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A saline transfusion technique for crayfish cns studies
Camp. Biochem. Physiol., 1974, Vol. 47A, pp, 118.5 to 1190. Pergamon Press. Printed in Great Britain
A SALINE TRANSFUSION TECHNIQUE CNS STUDIES ROBERT
FOR CRAYFISH
C. TAYLOR
Department of Zoology, University of Georgia, Athens, Georgia 30602, U.S.A. (Received 7 May 1973)
The effects of commonly used dissection techniques were tested by observing their effects on the activity of two interneurons. The control level was determined using chronic electrodes. 2. The major problem with the tested techniques was an anoxia-induced deterioration of the central nervous system. 3. A technique is described which will maintain near normal output for 8 or more hours.
Abstract-l.
INTRODUCTION NUMEROUS
investigators have observed the rapid functional deterioration of the crustacean CNS, apparently in response to anoxia induced by various dissection techniques. The difficulties were partially overcome by cannulation (Maynard, 1966, lobster; Pabst & Kennedy, 1968, crayfish; Sandeman, 1969, Curcinus) or blood replacement (de Vlieger & Wiersma, 1971). The other commonly used approach was implantation on large nerves (Rowell, 1963; Taylor, 1968; Wiersma & Yanagisawa, 1971). However, neither approach was entirely satisfactory. Effective cannulation was extremely difficult or impossible in some portions of the CNS due to the fineness and number of blood vessels serving a specific ganglia, and implantation did not have the necessary exactness to answer many questions. Recording with implanted electrodes produced fairly accurate control levels for specific interneurons, and could be used as a reference to measure the success of various dissection techniques. The effects of three widely used techniques were tested on two interneurons that responded to rapid displacements of the statocysts (WVR interneuron). The techniques tested included dorsal artery cannulation, evisceration and blood replacement, and decapitation followed by recording in iced saline. The study of the above three led to the development of the technique described in this paper which maintained the nervous system in a functional state for up to 8 hr and alive for over 36 hr. MATERIALS
AND METHODS
Procambarus clarkii was used throughout the study. The circulatory system was injected with India ink to observe its anatomy. The tail was forced into an extreme flexed position and a 24-gauge hypodermic needle, attached to 1185
ROBERTC. TAYLOR
1186
an ink-filled syringe, was inserted under the thoracic carapace and into the heart. A small amount of ink was injected, and after 2-3 min the crayfish was opened and drawings or photographs made. A few latex-injected specimen (Carolina Biological Supplies) were used for comparative purposes. The circulatory system within the brain was followed through serial sections; stained at room temperature with fast green FCF and naphthol yellow S (0.1% w/v, each dye) (Bryan, 1970), The blood vessels stained a bright yellow while the neural elements were various shades of green and blue. Recording techniques were the same as those previously reported (Taylor, 1968). RESULTS
AND DISCUSSION
A distinction must be made between functional and alive if a preparation is used while studying integration in the central nervous system. To make this distinction two criteria were used: (1) a normal appearing visual response in the connectives and (2) a constant threshold for the water-vibration receptor interneurons (WVR), determined with chronic electrodes. Figure 1 indicates the sensitivity of the second method. The WVR decreased in sensitivity following removal of two legs (a) and (d) and all eight legs (b) and (c) at a different rate for The depression had an approximate lag time, from the different temperatures. initial cut to the onset, of about 10 min.
I 0
\.
I
I
60
30 Time,
I
90
min
FIG. 1. Depressions in sensitivity produced by removal of two (a, d) and eight legs (b, c). The effect is shown for three different temperatures.
An implanted various dissection
electrode was used to monitor the health of the preparation while techiques were tried. Surprisingly, even dorsal artery cannula-
A SALINE TRANSFUSION
TECHNIQUE
FOR CRAYFISH CNS STUDIES
1187
tion produced a preparation with, at most, 30 min of near threshold response followed by a rapid increase in threshold and complete failure within 2 hr. The other two techniques were less successful. If the preparation deterioration, produced by the dissection technique, was caused by anoxia the same effects should have been also produced by recording in oxygen-depleted water. Anoxic conditions were produced by the continual bubbling of N, into the closed recording vessel. Chronic implanted animals were immobilized in the vessel and recordings made until the WVR stopped responding. During the tests, several similar effects were noted. Large interneurons were the first to stop responding, followed systematically by smaller and smaller cells. As a cells condition deteriorated the threshold and latency increased, in some cells the latency changed by more than 10 msec. The latency and response could be restored to a value near the control level by increasing the stimulus intensity but for only a short time. Simple responses, involving only one or two synapses, responded longer than did more complex chains. Sensory fibers survived long after the interneurons had stopped responding. Because anoxia appeared to be the principal problem, the anatomy of the cephalic circulatory system was determined (Fig. 2). Four large vessels, one dorsal, two lateral, and a ventral artery all supplied the brain. By noting, in some cases, where the injected ink stopped it was determined that all four arteries carried blood toward the brain. The two lateral arteries and the ventral artery anastomosed below the brain. It was, consequently, impossible to cut any one of the three without creating a low resistance leak through which the majority of the saline could pass. Dorsal artery cannulation did not maintain the brain because it only supplied the deeper mid-regions of the brain, the medial portions of the lateral neuropiles and the giant fibers, whereas the more lateral and ventral aspects of the brain were supplied by the lateral and ventral arteries as were the antennules and antennae. The complexity of the circulatory system was far greater than would be expected from many contemporary publications on the subject. The arteries, rather than dumping into the body cavity, divide and subdivide forming structures very similar in appearance to vertebrate capillaries. Similar complexities were observed in the crayfish opener muscle (Lang et al., 1971) and the crab brain (Sandeman, 1967). Every neuropile was covered by an extensive network of capillaries, and the giant cells received separate branches of small vessels which formed enlarged “feet” over the fiber. The thoroughness of circulatory innervation in the brain only punctuated the CNS susceptibility to anoxia. From the above evidence it was apparent that to study any integrative function an adequate supply of blood or oxygenated saline must be continuously supplied to the nervous system. A summary of one technique that produced near normal function in the brain for up to 8 hr follows. 1. As an anesthetic the crayfish were held overnight at 5°C. This tended to significantly reduce the size of the depressions. 2. The claws were removed by autotomy.
1188
ROBERTC. TAYLOR
2. The circulatory system on the right side of the brain when viewed from the posterior looking toward the rostrum. The brain and associated nerves are indicated by the dotted lines. Blood vessels that pass behind the brain are indicated by dashed lines and vessels that are visible by solid lines. ASN, antenna1 sensory nerve; C.F., car frontale; Conn., circumesophageal connective; D.B.V., dorsal blood vesssel; G. G., green gland; L.B.V., lateral blood vessel; O., optic nerve; O.M., oculo-motor nerve; S.E.G., supraesophageal ganglia; Teg., tegumentary nerve; V.B.V., ventral blood vessel. FIG.
3. The stomach was drained and washed with iced saline, or the crayfish were held unfed for 2 weeks. Regurgitation onto the exposed brain caused rapid death of CNS. 4. A portion of the carapace above the dorsal end of the heart, was removed in cold saline. 5. The maxillipeds and legs were cut off and the animal immediately placed in iced saline and the pericardium punctured. Saline must be pumped in the If the heart stopped the lateral aspects of the heart and out the cut appendages. tail were squeezed to induce one or two tailflips, which generally caused the heart to start pumping. The iced saline prolonged the clotting time of the blood and allowed the complete replacement of blood by the saline. Forty-five min were allowed for replacement, or until the fluid pumped from the legs was clear and lacked a milky appearance. 6. The exoskeleton was removed about the portion of the nervous system of interest, taking care not to injure any part of the surrounding circulatory system. 7. Best results were obtained if the preparation was kept in iced saline during the recording session, which tended to decrease the oxygen demand,
A SALINE TRANSFUSION TECHNIQUE FOR CRAYFISH CNS STUDIES
1189
8. The distilled water used in the saline was deionized to reduce the ionic content to less than 1-O ppm. Failure to do so reduced the life expectancy by as much as 50 per cent. This appeared to be a qualitative effect rather than a quantitative one as the preparation was not very sensitive to wide fluctuations in the standard ions used in crayfish saline. The success of the technique is indicated in Fig. 3. The output of the WR interneuronlmin was automatically plotted. The initial level was recorded at 9.20 a.m. A 7-hr section was removed and the record (at arrow) continues from
60r
40 .3 E 3 s . 6 2 20x 5 LL
o-
FIG. 3.
of l/set.
A plot of the number of responsesjmin to click stimuli delivered at a rate A 7-hour portion of the record was removed and the record starts again at the arrow. Time mark = 30 min.
5.30 p.m. The deterioration of the preparation is evident as the sensitivity decreased for the following hour. Similar success has been achieved with motor nerves during a study of the antenna1 resistance reflex, and with receptors (Taylor, 1973). The addition of nutrients to the saline would probably extend the life of the preparation. One further consideration needed to produce a heabhy preparation was a healthy crayfish. Generally animals were kept in the Iaboratory, in holding tanks, for at least 2 weeks and preferably 2 months. Animals with cloudy eyes and leas than average agressiveness invariably produced a preparation which died within 2 hr. Crowded crayfish were also less likely to produce a good preparation. Three large crayfish per 30 cm2 was maximum and two was closer to optimum.
1190
ROBERTC. TAYLOR
Acknowledgements-This work was supported by N.S.F. Grant No. GB-26469. thank Mr. J. Westbrook for technical assistance during portions of this work.
I
REFERENCES BRYANJ. H. D. (1970) An eosin-fast green-naphthol yellow mixture for differential staining of cytologic components in mammalian spermatozoa. Stain Technol. 45, 231-236. LANG F., ATWOODH. L. & MORIN W. A. (1972) Innervation and vascular supply of the crayfish opener muscle. Z. Zellforsch. mikrosk. Anat. 127, 189-200. MAYNARDD. M. (1966) Integration in crustacean ganglia. In Symposia of the Society for Experimental Biology, Vol. XX, pp. 111-149. Academic Press, New York. PABST H. & KENNEDY D. (1968) Cutaneous mechanoreceptors influencing motor output in the crayfish abdomen. Z. vergl. Physiol. 57, 190-208. ROWELL C. H. F. (1963) A method for chronically implanting stimulating electrodes into the brains of locusts and some results of stimulation. r. exp. Biol. 40, 271-284. SANDEMAND. C. (1967) The vascular circulation in the brain, optic lobes, and thoracic ganglia of the crab Carcinus. Proc. R. Sot. Lond. B 168, 82-90. SANDEMAND. C. (1969) The synaptic link between the sensory and motoneurones in the eye-withdrawal reflex of the crab. J. exp. Biol. 50, 87-98. TAYLOR R. C. (1968) Water-vibration reception: a neurophysiological study in unrestrained crayfish. Comp. Biochem. Physiol. 27, 795-805. TAYLOR R. C. (1973) The crayfish antennae and brain-I. The antenna and its receptors. (In press.) DE VLIECER T. A. & WIERSMAC. A. G. (1971) On the rhythmic discharges of the swimmeret motor neurons of the crayfish. Proc. Akuc. Wetenschappen (Amsterdam) C 74, 247-255. WIERSMAC. A. G. & YANAGISAWA L. (1971) On types of interneurons responding to visual stimulation present in the optic nerve of the rock lobster, Panulirus interruptus. J. Neurobiol. 2, 291-309. Key Word Index-Crayfish;
CNS; perfusion; anoxia.
A SALINE TRANSFUSION TECHNIQUE CNS STUDIES ROBERT
FOR CRAYFISH
C. TAYLOR
Department of Zoology, University of Georgia, Athens, Georgia 30602, U.S.A. (Received 7 May 1973)
The effects of commonly used dissection techniques were tested by observing their effects on the activity of two interneurons. The control level was determined using chronic electrodes. 2. The major problem with the tested techniques was an anoxia-induced deterioration of the central nervous system. 3. A technique is described which will maintain near normal output for 8 or more hours.
Abstract-l.
INTRODUCTION NUMEROUS
investigators have observed the rapid functional deterioration of the crustacean CNS, apparently in response to anoxia induced by various dissection techniques. The difficulties were partially overcome by cannulation (Maynard, 1966, lobster; Pabst & Kennedy, 1968, crayfish; Sandeman, 1969, Curcinus) or blood replacement (de Vlieger & Wiersma, 1971). The other commonly used approach was implantation on large nerves (Rowell, 1963; Taylor, 1968; Wiersma & Yanagisawa, 1971). However, neither approach was entirely satisfactory. Effective cannulation was extremely difficult or impossible in some portions of the CNS due to the fineness and number of blood vessels serving a specific ganglia, and implantation did not have the necessary exactness to answer many questions. Recording with implanted electrodes produced fairly accurate control levels for specific interneurons, and could be used as a reference to measure the success of various dissection techniques. The effects of three widely used techniques were tested on two interneurons that responded to rapid displacements of the statocysts (WVR interneuron). The techniques tested included dorsal artery cannulation, evisceration and blood replacement, and decapitation followed by recording in iced saline. The study of the above three led to the development of the technique described in this paper which maintained the nervous system in a functional state for up to 8 hr and alive for over 36 hr. MATERIALS
AND METHODS
Procambarus clarkii was used throughout the study. The circulatory system was injected with India ink to observe its anatomy. The tail was forced into an extreme flexed position and a 24-gauge hypodermic needle, attached to 1185
ROBERTC. TAYLOR
1186
an ink-filled syringe, was inserted under the thoracic carapace and into the heart. A small amount of ink was injected, and after 2-3 min the crayfish was opened and drawings or photographs made. A few latex-injected specimen (Carolina Biological Supplies) were used for comparative purposes. The circulatory system within the brain was followed through serial sections; stained at room temperature with fast green FCF and naphthol yellow S (0.1% w/v, each dye) (Bryan, 1970), The blood vessels stained a bright yellow while the neural elements were various shades of green and blue. Recording techniques were the same as those previously reported (Taylor, 1968). RESULTS
AND DISCUSSION
A distinction must be made between functional and alive if a preparation is used while studying integration in the central nervous system. To make this distinction two criteria were used: (1) a normal appearing visual response in the connectives and (2) a constant threshold for the water-vibration receptor interneurons (WVR), determined with chronic electrodes. Figure 1 indicates the sensitivity of the second method. The WVR decreased in sensitivity following removal of two legs (a) and (d) and all eight legs (b) and (c) at a different rate for The depression had an approximate lag time, from the different temperatures. initial cut to the onset, of about 10 min.
I 0
\.
I
I
60
30 Time,
I
90
min
FIG. 1. Depressions in sensitivity produced by removal of two (a, d) and eight legs (b, c). The effect is shown for three different temperatures.
An implanted various dissection
electrode was used to monitor the health of the preparation while techiques were tried. Surprisingly, even dorsal artery cannula-
A SALINE TRANSFUSION
TECHNIQUE
FOR CRAYFISH CNS STUDIES
1187
tion produced a preparation with, at most, 30 min of near threshold response followed by a rapid increase in threshold and complete failure within 2 hr. The other two techniques were less successful. If the preparation deterioration, produced by the dissection technique, was caused by anoxia the same effects should have been also produced by recording in oxygen-depleted water. Anoxic conditions were produced by the continual bubbling of N, into the closed recording vessel. Chronic implanted animals were immobilized in the vessel and recordings made until the WVR stopped responding. During the tests, several similar effects were noted. Large interneurons were the first to stop responding, followed systematically by smaller and smaller cells. As a cells condition deteriorated the threshold and latency increased, in some cells the latency changed by more than 10 msec. The latency and response could be restored to a value near the control level by increasing the stimulus intensity but for only a short time. Simple responses, involving only one or two synapses, responded longer than did more complex chains. Sensory fibers survived long after the interneurons had stopped responding. Because anoxia appeared to be the principal problem, the anatomy of the cephalic circulatory system was determined (Fig. 2). Four large vessels, one dorsal, two lateral, and a ventral artery all supplied the brain. By noting, in some cases, where the injected ink stopped it was determined that all four arteries carried blood toward the brain. The two lateral arteries and the ventral artery anastomosed below the brain. It was, consequently, impossible to cut any one of the three without creating a low resistance leak through which the majority of the saline could pass. Dorsal artery cannulation did not maintain the brain because it only supplied the deeper mid-regions of the brain, the medial portions of the lateral neuropiles and the giant fibers, whereas the more lateral and ventral aspects of the brain were supplied by the lateral and ventral arteries as were the antennules and antennae. The complexity of the circulatory system was far greater than would be expected from many contemporary publications on the subject. The arteries, rather than dumping into the body cavity, divide and subdivide forming structures very similar in appearance to vertebrate capillaries. Similar complexities were observed in the crayfish opener muscle (Lang et al., 1971) and the crab brain (Sandeman, 1967). Every neuropile was covered by an extensive network of capillaries, and the giant cells received separate branches of small vessels which formed enlarged “feet” over the fiber. The thoroughness of circulatory innervation in the brain only punctuated the CNS susceptibility to anoxia. From the above evidence it was apparent that to study any integrative function an adequate supply of blood or oxygenated saline must be continuously supplied to the nervous system. A summary of one technique that produced near normal function in the brain for up to 8 hr follows. 1. As an anesthetic the crayfish were held overnight at 5°C. This tended to significantly reduce the size of the depressions. 2. The claws were removed by autotomy.
1188
ROBERTC. TAYLOR
2. The circulatory system on the right side of the brain when viewed from the posterior looking toward the rostrum. The brain and associated nerves are indicated by the dotted lines. Blood vessels that pass behind the brain are indicated by dashed lines and vessels that are visible by solid lines. ASN, antenna1 sensory nerve; C.F., car frontale; Conn., circumesophageal connective; D.B.V., dorsal blood vesssel; G. G., green gland; L.B.V., lateral blood vessel; O., optic nerve; O.M., oculo-motor nerve; S.E.G., supraesophageal ganglia; Teg., tegumentary nerve; V.B.V., ventral blood vessel. FIG.
3. The stomach was drained and washed with iced saline, or the crayfish were held unfed for 2 weeks. Regurgitation onto the exposed brain caused rapid death of CNS. 4. A portion of the carapace above the dorsal end of the heart, was removed in cold saline. 5. The maxillipeds and legs were cut off and the animal immediately placed in iced saline and the pericardium punctured. Saline must be pumped in the If the heart stopped the lateral aspects of the heart and out the cut appendages. tail were squeezed to induce one or two tailflips, which generally caused the heart to start pumping. The iced saline prolonged the clotting time of the blood and allowed the complete replacement of blood by the saline. Forty-five min were allowed for replacement, or until the fluid pumped from the legs was clear and lacked a milky appearance. 6. The exoskeleton was removed about the portion of the nervous system of interest, taking care not to injure any part of the surrounding circulatory system. 7. Best results were obtained if the preparation was kept in iced saline during the recording session, which tended to decrease the oxygen demand,
A SALINE TRANSFUSION TECHNIQUE FOR CRAYFISH CNS STUDIES
1189
8. The distilled water used in the saline was deionized to reduce the ionic content to less than 1-O ppm. Failure to do so reduced the life expectancy by as much as 50 per cent. This appeared to be a qualitative effect rather than a quantitative one as the preparation was not very sensitive to wide fluctuations in the standard ions used in crayfish saline. The success of the technique is indicated in Fig. 3. The output of the WR interneuronlmin was automatically plotted. The initial level was recorded at 9.20 a.m. A 7-hr section was removed and the record (at arrow) continues from
60r
40 .3 E 3 s . 6 2 20x 5 LL
o-
FIG. 3.
of l/set.
A plot of the number of responsesjmin to click stimuli delivered at a rate A 7-hour portion of the record was removed and the record starts again at the arrow. Time mark = 30 min.
5.30 p.m. The deterioration of the preparation is evident as the sensitivity decreased for the following hour. Similar success has been achieved with motor nerves during a study of the antenna1 resistance reflex, and with receptors (Taylor, 1973). The addition of nutrients to the saline would probably extend the life of the preparation. One further consideration needed to produce a heabhy preparation was a healthy crayfish. Generally animals were kept in the Iaboratory, in holding tanks, for at least 2 weeks and preferably 2 months. Animals with cloudy eyes and leas than average agressiveness invariably produced a preparation which died within 2 hr. Crowded crayfish were also less likely to produce a good preparation. Three large crayfish per 30 cm2 was maximum and two was closer to optimum.
1190
ROBERTC. TAYLOR
Acknowledgements-This work was supported by N.S.F. Grant No. GB-26469. thank Mr. J. Westbrook for technical assistance during portions of this work.
I
REFERENCES BRYANJ. H. D. (1970) An eosin-fast green-naphthol yellow mixture for differential staining of cytologic components in mammalian spermatozoa. Stain Technol. 45, 231-236. LANG F., ATWOODH. L. & MORIN W. A. (1972) Innervation and vascular supply of the crayfish opener muscle. Z. Zellforsch. mikrosk. Anat. 127, 189-200. MAYNARDD. M. (1966) Integration in crustacean ganglia. In Symposia of the Society for Experimental Biology, Vol. XX, pp. 111-149. Academic Press, New York. PABST H. & KENNEDY D. (1968) Cutaneous mechanoreceptors influencing motor output in the crayfish abdomen. Z. vergl. Physiol. 57, 190-208. ROWELL C. H. F. (1963) A method for chronically implanting stimulating electrodes into the brains of locusts and some results of stimulation. r. exp. Biol. 40, 271-284. SANDEMAND. C. (1967) The vascular circulation in the brain, optic lobes, and thoracic ganglia of the crab Carcinus. Proc. R. Sot. Lond. B 168, 82-90. SANDEMAND. C. (1969) The synaptic link between the sensory and motoneurones in the eye-withdrawal reflex of the crab. J. exp. Biol. 50, 87-98. TAYLOR R. C. (1968) Water-vibration reception: a neurophysiological study in unrestrained crayfish. Comp. Biochem. Physiol. 27, 795-805. TAYLOR R. C. (1973) The crayfish antennae and brain-I. The antenna and its receptors. (In press.) DE VLIECER T. A. & WIERSMAC. A. G. (1971) On the rhythmic discharges of the swimmeret motor neurons of the crayfish. Proc. Akuc. Wetenschappen (Amsterdam) C 74, 247-255. WIERSMAC. A. G. & YANAGISAWA L. (1971) On types of interneurons responding to visual stimulation present in the optic nerve of the rock lobster, Panulirus interruptus. J. Neurobiol. 2, 291-309. Key Word Index-Crayfish;
CNS; perfusion; anoxia.