1P
PROCEEDINGS OF THE PHYSIOLOGICAL SOCIETY COPENHAGEN MEETING 25-26 April 1975
DEMONSTRATIONS Control of amylase secretion in the parotid gland BY 0. H. PETERSEN* and N. UEDA.* Institute of Medical Physiology C, University of Copenhagen, 2200 Copenhagen N, Denmark The existence of three kinds of receptor sites on parotid acinar cells has recently been established. Activation of cholinergic and adrenergic czreceptors results in K-release and cell membrane hyperpolarization and probably induces fluid secretion, while activation of fl-adrenoceptors causes adenyl cyclase activation, cell membrane depolarization and amylase secretion (Batzri & Selinger, 1973; Batzri, Selinger, Schramm & Robinowitch, 1973; Petersen & Pedersen, 1974). A newly developed automated fluorescence method for continuously monitoring amylase output from segments of pancreas (Matthews, Petersen & Williams, 1974) has been used for a study of mouse parotid enzyme output. A particular advantage of this method is that it allows us for the first time to compare the dynamic profile of amylase output following various types of stimulation in the same tissue. Fig. 1 shows the effects of stimulating the three different acinar receptors. The most powerful activation of amylase output is undoubtedly obtained by isoprenaline, but only following excitation of a-adrenoceptors or cholinergic receptors do we obtain a rapid rise of amylase secretion. It is possible that this has to do with the activation of fluid secretion obtained by ACh and adrenergic ax-excitation. Our results clearly show that ACh has a sustained effect on amylase secretion. * Present address: Department of Physiology, University of Dundee, DD1 4HN, Scotland.
PROCEEDINGS OF THE
2P
Isoprenaline 10-6'M.
a
.
Adrenaline 1O-6M
B.
,
A
E .so.re.aI
*.
Adrenaline---:O'~--R - ---J Adrenalifie Adrenaline 10oprenaLine 10'6M
Phentolam*ine 10-3 m._
Propranolol 5x10-'M
E
Acetylcholine 10-6M Atropine 1 4x10-6M
.
Fig. 1. A-E are consecutive traces from one experiment and should be read from right to left. The figures show the concentration of amylase in the effluent from the tissue flow cell. Calibration is shown in A. 20 mi interval between A and B. 35 min interval between and D. No interval between D and E. Total tissue wet weight 130 mg.
REFERENCES BATzRI, S. & SEINamGE, Z. (1973). J. biol. Chem. 248, 356-360. BATz~iL, S., SEiNiGER, Z., ScHRtAMM, M. & RoBlnowrrcH, M. R. (1973). J. biol. Chem. 248, 361-368.
Pae Fig.ws, E. K.,
& Wmaism se, 0. H.
,
J. A. (1974). Analyt. Biochem. 58,
155-160. PETERSEN, 0. H. & PEDERSEN, G. L. (1974). J. Membrane Biol. 16, 353-362.
Electrical coupling between pancreatic acinar cells By 0. H. PETERSEN.* Institute of Medical Physiology C, University of Copenhagen, 2200 Copenhagen N, Denmark Electrical coupling between epithelial cells has been extensively studied in Drosophila and Chironomws salivary glands (Loewenstein, 1966). In mammalian tissues it has been demonstrated in the liver (Penn, 1966). * Present address: Scotland.
DD( Department of Physiology, University of Dundee,
4HN,
3P PHYSIOLOGICAL SOCIETY, APRIL 1975 I have been able to insert two micro-electrodes into the same mouse or rat pancreatic acinus allowing a direct test of intra-acinar cell coupling. Fig. 1 shows the recording arrangement and an example of successful simultaneous recording with two intracellular electrodes directly demonstrating electrical coupling between two acinar cells.
2Opnh
Photo -40 _
:+10OmMV
b a,[ b
-30 Fig. 1. The upper part shows the arrangement of the micro-electrodes during the experiment. Below is shown a typical pen-recording from one pair of communicating acinar cells. The frequency of current pulse injection was lsec. Some time after stabilization of the membrane potentials the strength of the injected current pulses was increased, thereafter a photograph from a storage oscilloscope screen was taken. Calibration: horizontal, 20 msec; vertical, 10 mV and 10-9 A. Time marks in pen-recording occur at minute intervals.
Observations have been made on 10 communicating pairs of cells. The coupling ratio (current pulse-induced potential change in the cell of current injection divided by electrotonic potential change in the neighbouring cell) is 0-9 + 0-1 (s.E.). Acetylcholine added to the tissue bath depolarizes
4P PROCEEDINGS OF THE 4P both cells simultaneously and reduces the amplitude of the current-pulse induced potential deflexions similarly in both cells, directly demonstrating that ACh reduces acinar surface cell membrane resistance as previously suggested (Nishiyama & Petersen, 1974, 1975). REFERENCES
LOEwENSTEmIN, W. R. (1966). Ann. N.Y. Acad. Sci. 137, 441-472. NIsmYxA, A. & PETERSEN, 0. H. (1974). J. Phy8iol. 238, 145-158. NIsHYAMA, A. & PETERSEN, 0. H. (1975). J. Physiol. 244, 431-465. PENN, R. D. (1966). J. cell Biol. 29, 171-174.
An improved method for perfusion of the isolated rat pancreas
By N. UEDA.* Institute of Medical Physiology C, University of Copenhagen, 2200 Copenhagen N, Denmark It is generally accepted that pancreatic protein secretion occurs in the acinar cells, stimulated by acetylcholine (ACh), pancreozymin (CCK-Pz) and gastrin, whereas fluid and electrolyte secretion occurs along the entire duct system stimulated by secretin. Dockray (1972) has shown in rat pancreas in vivo that CCK-Pz evokes a marked fluid secretion. However, the effects of pure stimulants on fluid secretion in rat pancreas in vitro have not been studied. Fig. 1 shows a diagram of the preparation and the results of typical experiments. The effect of ACh, gastrin, CCK-Pz and caerulein were indistinguishable except in the presence of atropine which only abolished ACh-evoked secretion. The stimulating effect of pure secretin (Karolinska Institutet) on fluid secretion was only seen in the presence of extracellular C02/HCO3, whereas the effects of ACh, gastrin and CCK-Pz were independent of the presence of this buffer. These results, taken together with the recent observation by Sewell & Young (1974) that secretin-induced rat pancreatic juice is rich in bicarbonate whereas caerulein evoked secretion has a much lower bicarbonate content, shows that in the rat pancreas there are two different types of fluid secretion stimulated by different agonists. * Present address: Department of Physiology, University of Dundee, DD1 4HN, Scotland.
PPHYSIOLOGICAL SOCIETY, APRIL 1975
5P
O,~ ~ ~ ~ 4-0:O
Ol:C
Atropine 1 4x10-
I
Secretin (015 u/ml.)
0-6
tris
COJHC
0O*6
rCIc;
9
CCK-Pz x 10 5x10-4 5(u./ml.) ACh 10'nm
*
0
0
1
2 0 1 2 Time (hr) Fig. 1. Upper part shows diagram of the perfusion apparatus modified from Kanno (1972) and Ueda (1974). A, Medium perfusing the blood vessels; B, roller pump; C, cannula inserted into superior mesenteric artery; D, cannula inserted into coeliac artery; E, portal vein cannula; F, cannula for drainage of duodenal content; G, tissue bath; H, ligature of the proximal end of the common duct; I, ligature of pylorus; J, cannula for collecting pancreatic juice, K, heat exchanger; L, perfusion fluid for the tissue bath. Lower part shows the results of two typical experiments. The left part shows the effect of replacing the Krebs-bicarbonate solution by a C02/HC03-free Tris-buffered solution during secretin stimulation. The right panel shows the effect of ACh and CCK-Pz on fluid secretion from the isolated pancreas, in the presence or absence of atropine. This particular experiment was done using Krebs-bicarbonate solution, but similar results were obtained with Tris buffered solution. REFERENCES
DocnRAY, G. J. (1972). J. Physiol. 225, 679-692. KANno, T. (1972). J. Physiol. 226, 353-371. SEWELL, W. A. & YOUNG, J. A. (1974). Proc. Aut. Physiol. Pharmac. Soc. 5, 246-247. UEDA, N. (1974). J. Jap. Soc. Gaetroenterot. 71, 558-572. (In Japanese.)
6P PROCEEDINGS OF THE Combination of conventional mechanical kymograph, polythene roll flat-bed transport, electronic pen recorder and overhead projector
BY P. J. CANNON and T. P. MumLvrum. Department of Pharmacology, University College, Dublin The coupling of a specially built (i) flat-bed transport unit (recording paper replaced by polythene), (ii) high-gain preamplifier and (iii) motordriven spindle of an old-style Palmer kymograph has been demonstrated by Cannon & Mulvihill (1974), and is now shown adapted to a conventional overhead projector (Fordigraph). A 30/70 (v/v) drawing ink/methylated spirits mixture writes on a transparent polythene roll (cut from sheeting) transported across the Fresnel lens bed of the Fordigraph via two rollers built on to each side of its base. The pen-writing units of the George Washington (G.W.) or Harvard (or their equivalents) can be aligned and mounted on a rack, built by one of us (T.M.). The adapted G.W. recorder control unit offers the potentially wide variety of recordings (pressure gauge, e.c.g., heart rate, etc.) associated with conventionally expensive multi-channel (but no more sensitive) penrecording complete instruments. The polythene roll tracings are (i) readily photocopied and (ii) erased by rubbing with a damp cloth. In addition, the cost per ft. of conventional kymograph recording paper (11 in. wide) is 3-4 times that of a corresponding length of the polythene. We are grateful to the Medical Research Council in Ireland for support and to the firm of George Washington Ltd for transport assistance. REFERENCE
CANNoN, P. J. & MuLvImLL, T. P. (1974). J. Phy&il. 236, 5P.
Adaptation of the George Washington -(G.W.) pen recorder for overhead projection By P. J. CANNON and T. P. MULVIHILL. Department of Pharmacology, University College, Dublin Instead of combining conventional kymograph, flat-bed transport pen recorder and overhead projector (as shown separately at this meeting), the 400 MD2 G.W. two-channel recorder may itselfbe modified and achieve 'compactly' the same effect. The Fresnel lens base of the overhead projection system, surmounted by the double lens/mirror complex, is now interposed between the G.W.
PHYSIOLOGICAL SOCIETY, APRIL 1975 7P gearbox and galvanometer units. The lens/mirror complex is rotatable, thus throwing the image in any required horizontal direction. The height of the projected image is adjusted by the telescoping sections of the fixture which supports the lenses. The positions for feeding in and feeding back the polythene lie side-by-side at one end of the instrument, the polythene being run over a Perspex plate and roller placed over the Fresnel lens. Apart from achieving movement of the projected tracing from right to left, the polythene feed position is well clear of the pens and galvanometers at the opposite end of the recorder. The heat from the 600 W tungsten-halogen lamp, located within the G.W. recorder for overhead projection, is reduced by an in-built fan and controllable by a half-wave rectifier diode, so that the instrument may be used for reasonably long intervals of time for projection of ordinary slides, etc. As the flat-bed is ca. 17 x 10*5 cm2, the image projected reveals a greater length of tracing than is perceived on glancing at the paper recordings as they emerge from the unmodified G.W. instrument. Continuous closed circuit television observation of the preparation may proceed pars passu with the projected tracings. The advantages of overhead projection and 'slaving' may be compared as teaching procedures. The adaptability and cost-effectiveness of the demonstrated items suggest a relatively simple solution for routine teaching as well as for sophisticated demonstration problems. We are grateful to the Medical Research Council in Ireland for support and the firm of George Washington Ltd for transport assistance.
Effects of post-ganglionic sympathectomy relating to parotid 'degeneration secretion' in rats BY J. R. GARRETT* and A. THruxN. Department of Oral Pathology, King's College Hospital Dental School, London SE5 8RX, and Institute of Physiology, Lund, Sweden Delfs & Emmelin (1974) have recently found that degeneration activation after sympathetic ganglionectomy causes a small, but distinct, 'degeneration secretion' from parotid glands of anaesthetized rats, at times between 12 and 24 hr after sympathectomy. Previous studies (Harrop & Garrett, 1974; Garrett & Thulin, 1975) have shown that secretion of secretary granules from parotid glands of rats is essentially a sympathetic nerve function in vivo. It therefore seemed possible that structural changes *
Assisted by a travel grant from the Royal Society.
8P PROCEEDINGS OF THE may be discernible in the parotid acinar cells as a consequence of sympathetic 'degeneration secretion'. Rats were starved overnight and right superior cervical ganglionectomy was performed the next morning under Nembutal anaesthesia. Food was withheld from animals to be terminated 12 or 24 hr later, but animals left
A MH
B
Fig. 1. Parotid acini from the same animal 24 hr after right sympathectomy x 2900. A, Left control gland loaded with secretary granules. B, Right gland showing a marked depletion of secretary granules.
for 48 hr were allowed food until the night before tissues were removed. Both parotid glands were excised from each animal under Nembutal anaesthesia and tissues were prepared for formaldehyde-induced fluorescence and electron microscopy. The gland from the left unoperated side acted as a control in each instance.
9P PHYSIOLOGICAL SOCIETY, APRIL 1975 Macroscopically the sympathectomized and control glands appeared similarly whitish and opaque at 12 and 48 hr after sympathectomy. At 24 hr, however, the sympathectomized glands appeared more pink, watery and less opaque than the control glands. Formaldehyde-induced fluorescence showed no obvious differences in the numbers or appearances of adrenergic nerves in test and control glands at 12 hr after sympathectomy. However, very few nerves were detectable in the test gland after 24 hr and none was seen 48 hr after sympathectomy. Electron microscopy revealed very few degenerating axons 12 hr after sympathectomy. Osmiophilic degenerative changes were abundant in axons 24 hr after sympathectomy but they seemed to have disappeared by 48 hr. Acini were mostly loaded with secretary granules 12 hr after sympathectomy and appeared similar to those in control glands. At 24 hr, although the acini of the control glands were loaded with granules those of the test glands showed a big depletion of secretary granules (see Fig. 1). The appearances were similar to those seen after sympathetic nerve stimulation (Garrett & Thulin, 1975), which strongly suggests that there had been a recent sympathetic type secretion. These changes were most pronounced in the glands that showed the fewest fluorescent nerves. At 48 hr the parotid acinar cells were again loaded with secretary granules. The work confirms that, after sympathetic ganglionectomy in rats, neurotransmitter is lost from the adrenergic axons in the parotid glands mainly between 12 and 24 hr later, and this release of neurotransmitter induces pronounced structural changes in the acinar cells indicative of sympathetic 'degeneration secretion'. REFERENCES
DELFS, U. & EMns, N. (1974). J. Physiol. 234, 623-630. GARRErr, J. R. & TmLIN, A. (1975). J. Physiol. 246, 84-85P. HAmaoP, T. J. & GARRETr, J. R. (1974). Cell Tiome Re8. 154, 135-150.
Slaving oscillographic recorders for teaching purposes BY W. V. WEBER. Department of Physiology, Basic Medical Sciences Group, Chelsea College, University of London, SW3 6LX The kymograph, although revered by many generations of physiologists, suffers from a number of limitations which have been partly overcome in modem pen recorders. The almost frictionless bearing surfaces of modern galvanometers endow an improved frequency response on the pen recorders currently available. Austerity budgets and the current price inflation of all materials and animals as well as vastly increased student numbers require that economies
PROCEEDINGS OF THE loP be made without sacrificing educational principles. One way of achieving such economies is to operate a number of recorders in parallel, obtaining multiple records simultaneously at low cost, reproducing the desired biological parameters from a single animal preparation. It is virtually impossible to operate a number of kymographs in parallel, a severe limitation. A design modification introduced early on George Washington oscillographs (G.W.) facilitates such simultaneous parallel recordings to be made on G.W. and other pen recorders, U.V. recorders, oscilloscopes, FM tape Master unit
Amplified signal
Ink writing
Inkswitingrp
(0-6 V) detected at appropriate pin on
osc riograph
Slaved units
monitoring
|Socket\(90 Biological
ip
Di
a
coupler amplier Stored data _can be used for the repeat demonstrations
YWA*
O
s
F
tape
recorder \ lClosed circuit television
D
Computer
Fig. 1. Diagrammatic representation of slaving from a single channel oscillograph.
recorders, video recorders and computer terminals, as shown diagrammatically in Fig. 1. This facility was achieved by wiring the output of the driver amplifier to an appropriate pin of a monitoring socket, located on the deck of the G.W. oscillograph. The amplified signals vary within the range 0-6 V and standard calibration pulses can be fed into the system to calibrate the slaved units. The versatility of this system was demonstrated as one method of achieving a solution in structured course teaching and of doing sophisticated demonstrations to large numbers of students. If closed circuit television observation of the preparation could be used at the same time, students would benefit even more. The author wishes to thank Mr Ken Hore for his advice and help with operating the CCTV and George Washington Ltd for lending all the oscillographic equipment used in this demonstration.
PROCEEDINGS OF THE PHYSIOLOGICAL SOCIETY COPENHAGEN MEETING 25-26 April 1975
DEMONSTRATIONS Control of amylase secretion in the parotid gland BY 0. H. PETERSEN* and N. UEDA.* Institute of Medical Physiology C, University of Copenhagen, 2200 Copenhagen N, Denmark The existence of three kinds of receptor sites on parotid acinar cells has recently been established. Activation of cholinergic and adrenergic czreceptors results in K-release and cell membrane hyperpolarization and probably induces fluid secretion, while activation of fl-adrenoceptors causes adenyl cyclase activation, cell membrane depolarization and amylase secretion (Batzri & Selinger, 1973; Batzri, Selinger, Schramm & Robinowitch, 1973; Petersen & Pedersen, 1974). A newly developed automated fluorescence method for continuously monitoring amylase output from segments of pancreas (Matthews, Petersen & Williams, 1974) has been used for a study of mouse parotid enzyme output. A particular advantage of this method is that it allows us for the first time to compare the dynamic profile of amylase output following various types of stimulation in the same tissue. Fig. 1 shows the effects of stimulating the three different acinar receptors. The most powerful activation of amylase output is undoubtedly obtained by isoprenaline, but only following excitation of a-adrenoceptors or cholinergic receptors do we obtain a rapid rise of amylase secretion. It is possible that this has to do with the activation of fluid secretion obtained by ACh and adrenergic ax-excitation. Our results clearly show that ACh has a sustained effect on amylase secretion. * Present address: Department of Physiology, University of Dundee, DD1 4HN, Scotland.
PROCEEDINGS OF THE
2P
Isoprenaline 10-6'M.
a
.
Adrenaline 1O-6M
B.
,
A
E .so.re.aI
*.
Adrenaline---:O'~--R - ---J Adrenalifie Adrenaline 10oprenaLine 10'6M
Phentolam*ine 10-3 m._
Propranolol 5x10-'M
E
Acetylcholine 10-6M Atropine 1 4x10-6M
.
Fig. 1. A-E are consecutive traces from one experiment and should be read from right to left. The figures show the concentration of amylase in the effluent from the tissue flow cell. Calibration is shown in A. 20 mi interval between A and B. 35 min interval between and D. No interval between D and E. Total tissue wet weight 130 mg.
REFERENCES BATzRI, S. & SEINamGE, Z. (1973). J. biol. Chem. 248, 356-360. BATz~iL, S., SEiNiGER, Z., ScHRtAMM, M. & RoBlnowrrcH, M. R. (1973). J. biol. Chem. 248, 361-368.
Pae Fig.ws, E. K.,
& Wmaism se, 0. H.
,
J. A. (1974). Analyt. Biochem. 58,
155-160. PETERSEN, 0. H. & PEDERSEN, G. L. (1974). J. Membrane Biol. 16, 353-362.
Electrical coupling between pancreatic acinar cells By 0. H. PETERSEN.* Institute of Medical Physiology C, University of Copenhagen, 2200 Copenhagen N, Denmark Electrical coupling between epithelial cells has been extensively studied in Drosophila and Chironomws salivary glands (Loewenstein, 1966). In mammalian tissues it has been demonstrated in the liver (Penn, 1966). * Present address: Scotland.
DD( Department of Physiology, University of Dundee,
4HN,
3P PHYSIOLOGICAL SOCIETY, APRIL 1975 I have been able to insert two micro-electrodes into the same mouse or rat pancreatic acinus allowing a direct test of intra-acinar cell coupling. Fig. 1 shows the recording arrangement and an example of successful simultaneous recording with two intracellular electrodes directly demonstrating electrical coupling between two acinar cells.
2Opnh
Photo -40 _
:+10OmMV
b a,[ b
-30 Fig. 1. The upper part shows the arrangement of the micro-electrodes during the experiment. Below is shown a typical pen-recording from one pair of communicating acinar cells. The frequency of current pulse injection was lsec. Some time after stabilization of the membrane potentials the strength of the injected current pulses was increased, thereafter a photograph from a storage oscilloscope screen was taken. Calibration: horizontal, 20 msec; vertical, 10 mV and 10-9 A. Time marks in pen-recording occur at minute intervals.
Observations have been made on 10 communicating pairs of cells. The coupling ratio (current pulse-induced potential change in the cell of current injection divided by electrotonic potential change in the neighbouring cell) is 0-9 + 0-1 (s.E.). Acetylcholine added to the tissue bath depolarizes
4P PROCEEDINGS OF THE 4P both cells simultaneously and reduces the amplitude of the current-pulse induced potential deflexions similarly in both cells, directly demonstrating that ACh reduces acinar surface cell membrane resistance as previously suggested (Nishiyama & Petersen, 1974, 1975). REFERENCES
LOEwENSTEmIN, W. R. (1966). Ann. N.Y. Acad. Sci. 137, 441-472. NIsmYxA, A. & PETERSEN, 0. H. (1974). J. Phy8iol. 238, 145-158. NIsHYAMA, A. & PETERSEN, 0. H. (1975). J. Physiol. 244, 431-465. PENN, R. D. (1966). J. cell Biol. 29, 171-174.
An improved method for perfusion of the isolated rat pancreas
By N. UEDA.* Institute of Medical Physiology C, University of Copenhagen, 2200 Copenhagen N, Denmark It is generally accepted that pancreatic protein secretion occurs in the acinar cells, stimulated by acetylcholine (ACh), pancreozymin (CCK-Pz) and gastrin, whereas fluid and electrolyte secretion occurs along the entire duct system stimulated by secretin. Dockray (1972) has shown in rat pancreas in vivo that CCK-Pz evokes a marked fluid secretion. However, the effects of pure stimulants on fluid secretion in rat pancreas in vitro have not been studied. Fig. 1 shows a diagram of the preparation and the results of typical experiments. The effect of ACh, gastrin, CCK-Pz and caerulein were indistinguishable except in the presence of atropine which only abolished ACh-evoked secretion. The stimulating effect of pure secretin (Karolinska Institutet) on fluid secretion was only seen in the presence of extracellular C02/HCO3, whereas the effects of ACh, gastrin and CCK-Pz were independent of the presence of this buffer. These results, taken together with the recent observation by Sewell & Young (1974) that secretin-induced rat pancreatic juice is rich in bicarbonate whereas caerulein evoked secretion has a much lower bicarbonate content, shows that in the rat pancreas there are two different types of fluid secretion stimulated by different agonists. * Present address: Department of Physiology, University of Dundee, DD1 4HN, Scotland.
PPHYSIOLOGICAL SOCIETY, APRIL 1975
5P
O,~ ~ ~ ~ 4-0:O
Ol:C
Atropine 1 4x10-
I
Secretin (015 u/ml.)
0-6
tris
COJHC
0O*6
rCIc;
9
CCK-Pz x 10 5x10-4 5(u./ml.) ACh 10'nm
*
0
0
1
2 0 1 2 Time (hr) Fig. 1. Upper part shows diagram of the perfusion apparatus modified from Kanno (1972) and Ueda (1974). A, Medium perfusing the blood vessels; B, roller pump; C, cannula inserted into superior mesenteric artery; D, cannula inserted into coeliac artery; E, portal vein cannula; F, cannula for drainage of duodenal content; G, tissue bath; H, ligature of the proximal end of the common duct; I, ligature of pylorus; J, cannula for collecting pancreatic juice, K, heat exchanger; L, perfusion fluid for the tissue bath. Lower part shows the results of two typical experiments. The left part shows the effect of replacing the Krebs-bicarbonate solution by a C02/HC03-free Tris-buffered solution during secretin stimulation. The right panel shows the effect of ACh and CCK-Pz on fluid secretion from the isolated pancreas, in the presence or absence of atropine. This particular experiment was done using Krebs-bicarbonate solution, but similar results were obtained with Tris buffered solution. REFERENCES
DocnRAY, G. J. (1972). J. Physiol. 225, 679-692. KANno, T. (1972). J. Physiol. 226, 353-371. SEWELL, W. A. & YOUNG, J. A. (1974). Proc. Aut. Physiol. Pharmac. Soc. 5, 246-247. UEDA, N. (1974). J. Jap. Soc. Gaetroenterot. 71, 558-572. (In Japanese.)
6P PROCEEDINGS OF THE Combination of conventional mechanical kymograph, polythene roll flat-bed transport, electronic pen recorder and overhead projector
BY P. J. CANNON and T. P. MumLvrum. Department of Pharmacology, University College, Dublin The coupling of a specially built (i) flat-bed transport unit (recording paper replaced by polythene), (ii) high-gain preamplifier and (iii) motordriven spindle of an old-style Palmer kymograph has been demonstrated by Cannon & Mulvihill (1974), and is now shown adapted to a conventional overhead projector (Fordigraph). A 30/70 (v/v) drawing ink/methylated spirits mixture writes on a transparent polythene roll (cut from sheeting) transported across the Fresnel lens bed of the Fordigraph via two rollers built on to each side of its base. The pen-writing units of the George Washington (G.W.) or Harvard (or their equivalents) can be aligned and mounted on a rack, built by one of us (T.M.). The adapted G.W. recorder control unit offers the potentially wide variety of recordings (pressure gauge, e.c.g., heart rate, etc.) associated with conventionally expensive multi-channel (but no more sensitive) penrecording complete instruments. The polythene roll tracings are (i) readily photocopied and (ii) erased by rubbing with a damp cloth. In addition, the cost per ft. of conventional kymograph recording paper (11 in. wide) is 3-4 times that of a corresponding length of the polythene. We are grateful to the Medical Research Council in Ireland for support and to the firm of George Washington Ltd for transport assistance. REFERENCE
CANNoN, P. J. & MuLvImLL, T. P. (1974). J. Phy&il. 236, 5P.
Adaptation of the George Washington -(G.W.) pen recorder for overhead projection By P. J. CANNON and T. P. MULVIHILL. Department of Pharmacology, University College, Dublin Instead of combining conventional kymograph, flat-bed transport pen recorder and overhead projector (as shown separately at this meeting), the 400 MD2 G.W. two-channel recorder may itselfbe modified and achieve 'compactly' the same effect. The Fresnel lens base of the overhead projection system, surmounted by the double lens/mirror complex, is now interposed between the G.W.
PHYSIOLOGICAL SOCIETY, APRIL 1975 7P gearbox and galvanometer units. The lens/mirror complex is rotatable, thus throwing the image in any required horizontal direction. The height of the projected image is adjusted by the telescoping sections of the fixture which supports the lenses. The positions for feeding in and feeding back the polythene lie side-by-side at one end of the instrument, the polythene being run over a Perspex plate and roller placed over the Fresnel lens. Apart from achieving movement of the projected tracing from right to left, the polythene feed position is well clear of the pens and galvanometers at the opposite end of the recorder. The heat from the 600 W tungsten-halogen lamp, located within the G.W. recorder for overhead projection, is reduced by an in-built fan and controllable by a half-wave rectifier diode, so that the instrument may be used for reasonably long intervals of time for projection of ordinary slides, etc. As the flat-bed is ca. 17 x 10*5 cm2, the image projected reveals a greater length of tracing than is perceived on glancing at the paper recordings as they emerge from the unmodified G.W. instrument. Continuous closed circuit television observation of the preparation may proceed pars passu with the projected tracings. The advantages of overhead projection and 'slaving' may be compared as teaching procedures. The adaptability and cost-effectiveness of the demonstrated items suggest a relatively simple solution for routine teaching as well as for sophisticated demonstration problems. We are grateful to the Medical Research Council in Ireland for support and the firm of George Washington Ltd for transport assistance.
Effects of post-ganglionic sympathectomy relating to parotid 'degeneration secretion' in rats BY J. R. GARRETT* and A. THruxN. Department of Oral Pathology, King's College Hospital Dental School, London SE5 8RX, and Institute of Physiology, Lund, Sweden Delfs & Emmelin (1974) have recently found that degeneration activation after sympathetic ganglionectomy causes a small, but distinct, 'degeneration secretion' from parotid glands of anaesthetized rats, at times between 12 and 24 hr after sympathectomy. Previous studies (Harrop & Garrett, 1974; Garrett & Thulin, 1975) have shown that secretion of secretary granules from parotid glands of rats is essentially a sympathetic nerve function in vivo. It therefore seemed possible that structural changes *
Assisted by a travel grant from the Royal Society.
8P PROCEEDINGS OF THE may be discernible in the parotid acinar cells as a consequence of sympathetic 'degeneration secretion'. Rats were starved overnight and right superior cervical ganglionectomy was performed the next morning under Nembutal anaesthesia. Food was withheld from animals to be terminated 12 or 24 hr later, but animals left
A MH
B
Fig. 1. Parotid acini from the same animal 24 hr after right sympathectomy x 2900. A, Left control gland loaded with secretary granules. B, Right gland showing a marked depletion of secretary granules.
for 48 hr were allowed food until the night before tissues were removed. Both parotid glands were excised from each animal under Nembutal anaesthesia and tissues were prepared for formaldehyde-induced fluorescence and electron microscopy. The gland from the left unoperated side acted as a control in each instance.
9P PHYSIOLOGICAL SOCIETY, APRIL 1975 Macroscopically the sympathectomized and control glands appeared similarly whitish and opaque at 12 and 48 hr after sympathectomy. At 24 hr, however, the sympathectomized glands appeared more pink, watery and less opaque than the control glands. Formaldehyde-induced fluorescence showed no obvious differences in the numbers or appearances of adrenergic nerves in test and control glands at 12 hr after sympathectomy. However, very few nerves were detectable in the test gland after 24 hr and none was seen 48 hr after sympathectomy. Electron microscopy revealed very few degenerating axons 12 hr after sympathectomy. Osmiophilic degenerative changes were abundant in axons 24 hr after sympathectomy but they seemed to have disappeared by 48 hr. Acini were mostly loaded with secretary granules 12 hr after sympathectomy and appeared similar to those in control glands. At 24 hr, although the acini of the control glands were loaded with granules those of the test glands showed a big depletion of secretary granules (see Fig. 1). The appearances were similar to those seen after sympathetic nerve stimulation (Garrett & Thulin, 1975), which strongly suggests that there had been a recent sympathetic type secretion. These changes were most pronounced in the glands that showed the fewest fluorescent nerves. At 48 hr the parotid acinar cells were again loaded with secretary granules. The work confirms that, after sympathetic ganglionectomy in rats, neurotransmitter is lost from the adrenergic axons in the parotid glands mainly between 12 and 24 hr later, and this release of neurotransmitter induces pronounced structural changes in the acinar cells indicative of sympathetic 'degeneration secretion'. REFERENCES
DELFS, U. & EMns, N. (1974). J. Physiol. 234, 623-630. GARRErr, J. R. & TmLIN, A. (1975). J. Physiol. 246, 84-85P. HAmaoP, T. J. & GARRETr, J. R. (1974). Cell Tiome Re8. 154, 135-150.
Slaving oscillographic recorders for teaching purposes BY W. V. WEBER. Department of Physiology, Basic Medical Sciences Group, Chelsea College, University of London, SW3 6LX The kymograph, although revered by many generations of physiologists, suffers from a number of limitations which have been partly overcome in modem pen recorders. The almost frictionless bearing surfaces of modern galvanometers endow an improved frequency response on the pen recorders currently available. Austerity budgets and the current price inflation of all materials and animals as well as vastly increased student numbers require that economies
PROCEEDINGS OF THE loP be made without sacrificing educational principles. One way of achieving such economies is to operate a number of recorders in parallel, obtaining multiple records simultaneously at low cost, reproducing the desired biological parameters from a single animal preparation. It is virtually impossible to operate a number of kymographs in parallel, a severe limitation. A design modification introduced early on George Washington oscillographs (G.W.) facilitates such simultaneous parallel recordings to be made on G.W. and other pen recorders, U.V. recorders, oscilloscopes, FM tape Master unit
Amplified signal
Ink writing
Inkswitingrp
(0-6 V) detected at appropriate pin on
osc riograph
Slaved units
monitoring
|Socket\(90 Biological
ip
Di
a
coupler amplier Stored data _can be used for the repeat demonstrations
YWA*
O
s
F
tape
recorder \ lClosed circuit television
D
Computer
Fig. 1. Diagrammatic representation of slaving from a single channel oscillograph.
recorders, video recorders and computer terminals, as shown diagrammatically in Fig. 1. This facility was achieved by wiring the output of the driver amplifier to an appropriate pin of a monitoring socket, located on the deck of the G.W. oscillograph. The amplified signals vary within the range 0-6 V and standard calibration pulses can be fed into the system to calibrate the slaved units. The versatility of this system was demonstrated as one method of achieving a solution in structured course teaching and of doing sophisticated demonstrations to large numbers of students. If closed circuit television observation of the preparation could be used at the same time, students would benefit even more. The author wishes to thank Mr Ken Hore for his advice and help with operating the CCTV and George Washington Ltd for lending all the oscillographic equipment used in this demonstration.
