stimulus is being presented to the otolith organs. This makes it possible to assess the role of the labyrinth in initiating those reactions to overbalancing by which the upright posture is maintained. We use a novel technique for displaying the relevant parameters. A subject stands on a force-plate, each end of which rests on a cantilever-beam load-cell (Strainstall Ltd.) whose output is independent of the point of loading along the length of the beam. If the distance between the load-cells is 2a, and their upthrust signals are A and B respectively, then a(A - B)/(A + B) gives the displacement of the resultant from the midpoint and (A + B) gives the magnitude, independent of its position. An additional load-cell gives the horizontal component of thrust. The strain-gauge signals are combined by digital computer (PDP8/I, Digital Equipment Co.) and displayed on an oscilloscope screen as a moving arrow that indicates the magnitude, direction and point of application of the upthrust transmitted from the force-plate to the subject's feet. Additional markers, displayed at arbitrary vertical positions, indicate horizontal excursions of the skull, hips and platform, head displacements being measured by ultrasonic rangefinder to avoid giving proprioceptive cues.
PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 9P Coherent calibrations for horizontal displacements are obtained by rolling a weighted trolley along the force-plate. Calibrations for force depend on the use of a light strut as a jib that stands on the force-plate and is constrained by a horizontal tether to support a weight. The inclination of the jib is measured, and the digital signals for upthrust and for horizontal force are adjusted to be in the appropriate ratio. The platform carrying the force-plate is driven horizontally in a preset pattern of accelerations so chosen that the subject is unable to predict changes in the motion. The recorded data can be stored and replayed at different speeds. The time-courses of the variables can also be displayed in more conventional fashion with links to specific snapshot appearances of the vector display. Some results, on hopping and overbalancing, are presented elsewhere (Roberts, 1977b). REFERENCES ROBERTS, T. D. M. (1975). Chapter 27 in Scientific Foundatiom of Otolaryngology, eds. HIN;CHOLiFE, R. & HBARRisON, D. London: Heinemann. ROBERTS, T. D. M. (1977a). The relationship of gravity to vestibular studies. Life Science Research in Space. Paris: European Space Agency. ROBERTS, T. D. M. (1977b). Neurophysiology of Postural Mechanism, 2nd ed. London: Butterworths.
A simple model of the circulation for students BY K. F. HosIE. Department of Veterinary Phyaiology, Univer8ity of Gaogow, Glagow 012 8QQ The model is intended to demonstrate (a) the relationship between cardiac output, total peripheral resistance and arterial mean blood pressure, and (b) the dependence of capillary pressure on arterial and venous pressures and the pre- to post-capillary resistance ratio. It is constructed as an easily portable permanent assembly and consists of a calibrated variable speed roller pump supplying water to a single circulation. As the output of the pump is constant at a given speed of rotation, Starling's law of the heart is not represented. From a distensible large-bore arterial conduit arise five parallel tubes each of which contains a pair of hydraulic resistors of fixed value. These resistors are bundles of fine-bore tubes and have linear pressure/flow relationships up to a head of at least 250 mmHg. The resistance of the five pairs of resistors have precise values of 2, 3, 4, 5 and 6 arbitrary units and the pre- to postcapillary resistance ratios are 1:1, 2:1, 3:1, 4:1 and 2:1 respectively. Any other resistance values could be simply and accurately obtained by making the resistors of appropriate length. Four of the tubes can be shut 18-2
PROCEEDINGS OF THE lop off or have their resistances increased by means of adjustable clips; the fifth always remains open and of fixed value. All five tubes discharge into a single venous conduit which returns to the pump. Mean pressure in the arterial conduit or between any pair of resistors can be selected by a six-port rotary valve for measurement on a damped mercury manometer. If the pressure exceeds about 200 mmTg the mercury column actuates a latching relay which switches off the pump motor. Re-starting the motor requires manual switching and draws attention to the need to correct maladjustments of pump output (motor speed) and peripheral resistance. No attempt has been made to provide automatic regulation of arterial pressure. Venous pressure is measured by a water manometer which serves also as the venous compliance. Apart from the hydraulic resistors and the arterial and venous manifolds, all of which are easily fabricated, and a minor modification to the motor speed-controller, the components of the model are readily available commercially. Full constructional details are available from the author. A model for demonstrating the measurement of cardiac output by indicator dilution BY K. F. HosrE. Department of Veterinary Physiology, Univer8ity of Gltgow, Glawgow G12 8QQ Water is pumped from a beaker to another by a variable output roller pump through a glass labyrinth made by packing a large pipette bulb with glass beads. The progressive dispersion of a bolus of dye injected upstream is easily seen and the time-course of dye concentration at a downstream site is displayed using a photo-detector and a direct-writing recorder. This arrangement allows comparison of dilution curves produced by a single passage of indicator at different flow rates. Replacement of the beakers by a second labyrinth, so that continuous circulation occurs, allows the effect on the recorded dilution curve of repeated passage of indicator to be shown. By using continuous infusion of dye the model can also be used to demonstrate the Fick principle. Low X-ray background in energy dispersive microanalysis of ultrathin sections BY W. H. BIDDLECOMBE, D. W. DEMPSTER, H. Y. ELDER, W. A. P. NIcHOLSON* and B. W. ROBERTSON.* Department8 of Phygiology and *Natural Philo8ophy, Univer8ity of Glasgow, Galgow G12 8QQ The performance of all commercially available systems combining energy-dispersive X-ray microanalysis (EDX) and transmission electron
lip PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 microscopes (TEM) is degraded by high X-ray background and instrumentally generated elemental peaks. The requirements for maximum quantitative accuracy include an optimal signal/noise ratio and freedom from spurious peaks. Steps taken to achieve this in our system (JEOL 100C TEM with STEM and Link Systems 290 EDX) were summarized. A
P/B of Ca K, 50:1 in central channel
key Fig. 1.
A carbon-coated (graphite in isopropanol) aluminium tube was inserted in the lower pole piece and a shortened objective aperture rod is used. The decontaminator was replaced by one of carbon-coated aluminium incorporating a carbon aperture above the specimen for collimation and reduction of back scattered electrons (Fig. 1A). Side-entry specimen rods have been redesigned with aluminiium or Perspex tips to reduce the X-ray background. The tips include a 1 mm carbon plate thinned to < 0e25 mm around the specimen area. Dry-cut, ultratbin sections, resin embedded or cryo-cut and freeze-dried (Sorvall cryo-ultramicrotome), are mounted on formvar films over one-hole mounts (Nicholson &; Schreiber, 1975) and carbon coated. Fig. 1 shows
PROCEEDINGS OF THE 12P spectra obtained from an ultrathin section of cat dental enamel in resin (B) and a carbon-coated formvar film (C). A high accelerating voltage (e.g. 80 kV) was used since, although the electron mean free path increases with voltage; so does the peak to background ratio. Specimens were tilted 300 towards the detector; it is of no advantage to keep the specimen horizontal as the take-off angle is not critical with ultrathin sections and high voltage. A cold stage for analysis of frozen hydrated ultrathin sections has also been made (Nicholson, Robertson, Hutchinson, Ferrier & Biddlecombe, 1977). The resolution (FWIMi 148 eV at Mn K.) of the 30 mm2 Si (Li) detector (Kevex Corporation) and the energy calibration were measured in situ with a specially constructed side-entry rod containing two 4 U(Ci sources, 65Fe and 57Co. Further improvements in detector and microscope collimation remain to be made. We are grateful to M.R.C. for provision of microanalysis facilities and to Professor T. E. Hutchinson (Seattle) for provision of the detector. REFERENCES
NICHOLsON, W. A. P., ROBERTSON, B. W., HUTCHINSON, T. E., FERRIER, R. P. & BIDDLECOMBE, W. H. (1977). Proc. EMAG 77, lOP Conf. Ser., ed MiSELL, D. L., Institute of Physics. NIcHoLsoN, W. A. P. & SCHREIBER, J. (1975). J. Mcro copie 22, 169-176.
Exploration of Graafian follicle of mouse ovary with microelectrodes BY 0. F. HuTTER and C. MCCAIG,* Institute of Phy8iology, University of Glasgow, Glasgow G12 8QQ A stepmotor-driven micro-electrode advanced through the wall of the Graafian follicle of isolated mouse ovaries, or of exteriorized ovaries with largely intact blood supply, records cellular potentials (- 10 to -65 mV) especially as it passes through the theca internal and granulosa layers. Intra-antral potentials are small and variable but tend to be a few mV positive especially in animals treated with pregnant mare serum and human chorionic gonadotrophin to induce pre-ovulatory maturation. With one micro-electrode injecting current into the antrum and a second recording the voltage difference between the antral fluid and the outside of the ovary, the resistance of the follicular wall may be measured. If the voltage electrode is advanced steadily across the follicular wall step increases are observed, suggesting the existence of cell layers forming resistive barriers to current flow. *
PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977
Micro-electrode recording from a tethered locust BY M. D. BURNS and R. WILLIAMSON. Department of Zoology, Univer8ity of Glaegow, Glasgow G12 8QQ The use of dye-filled micro-electrodes to stimulate and record from insect central neurones has so far been confined to severely restrained animals. A method was demonstrated for recording from marked neurones while allowing the animal freedom to display a wide variety of behaviours. The locust is tethered by gluing a steel rod to the dorsal surface, and a light expanded Perspex drum, which is free to rotate and counterbalanced to provide an upthrust equivalent to the animal's weight, is placed beneath. The C.N.S. is exposed by removing part of the dorsal cuticle of the anterior thorax, and a waxed steel platform is positioned to support the ventral nerve cord against movement. One or more micro-electrodes may then be inserted into the cord. This technique is being used to monitor the traffic passing between ganglia in marked neurones during or preceding particular motor outputs, to examine the processing of sensory inputs and the capability of single neurones to trigger specific motor outputs. Microprocessor analyser for neurophysiological data BY M. D. BURNS. Department of Zoology, Univereity of Glasgow, Gaegow G12 8QQ The advent of microprocessors has made it possible to construct a cheap laboratory computer for the on-line analysis of data. Many kits and ready-built cards are available to reduce the time investment in construction, but the development of programs may be very time-consuming. To reduce programming time the microprocessor used here is the Intersil IM6100, which uses the same instruction set as the PDP-8. Although the architecture and instructions are somewhat dated, the ready availability of PDP-8s in most universities means that they can be used to assemble and debug programs for the microprocessor. There is also a large library of PDP-8 subroutines, many of which can be directly transferred to the microprocessor. The analyzer shown here was designed to function as a pre-programmed instrument in which all operations are controlled by front panel push buttons, but in which the function performed can be changed by loading a different program. Programs are read in via a paper-tape reader, while pulses representing neurophysiological spikes are fed in directly. Initially
PROCEEDINGS OF THE 14P the analyser will be used to construct real time PST histograms and spike interval histograms and display them on an oscilloscope screen. It will also be used as a data logger to punch the times of occurrence of different identified spikes on to paper tape for subsequent analysis. Design and construction was limited to the cabinet, the power supplies and the interface electronics. The total component cost was about £600 (1977). The machine presently has only 1-25 K of memory but this can be expanded to 4 K with additional memory cards. Other facilities such as A/D converter inputs for signal processing and averaging and a teletype port can be easily added. Measurement of hormonal activity in the human foetal hypothalamus by bioassay BY NORMA C. CAMPBELL and D. P. GILMORE. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ In order to determine the presence of releasing hormones in human foetal hypothalamic tissue a sensitive bioassay technique has been developed. Mature female Sprague-Dawley rats which have been ovaries. tomized at least 3 weeks beforehand are used. To sensitize their pituitary to the effects of hypothalamic hormones the rats are primed with a subcutaneous injection of 50 pug oestradiol benzoate and 25 mg progesterone in 1 ml. corn oil 72 hr before use. The rats are anaesthetized with urethane (1.7 g/kg) and left for 45 min to allow serum hormone levels to stabilize. A sample of blood (1 ml.) is then withdrawn from the femoral vein for determination of the resting levels of circulating anterior pituitary hormones. A known amount of synthetic hypothalamic hormone or an aliquot of the extract to be tested is injected into the carotid artery and two further 1 ml. blood samples collected after 15 and 30 min. The blood samples are centrifuged and the separated serum snap frozen and stored at -15 0C for subsequent measurement by radioimmunossay of the anterior pituitary hormones present. Dose-response curves are constructed by injecting varying concentrations of synthetic luteinizing hormone releasing hormone (LHRH) and thyrotrophin releasing hormone (TRH) into groups of rats. Foetal specimens are obtained from hysterotomy terminations and hypothalamic and cortical extracts prepared by homogenization of the tissue in acid solution (McNeilly, Gilmore, Dobbie & Chard, 1977). The neutralized supernatant is injected into the carotid. Extracts of cortical tissue are used as controls. We have been able in this way to demonstrate the biological activity
PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 15P of LHRH in the mid-term foetal brain. As regards the control of prolactin release, the indication is that up to the 15th week of gestation the hypothalamus is either devoid of activity, or contains only releasing activity for this hormone. REFERENCE
McNEILLY, A. S., GILMORE, D., DOBBIE, G. & CHAiW, T. (1977). J. EndoCr. 73, 533-534.
Pre- and post-ganglionic stimulation of the rat vas deferens in situ By G. J. CONNELL, J. R. DOCHERTY and J. C. MCGRATH. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ Male Wistar rats, anaesthetized with halothane, are pithed using a rod electrode (Gillespie, MacLaren, Marshall & Pollock, 1970; Gillespie, MacLaren & Pollock, 1970). Respiration is maintained with 02 at 60 strokes/min and stroke volume adjusted to maintain end-tidal C02 at 4 % (Clanachan & McGrath, 1976). Carotid arterial pressure is monitored. After opening the abdomen, a thread is attached to the epididymal end of the vas deferens and the vas separated from the epididymis. The vas is then drawn through an electrode assembly in the form of a tube slightly longer than the vas itself, and the attached thread fixed at an initial resting tension of 0 5-1.0 g to a Grass FTO3 isometric tension transducer. The electrode assembly is moulded from polyester resin with loops of silver wire embedded in its inner wall at intervals of 10 mm. The inner diameter of the tube is approximately 1P5 mm so as to fit closely the wide prostatic part of the vas without occluding the blood supply. The vas, when drawn through this assembly, is gently constricted at the prostatic end but free to 'contract' isometrically along the rest of its length. The space between the vas and the inside of the tube is filled with normal saline to maintain electrical conductivity between tissue and electrodes and the abdomen filled with warmed liquid paraffin which serves both to prevent drying of the tissues and to insulate the electrode assembly from the animal other than via the vas. Rectal temperature is maintained at 37*5 + 0 5 0C. Since the nerve-induced contractile response in the vas is critically dependent on temperature both in vitro and in situ (Birmingham & Freeman, 1976; McGrath, 1977), the temperature of the liquid paraffin is similarly maintained. The sympathetic outflow to the vas is stimulated with supramaximal pulses (0-1-1.0 msec) through the pithing rod electrode at L1-2, a silver strip having been inserted subcutaneously dorsal to the vertebral column to act as an indifferent electrode. Skeletal muscle twitching is prevented by gallamine 10 mg/kg. Single pulse stimulation is employed throughout
PPROCEEDINGS OF THE 16P (McGrath, 1977) to avoid complication by feed-back processes which modify neurotransrission in this tissue (Stjarne, 1973). Single maximal pulses can also be applied through the electrode assembly around the vas between the loop electrode at the prostatic end and either the indifferent electrode or a loop electrode further along the vas. In this in aitu preparation, stimulation of the vertebral outflows and through the loop electrodes produces comparable maximal responses. After treatment with hexamethonium (5 mg/kg) only the response to stimulation of the vertebral outflow is modified. In the in vitro preparation so far used, by contrast, hypogastric nerve stimulation produces responses that are smaller than those produced by electrodes around the vas (Birmingham & Freeman, 1976). It may be that in situ fibres are stimulated in addition to those reaching the vas via the hypogastric nerve, or that in vitro ganglionic transmission is incomplete. REFERENCES BMINGHAM, A. T. & FREEMAN, A. M. (1976). J. Physiol. 256, 747-759. CLAcHa, A. S. & McGnATH, J. C. (1976). Br. J. Pharmac. 58, 247-252. GLLEsPIE, J. S., MACLARE, A., MD hALL, R. W. & PoLLOCK, D. (1970). J. Physiol. 211, 11-12P. Giuxspr, J. S., MAcLARE, A. & PoLLocK, D. (1970). Br. J. Pharnac. 40, 257-267. McGRuTH, J. C. (1977). J. Phy8iol. 270, 52-53P. STJARNE, L. (1973). Eur. J. Pharmac. 22, 233-238.
Human bladder and urethral function BY E. S. GLEN and D. ROWAN. Urodynamic Service, Southern General Hospital, Glasgow G51 4TF, and Department of Clinical Phys8ic and BioEngineering, 11 West Graham Street, Gasgow G4 9LF Human bladder and urethral sphincter mechanisms are not yet fully understood, far less the alterations resulting from pathogical processes. The following urodynamic and static techniques of recent development were demonstrated, together with the equipment. Continuoni cystometry. This is more accurate than the older technique of incremental filling and intermittent recording of intravesical pressure. Two methods were shown employing (i) a double lumen urethral catheter developed to permit subsequent measurement of urethral closure pressure profile, (ii) a urethral catheter for filing the bladder, and a cannula inserted suprapubically into the bladder to measure intravesical pressure during micturition once the urethral catheter has been removed. Bladder-emptying flow-rate measurement. A Disa flowmeter is used. The urine stream strikes a rotating disk. The amount of electrical energy required to maintain disk velocity is proportional to the urine mass flow-rate
17P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 and this is recorded. The results require careful interpretation since they depend on the volume of urine voided and the direction of the stream of urine as it strikes the flowmeter funnel. Bladder-emptying flow-rate measurement combined uwtth intravesical and intrarectal pressure measurements during radiological micturating cystography. The intrarectal pressure is taken to indicate intra-abdominal pressure and is automatically subtracted electronically from intravesical pressure to give a value for detrusor pressure. The pressure recordings are displayed on a videotape monitor side by side with a simultaneous recording of the micturating cystogram, using a radio opaque fluid in the bladder. Several samples were shown. Obstruction to emptying may be due to stenosis, e.g. stricture, or failure of co-ordination in detrusor-urethral mechanisms. High intravesical pressures during attempted voiding may cause reflux into ureters. Urethral closure pressure profile measurement. A specially designed double lumen urethral catheter, withdrawal platform and recording apparatus was demonstrated. This equipment is used for diagnostic cystometry and urethral closure pressure profile measurement in the conscious patient. It is necessary to measure intravesical pressure continuously while the catheter is moved along the urethra to measure the urethral profile. If detrusor activity occurs, the urethral profile is affected. Detrusor activity simulates or may even produce the voiding reflex with obvious changes in the urethra. The volume of fluid in the bladder can affect the urethral profile. The normal response is for the functioning urethral length, and the pressure along that length, to increase in value or at least stay static. Weakness of the pelvic and perineal muscles tends to reverse this, producing deteriorating profile values. The role of the anatomically defined urethral sphincters is not clear. Long-term patient-operated electronic muscle stimulators to control urinary incontinence. The main types in use are (i) an anal plug electrode, (ii) vaginal electrodes - these two require no surgery - and (iii) a radio receiver implanted in the abdominal wall with buried electrodes secured to the pubococcygeus muscle. The receiver is activated by an external batterypowered transmitter. Bladder and urethral function may be affected by many pathological processes that first manifest themselves also in other ways, e.g. pressure on the spinal cord by prolapsed intervertebral disks. Application of the techniques demonstrated now suggests that some incontinent individuals and some with poor voiding ability have a neurological disorder of bladder and/or urethra without recognizable neurological defects elsewhere. This has important clinical implications.
A8PPROCEEDINGS OF THE
Ganglion cell recordings from the exposed frog retina in situ By J. D. MORRISON. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ Action potentials have been recorded extracellularly from the terminals of optic nerve fibres in the optic tectum of frog with the eye intact (Maturana, Lettvin, McCulloch & Pitts, 1960) or directly from the retina in the excised eyecup (Barlow, 1953). Generally investigators have used relatively coarse tungsten electrodes which select the larger cell bodies and axons (Stone, 1973). In this demonstration, recordings are made with fine glass micropipettes from the exposed retina in situ of Rana temrporaria. The dissection, which entails removal of the eyelids, aqueous humour, lens and vitreous body (Morrison, 1975) minimizes disturbance to the retina. The circulation may be maintained intact and the entire retinal surface is available for investigation. At present a pithed preparation is employed, but in principle the preparation also permits simultaneous recordings from the tectum. The body of the frog is kept wet in a shallow bath of saline and is oxygenated by a stream of moist 95 % 02/5 % C02. The micro-electrodes are pulled from borosilicate glass 2-0 mm o.d. and 1-2 mm i.d. which contains a microfilament (Clark Electromedical Instruments) and are filled by capillarity with 4 M-NaCl. Tip diameters are about 0 3 Am and impedances exceed 20 MD. These micro-electrodes permit isolation of single units and reduce bias towards larger cells. The recordings are extracellular. An insulated silver wire electrode 100 Am diameter monitors the electroretinogram. The preparation remains viable for 4-12 hr. When searching for units the micro-electrode is inserted into the retina. A green light-emitting diode flashed manually provides stimulation. Spike activity is recorded from the superficial layers and suggests it arises from ganglion cells. Axon and soma recordings may be readily distinguished: a small light spot moved across the electrode tip will evoke a response in the latter case since the receptive field centre is located at the micro-electrode tip. A unit may be held for up to 2 hr. The retina is stimulated by an optical stimulator which has two beams combined by a beamsplitter. Light from a tungsten halogen source (Barr & Stroud, LS1O) is transmitted to the optical stimulator by two fibreoptic light guides. The lamp is run from a stabilized d.c. supply. The light beams are corrected to daylight with a Kodak 80A filter. Their beam intensities are attenuated with calibrated neutral density filters. Maximum intensity measured directly by a photodiode is 5000 lux. The diameter of the projected spot may be varied from 0-3 to 6-0 mm. The duration of the opening of the shutter to allow the beam to project onto the retina is
PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 19P controlled by a Digitimer and the stimulus duration is monitored by a photodiode. Measurement of light output showed drift to be less than 3 % per hr and random noise to be less than + 0 5 %. The observed variability in the ganglion cell response is much greater than this and so is not attributable to fluctuations in light output. The source of the variability must be within the retina itself. Preliminary results reveal a direct proportionality between mean spike total and its standard deviation in some units. In other units, when the mean interspike interval was calculated for each response, the mean ofthese values was directly proportional to its standard deviation (cf. Werner & Mountcastle, 1963). These relationships may be important in understanding the relative efficacies of the trains of action potentials which reach the optic tectum. REFERENCES BAmLow, H. B. (1953). J. Physiol. 119, 58-68. MATURAA, H. R., LErrvn, J. Y., MCCUIMoCH, W. S. & J. gen. Phygiol. 43, suppi., 129-175. MoRRmSoN, J. D. (1975). Viaion Re8. 15, 1339-1344. SToNE, J. (1973). J. Neurophypiol. 36, 1071-1079.
W. H. (1960).
WERNER, G. & MouNTcssTLE, V. B. (1963). J. Neurophysiol. 26, 958-977.
Self-instruction material in physiology BY 0. HOLMES, SRmLA M. JENNETT and R. ORCICRDSON, Institute of Physiology, University of Glasgow, Glasgow (12 8QQ As a supplement to more conventional teaching methods, self-instruction programmes are used in Glasgow as part of the 2nd M.B. and B.D.S. courses. Students use the material in designated periods in the timetable and in their spare time. Each programme is designed to allow students to reach specified learning objectives. Programmes which have been produced in the Department are used to provide back-up theoretical material for lectures and practical classes. In all cases, the student is encouraged to check up on his own progress during the programme. Titles in the form of question-and-answer booklets include: Flow (pressure and resistance in tubes); 02 supply to tissues; Respiratory gases; Introduction to blood; Colorimetric estimation of haemoglobin; Light and colour. Other programmes, based on a tape-booklet format, have been produced on: Introduction to statistics; Mastication. We are assessing the use and usefulness of the material by asking students to fill in a questionnaire at the end of each study session. The material is housed in a library and this was open for inspection during the meeting. Members wishing to demonstrate any of their own selfinstruction material were invited to add their material to the demonstration.
A0PROCEEDINGS OF THE
A technique for perusing the cerebrospinal fluid spaces of the cat from lateral ventricle via the cisterna magna to the cortical subarachnoid space BY Mnt-Oru LIEw, B. R. MACKENNA and J. A. G. WArT, Institute of Physiology, University of Glagow, Glagow G12 8QQ, and Gartnavel Royal Hospital, Glagow, Scotland Perfusion techniques of the cerebrospinal fluid (c.s.f.) spaces have been mainly from the lateral ventricles of the brain to the cisterna magna. Thus the contents of the perfusate collected do not include substances released from the cerebral cortex. We have therefore developed a technique for perfusing from the lateral ventricle not only to the cisterna magna but in addition over the cerebral cortex through the subarachnoid space. It is hoped to study, by this technique, the contribution of acid metabolites of 5-hydroxytryptamine and dopamine from the cerebral cortex. Adult cats (2.5-3.5 kg) anaesthetized with sodium pentobarbitone were used. The temporal muscles and periosteum overlying the vault ofthe skull were reflected laterally. At a point 5 mm caudal to the bregma and 3-5 mm lateral to the mid line a hole was drilled vertically with a 2 mm drill. The opening was tapped with a 5 BA tap and a stainless-steel guide tube screwed firmly in place. This guide tube enabled the accurate insertion of a needle into the lateral ventricle. A second hole 5 mm in diameter was also drilled over the parietal cortex to expose the dura. A cross-wise slit was made in the dura and arachnoid membrane. A metal cannula (1.3 mm depth, 3-5 mm diameter) was inserted at right angles to the skull through the slit to sit in the subarachnoid space without coming into contact with the underlying cortical tissue. The cannula was held in place with cold acrylic resin. The perfusion fluid was driven by a Watson Marlow flow inducer (MHRE-7 delta type) in a fashion similar to that of Moir and Dow (1970). The outflowing c.s.f. was collected at the level of the external auditory meatus via a silicone tube attached to the subarachnoid cannula. With this technique, perfusion with artificial c.s.f. of the ventriculocortical subarachnoid space for 4-6 hr is possible. To establish that during perfusion no artificial connexion has developed between the subarachnoid and subdural spaces, and so enabled the perfusing fluid to exchange directly with blood via the dural capillaries, two studies were made in two different groups of cats. Perfusion was carried out either from lateral ventricle to cisterna magna or from lateral ventricle to cortical subarachnoid space. In both groups of cats MNaCl or 51Cr(EDTA) was introduced into the blood and held at a constant level. The inflow rate of artificial c.s.f. was held at 20 #1l./min. The degree of penetration of these substances into the artificial c.s.f. was noted. There
21P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 was little difference in the amount of MNaCl or 51Cr(EDTA) in the c.s.f. of the two groups, suggesting that no irregularity in their penetration into the c.s.f. in the subarachnoid space had occurred. This work was supported by a grant from the Medical Research Council. REFERENCE Mom, A. T. B. & Dow, R. C. (1970). J. apple. Phyeiol. 28, 628-529.
A method for studying the length-tension relationships of the jaw muscles of the cat BY B. R. MACKENNA and K. S. TURKER.* Institute of Phy8iology, Univer8ity of Glawgow, Glwagow G12 8QQ The length-tension relationships of jaw muscles have not been extensively studied because of three main difficulties. The first problem is to construct a rigid head holder which keeps the head in a fixed position without interfering with the mouth. The second is to devise a method whereby the jaw can be opened and closed freely but also can be fixed in any desired natural position so that the forces exerted by the jaw muscles on the mandible can be recorded. The third is to obtain access to the nerves to the jaw muscles without serious damage to the muscles being studied. Because of the latter problem all previous length-tension studies of jaw muscles have been carried out by stimulating the muscles directly. The cat was chosen for the present studies because it was thought that access could be gained to the nerve to masseter muscle by approaching it from above and deep to the zygomatic arch and thus avoid damage to the muscle itself. To hold the head of the cat in a fixed position we constructed a frame and a head holder. The frame was constructed of Handy-angle Speed Frame and measured 89 cm in length, 49 cm in width and 36 cm in height. There was an additional central longitudinal bar fitted along the top to which the head holder was attached. The head holder consisted of two inverted U-shaped bars, one of which was large enough to fit over the cat's ears. A pin was then pushed through a hole in each end of this U-bar into each middle ear of the cat and fixed in position with locking screws. The second U-shaped bar was smaller and had two pointed screws at each end. When this U-bar was inverted over the cat's snout the points of the screws could be screwed towards one another and the points thus tightened on to either side of the orbital part of the frontal bone. Both the *
Turkish Government scholar.
PROCEEDINGS OF THE 22P U-shaped bars of the head holder were then attached to the central bar at the top of the frame. A hole was drilled completely through the lower jaw in the region of the mental foramen. A square pin was pushed through this hole and fixed in position with cold curing acrylic resin. Another U-shaped bar was used to connect the ends of this pin- to a tension transducer (Grass FT03C). The tension transducer was fixed by a universal joint to an upright which was screwed to the operating table. With this arrangement it was possible to open and close the jaw freely to any desired position and keep the angle between the occlusal plane and the recording part of the transducer constant. The gap between the incisors was measured using a potentiometer attached to the pin in the lower jaw. The nerve to the masseter was stimulated using guarded silver wire electrodes. The jaw muscles were directly stimulated using bipolar wire electrodes. With this set-up we have been able to study the effect of jaw position on the maximum force developed by all the jaw muscles when stimulated directly, make a comparison between the effect of direct stimulation of the masseter muscle with the effect of nerve stimulation and study the effect of jaw position on the jaw reflexes.
Morphometric analysis by computerized planimetry BY W. H. BIDDLECOMBE, D. W. DEMPSTER, H. Y. ELDER, J. K. KERR, V. A. Moss, L. D. PEACHEY* and N. C. SPURwAY. Department of Phy8iology, Univer8ity of Gla.gow, Scotland, and *Department of Biology, University of Pennwylvania, Philadelphia, U.S.A. A computer records and processes co-ordinates derived from the analogue output of a manually operated planimeter. Given a computer with available off-peak time, the planimeter and control unit are relatively inexpensive. For many morphometric applications the system has advantages over other methods. Images from prints or a projection microscope can be analysed. Profiles of the features analysed can be recorded on paper or a storage oscilloscope. The method gives greater accuracy from fewer images in less time than conventional grid sampling stereometry. It does not compete with fully automated image analysis systems for speed in counting and measuring image features which are easily machine discriminated (e.g. stained nuclei in an unstained background). However it combines the advantages of human discrimination (e.g. smooth, ve. rough, endoplasmic reticulum) with immediate computer handling of the data.
23P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1977 The device is a development from an earlier instrument (Peachey, 1971). Voltage outputs from two precision potentiometers represent the radial and angular co-ordinates traced by the hand-held stylus of a planimeter; these are digitalized, for processing by PDP-8 or NOVA computers, at a calibration appropriate to the magnification chosen (Fig. 1). Co-ordinates are sampled at linear intervals preset by the operator. Line lengths, areas and perimeters can be measured to mainly operator error of