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Aplysia eye: modulation by efferent optic nerve activity
Brain Research, 115 (1976) 501-505 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Aplysio J. L.
501
eye: modulation by efferent optic nerve activity
LUBORSKY-MOORE and J. W. JACKLET
Department of Anatomy, Medical Universityof South Carolina, Charleston, S.C., 29401 and Department of Biological Sciences, State University of New York, Albany, N.Y. 12222 (U.S.A.)
(Accepted July 6th, 1976)
The results of previous studies 1,s suggested to us that some of the chemical synapses observed in the neuropile of the Aplysia eye7 (Luborsky-Moore and Jacklet, in preparation) belong to efferent optic nerve fibers. These studies show that when the eye and optic nerve are isolated a regular pattern of compound action potentials (CAPs) is observed in the optic nerve. But, when the eye is attached to the cerebral ganglion, the CAP activity is less regular and has a lower frequency1,s. A similar inhibition of the isolated eye is produced by exogenously applied acetylcholinea. In this study we compared the optic nerve activity of detached (cut optic nerve) eyes and attached (intact optic nerve) eyes in the presence of D-tubocurarine, which blocks acetylcholine action a and found that n-tubocurarine blocked the inhibition of the attached eye. We also filled the optic nerve with cobalt chloride and found that cells in the cerebral ganglion have axons in the optic nerve. This suggests that efferent impulses modulate the neural output of the eyes. Aplysia californica (Pacific Bio-Marine, California) were kept in artificial seawater tanks at 15 °C and were subjected to a light-dark cycle (LD, 12:12). The eye preparations were maintained in darkness in artificial seawater or culture medium 6 at 15 °C. Optic nerve activity was monitered with a suction electrode (polyethylene tubing containing a chlorided silver wire) and recorded on a Grass polygraph. When recording from the attached eye, the cerebral ganglion was placed in a separate compartment within the recording chamber, and the small gap in the ganglion compartment (containing a fraction of the nerve) sealed with vasoline. The chamber was kept in a light tight box and the D-tubocurarine (Lilly) in seawater was applied to the eye compartment. Optic nerve activity was recorded for 2-4 days from each of 15 preparations. Since the eyes exhibit a circadian rhythm of CAP frequency1,5,6, the effect of D-tubocurarine on each preparation was tested for 2 h at about the same time on successive days. The eye responds to light with a characteristic increase in the number of CAPs4; the light response was tested before, during and after drug application. The optic nerve was filled by diffusion by placing the cut end of the nerve in 2 M cobaltous chloride for 12-24 h. The cobalt was precipitated with 3 9/o ammonium
502 sulfate. Ten cerebral ganglia were fixed in f o r m a l i n ( f o r m a l i n : seawater, 1 : 7), dehydrated, cleared and p h o t o g r a p h e d as whole m o u n t s with P o l a r o i d type 55 film. The s p o n t a n e o u s c o m p o u n d a c t i o n p o t e n t i a l s ( C A P s ) recorded in the d a r k from the two types o f p r e p a r a t i o n s used in this study are shown in Fig. 1. C A P s f r o m b o t h p r e p a r a t i o n s frequently occurred in groups. There was a relatively c o n s t a n t interval between g r o u p s o f C A P s f r o m the d e t a c h e d eye (Fig. IA) while the C A P s from the a t t a c h e d eye (Fig. 1B) were separated by variable time intervals. Optic nerve potentials t h a t were smaller in a m p l i t u d e (10-40/~V) than the C A P s (100-200 #V) were interspersed in the recordings f r o m the a t t a c h e d eye a n d were n o t seen in recordings from the detached eyes indicating that the low a m p l i t u d e activity was originating in the ganglion. These small potentials were m o s t frequent d u r i n g inhibition o f the eye when the n u m b e r o f C A P s was low.
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Fig. 1. Compound action potentials recorded extracellularly from the detached eye optic nerve (A) and the attached eye optic nerve (B). Efferent activity was only evident in recordings from the attached eye (e.g., between the two points of line B) and was coincident with inhibition of the CAPs. CAP amplitude is about 100/~V. Fig. 2. The effect of D-tubocurarine applied to the eye alone on the CAP pattern from the attached eye. A: control record before D-tubocurarine. B: 10 -4 M D-tubocurarine. C: control record after D-tubocurarine. CAP amplitude is about 150/~V.
503 However, in D-tubocurarine the CAP pattern of the attached eye became more regular and resembled that of the detached eye (Fig. 2). When the D-tubocurarine was removed, the CAP pattern again became irregular. The threshold concentration o~" D-tubocurarine needed to obtain this effect was about 10-8 M, while the most effective concentration was about 10-4 M. D-Tubocurarine did not change the characteristic pattern of CAPs from the detached eye. Also, when 10-4 M D-tubocurarine was applied to the cerebral ganglion alone there was no effect on the efferent activity indicating that the effect of D-tubocurarine was occurring specifically at the eye. In addition, both types of preparations responded to light with a similar pattern and number of CAPs, indicating that inhibition is not effective during the light response. After the optic nerve was filled with cobalt, cobalt filled fibers could be seen arising from cells in the cerebral ganglion, continuing toward the caudal quadrant of the ganglion or ending upon entering the ganglion. Only ipsilateral structures filled with cobalt; no contralateral deposits of cobalt were detected. The cells that filled (Fig. 3) were 20-30 # m in diameter. Six to seven cells were located at the base of each rhinophore nerve (at the anterior edge of the ganglion) and 3-4 cells were located in the left rhinophore nerve. The position of these cells relative to each other varied slightly from one ganglion to another. A small cell (10/~m) along the midline of the ganglion, with several branching processes, also filled with cobalt.
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Fig. 3. A: dorsal view of a cerebral ganglion whose left optic nerve (ON) was filled with cobalt chloride. The cells (arrows) in the rhinophore nerve (RN) and cells (arrows) at the base of the rhinophore nerve that filled with cobalt are shown. B: enlarged view of the RN to show the processes of cobalt filled cells (arrows).
504 Chemical synapses 7 (Luborsky-Moore and Jacklet, in prep.) have been observed in the .4plysia eye, but the results of previous studies indicated that they are not involved in retinal cell interactions. The eye contains both receptor cells and secondary (non-receptor) cellsa. CAP production, which reflects secondary cell activity4, 5, and the light response which represents receptor cell activation 4, are thought to involve electrical junctions 6. The results of this study indicate that the eye is subject to efferent control by chemical synapses because D-tubocurarine blocked the inhibition of optic nerve activity in the attached eye preparation. D-Tubocurarine has been shown to block the inhibitory action ofacetylcholine in the central nervous system 9 and the eye3 of Aplysia. However, it also blocks excitatory and inhibitory actions of serotonin and excitatory actions of dopamine and acetycholine2. Since the efferent activity results in inhibition of the eye, and serotonin (Luborsky-Moore, unpublished observations) and dopamine 3 result in excitation of the eye, the D-tubocurarine is probably acting at a cholinergic type of synapse. Thus, while the preliminary results of this pharmacological study are suggestive, further pharmacological, biochemical and physiological studies are required to establish that inhibition of the eye is due to acetylcholine released by efferent impulses in axons of cerebral ganglion cells. The results of this study also confirm that neurons in the cerebral ganglion send axons to the eye. However, the anterior position of the cells that filled with cobalt do not correspond to those identified by Rossner 8. Using intracellular recordings he described a group of cells in the medial and caudal areas of the ganglion that responded to light or electrical stimulation of the eye. It is possible that (1) all the cells with axons in the optic nerve did not fill with cobalt due to limitations of the technique and/or (2) some of the filled cells are driven by other cerebral ganglion cells that do not have axons in the optic nerve, and (3) the relatively small cobalt filled cells could be overlooked in intracellular studies. The second possibility is supported by the observation that stimulation of the rhinophore nerves, the tentacular nerves or the contralateral optic nerve inhibits the efferent activity 1. Thus, axons from these nerves may synapse directly with the cobalt filled cells or with autoactive interneurons that drive the cobalt filled cells. The similarity of the light responses of the detached and attached eyes suggests that the receptor cells have a strong synchronizing effect on the secondary cells. We suggest that the efferent fibers act upon the secondary ceils and not on the receptor cells because: (1) the light responses of the attached and detached eyes were similar showing that the receptor cells have not been inhibited, (2) the inhibition of CAP activity occurred in the dark when receptor cells are not active, and (3) a biphasic response is seen in the secondary cells when the optic nerve is stimulated a which could result from simultaneous stimulation of the secondary cell axon and an efferent axon with a synapse onto the secondary cell. In summary, this study provides evidence for efferent control of the eye by the following two criteria: (1) the presence of efferent axons in the optic nerve, and (2) the block of inhibition of the attached eye with D-tubocurarine. Furthermore, these results suggest that some of the chemical synapses in the eye belong to efferent nerves.
505 Supported by N . I . H . Traineeship G r a n t G M 0 2 0 1 4 (JLM) a n d N . I . H . G r a n t NS08443 (JWJ).
1 Eskin, A., Properties of the Aplysia visual system: in vitro entertainment of the circadian rhythm and centrifugal regulation of the eye, Z. vergL PhysioL, 74 (1971) 353-371. 2 Gerschenfeld, H. M., Chemical transmission in invertebrate central nervous systems and neuromuscular junctions, Physiol. Rev., 53 (1973) 2-119. 3 Jacklet, J. W., Synchronized neuronal activity and neurosecretory function of the eye of Aplysia, Proc. 24th Int. Union. PhysioL Sci., 7 (1968) 213. 4 Jacklet, J. W., Electrophysiological organization of the eye of Aplysia, J. gen. Physiol., 53 (1969) 21-42. 5 Jacklet, J. W., Neuronal population interactions in a circadian rhythm in Aplysia. In Neurobiology of the Invertebrates, Tihany (1971) (1973) pp. 363-380. 6 Jacklet, J. W., The circadian rhythm in the eye of Aplysia: effects of low Ca 2+ and high Mg~+, J. comp. Physiol., 87 (1973) 329-338. 7 Jacklet, J. W., Alvarez, R. and Bernstein, B., Ultrastructure of the eye of Aplysia, J. Ultrastruct. Res., 38 (1972) 246-261. 8 Rossner, K. L., Central projections of the Aplysia visual system, Comp. Biochem. PhysioL, 48 (1974) 609-615. 9 Tauc, L., Gerschenfeld, H. M., A cholinergic mechanism of inhibitory synaptic transmission in a molluscan nervous system, J. NeurophysioL, 25 (1962) 236-262.
Aplysio J. L.
501
eye: modulation by efferent optic nerve activity
LUBORSKY-MOORE and J. W. JACKLET
Department of Anatomy, Medical Universityof South Carolina, Charleston, S.C., 29401 and Department of Biological Sciences, State University of New York, Albany, N.Y. 12222 (U.S.A.)
(Accepted July 6th, 1976)
The results of previous studies 1,s suggested to us that some of the chemical synapses observed in the neuropile of the Aplysia eye7 (Luborsky-Moore and Jacklet, in preparation) belong to efferent optic nerve fibers. These studies show that when the eye and optic nerve are isolated a regular pattern of compound action potentials (CAPs) is observed in the optic nerve. But, when the eye is attached to the cerebral ganglion, the CAP activity is less regular and has a lower frequency1,s. A similar inhibition of the isolated eye is produced by exogenously applied acetylcholinea. In this study we compared the optic nerve activity of detached (cut optic nerve) eyes and attached (intact optic nerve) eyes in the presence of D-tubocurarine, which blocks acetylcholine action a and found that n-tubocurarine blocked the inhibition of the attached eye. We also filled the optic nerve with cobalt chloride and found that cells in the cerebral ganglion have axons in the optic nerve. This suggests that efferent impulses modulate the neural output of the eyes. Aplysia californica (Pacific Bio-Marine, California) were kept in artificial seawater tanks at 15 °C and were subjected to a light-dark cycle (LD, 12:12). The eye preparations were maintained in darkness in artificial seawater or culture medium 6 at 15 °C. Optic nerve activity was monitered with a suction electrode (polyethylene tubing containing a chlorided silver wire) and recorded on a Grass polygraph. When recording from the attached eye, the cerebral ganglion was placed in a separate compartment within the recording chamber, and the small gap in the ganglion compartment (containing a fraction of the nerve) sealed with vasoline. The chamber was kept in a light tight box and the D-tubocurarine (Lilly) in seawater was applied to the eye compartment. Optic nerve activity was recorded for 2-4 days from each of 15 preparations. Since the eyes exhibit a circadian rhythm of CAP frequency1,5,6, the effect of D-tubocurarine on each preparation was tested for 2 h at about the same time on successive days. The eye responds to light with a characteristic increase in the number of CAPs4; the light response was tested before, during and after drug application. The optic nerve was filled by diffusion by placing the cut end of the nerve in 2 M cobaltous chloride for 12-24 h. The cobalt was precipitated with 3 9/o ammonium
502 sulfate. Ten cerebral ganglia were fixed in f o r m a l i n ( f o r m a l i n : seawater, 1 : 7), dehydrated, cleared and p h o t o g r a p h e d as whole m o u n t s with P o l a r o i d type 55 film. The s p o n t a n e o u s c o m p o u n d a c t i o n p o t e n t i a l s ( C A P s ) recorded in the d a r k from the two types o f p r e p a r a t i o n s used in this study are shown in Fig. 1. C A P s f r o m b o t h p r e p a r a t i o n s frequently occurred in groups. There was a relatively c o n s t a n t interval between g r o u p s o f C A P s f r o m the d e t a c h e d eye (Fig. IA) while the C A P s from the a t t a c h e d eye (Fig. 1B) were separated by variable time intervals. Optic nerve potentials t h a t were smaller in a m p l i t u d e (10-40/~V) than the C A P s (100-200 #V) were interspersed in the recordings f r o m the a t t a c h e d eye a n d were n o t seen in recordings from the detached eyes indicating that the low a m p l i t u d e activity was originating in the ganglion. These small potentials were m o s t frequent d u r i n g inhibition o f the eye when the n u m b e r o f C A P s was low.
1, //I II//
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,~J
•
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!( IJif
l........................................ f. . f l
,, 1t/1111111tl 1t1111[[[ II JW.... ~......II.....IIt.... JlI Jll
III lit.... /III ... llllllI1 ....Illl
Fig. 1. Compound action potentials recorded extracellularly from the detached eye optic nerve (A) and the attached eye optic nerve (B). Efferent activity was only evident in recordings from the attached eye (e.g., between the two points of line B) and was coincident with inhibition of the CAPs. CAP amplitude is about 100/~V. Fig. 2. The effect of D-tubocurarine applied to the eye alone on the CAP pattern from the attached eye. A: control record before D-tubocurarine. B: 10 -4 M D-tubocurarine. C: control record after D-tubocurarine. CAP amplitude is about 150/~V.
503 However, in D-tubocurarine the CAP pattern of the attached eye became more regular and resembled that of the detached eye (Fig. 2). When the D-tubocurarine was removed, the CAP pattern again became irregular. The threshold concentration o~" D-tubocurarine needed to obtain this effect was about 10-8 M, while the most effective concentration was about 10-4 M. D-Tubocurarine did not change the characteristic pattern of CAPs from the detached eye. Also, when 10-4 M D-tubocurarine was applied to the cerebral ganglion alone there was no effect on the efferent activity indicating that the effect of D-tubocurarine was occurring specifically at the eye. In addition, both types of preparations responded to light with a similar pattern and number of CAPs, indicating that inhibition is not effective during the light response. After the optic nerve was filled with cobalt, cobalt filled fibers could be seen arising from cells in the cerebral ganglion, continuing toward the caudal quadrant of the ganglion or ending upon entering the ganglion. Only ipsilateral structures filled with cobalt; no contralateral deposits of cobalt were detected. The cells that filled (Fig. 3) were 20-30 # m in diameter. Six to seven cells were located at the base of each rhinophore nerve (at the anterior edge of the ganglion) and 3-4 cells were located in the left rhinophore nerve. The position of these cells relative to each other varied slightly from one ganglion to another. A small cell (10/~m) along the midline of the ganglion, with several branching processes, also filled with cobalt.
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Fig. 3. A: dorsal view of a cerebral ganglion whose left optic nerve (ON) was filled with cobalt chloride. The cells (arrows) in the rhinophore nerve (RN) and cells (arrows) at the base of the rhinophore nerve that filled with cobalt are shown. B: enlarged view of the RN to show the processes of cobalt filled cells (arrows).
504 Chemical synapses 7 (Luborsky-Moore and Jacklet, in prep.) have been observed in the .4plysia eye, but the results of previous studies indicated that they are not involved in retinal cell interactions. The eye contains both receptor cells and secondary (non-receptor) cellsa. CAP production, which reflects secondary cell activity4, 5, and the light response which represents receptor cell activation 4, are thought to involve electrical junctions 6. The results of this study indicate that the eye is subject to efferent control by chemical synapses because D-tubocurarine blocked the inhibition of optic nerve activity in the attached eye preparation. D-Tubocurarine has been shown to block the inhibitory action ofacetylcholine in the central nervous system 9 and the eye3 of Aplysia. However, it also blocks excitatory and inhibitory actions of serotonin and excitatory actions of dopamine and acetycholine2. Since the efferent activity results in inhibition of the eye, and serotonin (Luborsky-Moore, unpublished observations) and dopamine 3 result in excitation of the eye, the D-tubocurarine is probably acting at a cholinergic type of synapse. Thus, while the preliminary results of this pharmacological study are suggestive, further pharmacological, biochemical and physiological studies are required to establish that inhibition of the eye is due to acetylcholine released by efferent impulses in axons of cerebral ganglion cells. The results of this study also confirm that neurons in the cerebral ganglion send axons to the eye. However, the anterior position of the cells that filled with cobalt do not correspond to those identified by Rossner 8. Using intracellular recordings he described a group of cells in the medial and caudal areas of the ganglion that responded to light or electrical stimulation of the eye. It is possible that (1) all the cells with axons in the optic nerve did not fill with cobalt due to limitations of the technique and/or (2) some of the filled cells are driven by other cerebral ganglion cells that do not have axons in the optic nerve, and (3) the relatively small cobalt filled cells could be overlooked in intracellular studies. The second possibility is supported by the observation that stimulation of the rhinophore nerves, the tentacular nerves or the contralateral optic nerve inhibits the efferent activity 1. Thus, axons from these nerves may synapse directly with the cobalt filled cells or with autoactive interneurons that drive the cobalt filled cells. The similarity of the light responses of the detached and attached eyes suggests that the receptor cells have a strong synchronizing effect on the secondary cells. We suggest that the efferent fibers act upon the secondary ceils and not on the receptor cells because: (1) the light responses of the attached and detached eyes were similar showing that the receptor cells have not been inhibited, (2) the inhibition of CAP activity occurred in the dark when receptor cells are not active, and (3) a biphasic response is seen in the secondary cells when the optic nerve is stimulated a which could result from simultaneous stimulation of the secondary cell axon and an efferent axon with a synapse onto the secondary cell. In summary, this study provides evidence for efferent control of the eye by the following two criteria: (1) the presence of efferent axons in the optic nerve, and (2) the block of inhibition of the attached eye with D-tubocurarine. Furthermore, these results suggest that some of the chemical synapses in the eye belong to efferent nerves.
505 Supported by N . I . H . Traineeship G r a n t G M 0 2 0 1 4 (JLM) a n d N . I . H . G r a n t NS08443 (JWJ).
1 Eskin, A., Properties of the Aplysia visual system: in vitro entertainment of the circadian rhythm and centrifugal regulation of the eye, Z. vergL PhysioL, 74 (1971) 353-371. 2 Gerschenfeld, H. M., Chemical transmission in invertebrate central nervous systems and neuromuscular junctions, Physiol. Rev., 53 (1973) 2-119. 3 Jacklet, J. W., Synchronized neuronal activity and neurosecretory function of the eye of Aplysia, Proc. 24th Int. Union. PhysioL Sci., 7 (1968) 213. 4 Jacklet, J. W., Electrophysiological organization of the eye of Aplysia, J. gen. Physiol., 53 (1969) 21-42. 5 Jacklet, J. W., Neuronal population interactions in a circadian rhythm in Aplysia. In Neurobiology of the Invertebrates, Tihany (1971) (1973) pp. 363-380. 6 Jacklet, J. W., The circadian rhythm in the eye of Aplysia: effects of low Ca 2+ and high Mg~+, J. comp. Physiol., 87 (1973) 329-338. 7 Jacklet, J. W., Alvarez, R. and Bernstein, B., Ultrastructure of the eye of Aplysia, J. Ultrastruct. Res., 38 (1972) 246-261. 8 Rossner, K. L., Central projections of the Aplysia visual system, Comp. Biochem. PhysioL, 48 (1974) 609-615. 9 Tauc, L., Gerschenfeld, H. M., A cholinergic mechanism of inhibitory synaptic transmission in a molluscan nervous system, J. NeurophysioL, 25 (1962) 236-262.