JOURNAL OF NEUROBIOLOGY, VOL. 8, NO. 3 , PP. 273-299
Neurophysiological Mechanisms Involved in Photo-Entrainment of the Circadian Rhythm from the Aplysia Eye ARNOLD ESKIN Biology Department, Rice Uniuersity, Houston, Texas 77001 Receiued I7 Nouember 1976; revised I9 January 1977. SUMMARY
An attempt was made to identify the neurophysiological processes involved in entrainment of the circadian rhythm of spontaneous optic nerve potentials from the Aplysia eye by determining whether pharmacological agents or ion substitutions could block phase shifts produced by single light pulses. Knowing which physiological processes are involved in entrainment should help define the morphological pathway traveled by entrainment information. A secretory step does not appear to be involved in the flow of entrainment information from the environment to the circadian oscillator. A treatment (HiMg LoCa) capable of inhibiting secretion did not interfere with phase shifting by light. Furthermore, treating eyes with putative transmitters or extracts of eyes did not phase shift the free running rhythm. Also, the phase shifting information is not translated into action potentials before reaching the oscillator since TTX-HiMg LoCa solutions did not block the light-induced phase shift. The photoreceptor potential does seem to be important for light-induced phase shifts. A correlation was found between the effects of treatments on the ERG and their effects on the light-induced phase shift. Solutions which decreased the ERG by 90% or more blocked phase shifting whereas solutions which decreased the ERG by less than 74% had no effect on phase shifting by light. The results from these studies are consistent with two pathways for the flow of phase shifting information to the circadian oscillator. The circadian oscillator may be associated with receptor cells and the entrainment pathway would include a step involving the photoreceptor potential. Alternatively, the circadian oscillator may be associated with secondary cells and receive entrainment information via the photoreceptor potential and passive spread of current through a gap junction. Higher order cells than second-order ones are probably not involved in the entrainment pathway. INTRODUCTION
The circadian rhythm of spontaneous nerve activity from the isolated eye of Aplysia californica can be photoentrained in vitro (Eskin, 1971). Therefore, 273 01977 by John Wiley & Sons, Inc.
274
ESKIN
the mechanisms responsible for photo-entrainment can be studied in the isolated eye. Several general steps must be involved in the entrainment process as entrainment information flows from the environment to the oscillator mechanism responsible for the eye rhythm. The environmental information must first be absorbed by a photopigment. The information is then transduced and coded into a form which can be propagated to the circadian oscillator. After propagation, the information must be decoded by the oscillator resulting in a phase shift of the rhythm. Different processes such as action potentials or synaptic transmission could be involved in the flow of information along this pathway. The two possible general locations for the circadian oscillator in the eye are the photoreceptor cells and nonphotoreceptor second-lor higher order neurons (assuming that support or glial cells are not involved). If the circadian oscillator is associated with the receptors, the flow of phase shifting information to the oscillator may or may not involve an electrical membrane event. If second- or higher order neurons are involved, the phase shifting information may be propagated electronically (passively) or via action potentials and by either secretory or electrical junctions between cells. An attempt has been made to identify the physiological processes involved in entrainment of the rhythm from the eye by determining whether pharmacological agents or ion substitutions could block phase shifts produced by single light pulses. Knowing which physiological processes are involved in entrainment should help define the morphological pathway traveled by entrainment information. The present results indicate that for phase shifting by light a membrane event is required, action potentials are not required, and chemical synaptic transmission and neurosecretion are not required. Several locations of the oscillator are consistent with these results. The oscillator may be associated with receptor cells and require an electrical membrane event for entrainment. Alternatively, the oscillator may be associated with second-order cells. In this case, the entrainment information would propagate electrotonically and be transmitted between cells by electrical junctions. MATERIALS AND METHODS Aplysia californica were obtained from Pacific Biomarine (Venice, California) and maintained on a 12:12 light-dark cycle in aquaria filled with Instant Ocean Sea Water a t 15°C. The eyes with optic nerves attached were removed within two hours before the onset of dark and placed in 20 ml Nalgene beakers containing two polyethylene tubes embedded in 10 ml of Sylgard (Dow Corning). The optic nerve was pulled into one of these tubes (PE 20) using suction. The suction was released and a platinum wire (platinum-blacked) was lowered into the tube to record the optic nerve impulses. ERGS were recorded using suction electrodes attached to the surface of the eye in the vicinity of the cornea. The impulses and ERGS were amplified and recorded using a Grass polygraph. The Nalgene beakers were partially submerged in a temperature regulated bath which maintained the eyes within a range of 0.4'C throughout an experiment. The average temperature of different experiments varied from 14.5 to 15.5OC. The beakers containing the eyes were housed in light-tight boxes. The light pulses were produced by a Bausch and Lomh microscope illuminator (6.5 V) centered 20 in. above the eyes. The lucite bath containing the beakers was wrapped with aluminum foil to reflect the light back toward the eyes. The temperature in the solutions surrounding the eyes increased by less than 1°C during the light pulses.
ENTRAINMENT MECHANISMS OF APLYSIA EYE RHYTHM 275 An attempt to determine the physiological processes involved in phase shifting by light pulses was made by examining which processes were necessary for such a phase shift to occur. Experimental solutions which interfere with physiological processes were administered to the eye one-half hour before the eye was exposed to light. The eye was then exposed to light for six hours a t CT 18-24 (CT 00 is the last dawn seen by the intact animal) and returned to constant dark for the remainder of the experiment (3 days). Within ten minutes after the end of the light pulse the experimental solution was withdrawn and the eyes were rinsed, and exposed once again to the control solution. The experimental solutions had to satisfy certain criteria for use in these experiments. Any effects of the treatments on impulse activity from the eye had to he reversible. In addition, the experimental solutions themselves could not produce phase shifts in the rhythm when presented in the absence of light. If any solution produced phase shifts, the interpretation of results regarding its ability to interfere with the phase shift produced by light would he difficult. Thus, before each of the different attempts to block phase shifting by light, a group of eyes was exposed to the different treatments for 6.5 hrs a t C T 17.5-24 to determine if the solution itself caused phase shifts. The experiments were performed by administering the experimental solution to one eye while the other eye from the same animal served as the “dark” control in the experiments with no light, and the “light” control in the experiments with light pulses. The phase shifts (Ab) produced by exposing eyes only to the experimental solutions were determined by the difference in phase (defined as the peak of the rhythms) between the experimental and control eyes (positive differences are advances and negative differences are delays). The blocking effect of a solution on the phase shift produced by light was determined by the difference ( D )in phase between the light control eye and the eye receiving the experimental solution plus light. (Positive differences indicate that the experimental eye was phase shifted by the light more than the control eye and negative differences indicate that the experimental solution had a blocking effect on phase shifting by light.) All Ag’s and D’s in the text were calculated using data obtained during the third cycle of the rhythm after the experimental treatment (see Fig. 2). The control solution (ASW) contained Instant Ocean Sea Water, 30 mM HEPES buffer, and 100 units/ml of penicillin and 100 mcg/ml of streptomycin. The experimental solutions and ion substitutions also contained this same amount of buffer and antibiotic. The pH of all solutions was 7.85 (adjusted using Ca (0H)z for the LoNa-Mannitol solutions and NaOH for all other solutions) and all solutions were filtered (Millipore, 0.22 pm) before their use. The drags were added directly to ASW. The following were obtained from Sigma: Procaine-HCL, Mephenesin, Glycine, yamino-n-butyric acid (GABA), L-glutamic acid (monosodium salt), Tetrodotoxin ( T T X ) ,Acetylcholine chloride, choline chloride, 5 Hydroxytryptamine creatinine sulfate (Serotonin), creatinine sulfate, DL-octopamine hydrochloride, L-aspartic acid, 3-Hydroxytyramine-HCL (Dopamine), Ethyleneglycol his(@-Aminoethylether)N,N’-tetraacetic acid (EGTA),Histamine (free base), and L-Ascorbic acid. The basic solutions with their ion substitutions are listed in the table. The low sodium solutions had sodium reduced from 463 mM to either 70 (LoNa-70 mM), or 7 mM (LoNa-7 mM). Tris (pH 7.85 at 15”C),choline, mannitol (D-mannitol), cesium, and lithium were used as substitutes for Na+ or NaCl. The amount of mannitol required to maintain the LoNa (7 mM-Mannitol solution isotonic with ASW was determined using an osmometer (Advanced Instruments). No Hepes buffer was added to the solutions containing Tris. Mg2+ was increased 2.5 times an&Ca2+decreased by lo2 times in the HiMg LoCa solution. The molarity of this solution was normalized by reducing NaCl. Ca2+was buffered with EGTA in some of the reduced Ca2+solutions and the concentration of Ca2+ was calculated after Portzehl, Caldwell, and Ruegg (1964). The solutions had various amounts of EGTA, Ca2+and Na+, the concentrations of the other ions remaining constant. The recipe for EGTA M ) is given in the table. The amounts of Ca2+ and EGTA added, respectively, I (Ca2+= 1.3 X to the other solutions were: EGTA I1 (Ca2+ = 6.4 X 10W7M ) - 5,6 mM; EGI’A I11 (Ca2+ = 1.4 X M ) - 10, 10.05 mM: EGTA IV (Ca2+ = 4.5 X lo-* M ) - 10.5,10.05 mM. In the high Ca2+ solution (HiCa),the Ca2+ concentration was increased 6 times and Na+ was reduced an appropriate amount. Na acetate and Na propionate were substituted for NaCl in the chloride substitution experiments. Solutions were made hypotonic by diluting ASW with an appropriate amount of deionized water and were made hypertonic by adding 0.5 M mannitol to ASW. The solutions surrounding the eyes were completely exchanged by coupling a polyethylene tube, with an attached 10 ml syringe, outside of the box, to the other polyethylene tube (PE 150) embedded
276
ESKIN
Fig. 1. An advance phase shift produced by a single 6-hr light pulse. The optic nerve impulse rhythms in all figures are from two isolated eyes, both from the same animal. One eye, the control, was in constant darkness throughout the experiment. The other eye was exposed to 6 hours of light during the time shown by the closed bar a t the bottom of the graph. Before and after the light pulse, this eye was in constant darkness. The open bars a t the bottom of the graphs represent the projected light portion (12 hours) of the light-dark cycle used to entrain the intact animal. All of the eyes were dissected and then placed in light-tight boxes a t the end of a light period. The data from this time to the projected onset of light shown in the figures were recorded, but they are not shown in any of the figures. in the Sylgard in the Nalgene beaker. The experimental eyes were rinsed twice (10 ml each time) with the experimental solutions before remaining in these solutions. At the end of treatments, the experimental solutions were withdrawn and the eyes were rinsed 6 to 9 times (10 ml each time) with filtered Instant Ocean Sea Water before being returned to ASW. The control eyes were rinsed one time with ASW at the beginning of treatments and were rinsed one time each with filtered Instant Ocean Sea Water and ASW a t the end of the treatments.
RESULTS
Phase s h i f t by light Eyes were exposed to single 6-hr light pulses at different phases of the rhythm during the first 24 hours of isolation to determine at which phase the largest phase shift would occur. The largest phase shifts were produced by light during C T 18-24. All experiments were performed a t this phase. At this phase, the light produced a 3.21 f 0.31-hr advance phase shift on the second day after the light pulse and a 3.28 f 0.45-hr phase shift on the third day after the light pulse (Fig. 1). (All f values are 95% confidence intervals). These average phase shifts were obtained by subtracting the mean phase of the “light” controls ( N = 36) from the mean phase of the “dark” controls ( N = 40) used in the experiments presented below.
Treatments which a f f e c t membrane potential changes
A variety of membrane conductance or potential changes might be involved in carrying the phase shifting information to the circadian oscillator. The amplitudes of these potential changes are a function of the ionic gradients for Na+,
ENTRAINMENT MECHANISMS OF APLYSIA EYE RHYTHM 277
K+, C1-, and Ca2+depending upon the specific currents involved in producing a particular potential change. The Aplysia photoreceptor potentials might vary with the concentration of Na+
Neurophysiological Mechanisms Involved in Photo-Entrainment of the Circadian Rhythm from the Aplysia Eye ARNOLD ESKIN Biology Department, Rice Uniuersity, Houston, Texas 77001 Receiued I7 Nouember 1976; revised I9 January 1977. SUMMARY
An attempt was made to identify the neurophysiological processes involved in entrainment of the circadian rhythm of spontaneous optic nerve potentials from the Aplysia eye by determining whether pharmacological agents or ion substitutions could block phase shifts produced by single light pulses. Knowing which physiological processes are involved in entrainment should help define the morphological pathway traveled by entrainment information. A secretory step does not appear to be involved in the flow of entrainment information from the environment to the circadian oscillator. A treatment (HiMg LoCa) capable of inhibiting secretion did not interfere with phase shifting by light. Furthermore, treating eyes with putative transmitters or extracts of eyes did not phase shift the free running rhythm. Also, the phase shifting information is not translated into action potentials before reaching the oscillator since TTX-HiMg LoCa solutions did not block the light-induced phase shift. The photoreceptor potential does seem to be important for light-induced phase shifts. A correlation was found between the effects of treatments on the ERG and their effects on the light-induced phase shift. Solutions which decreased the ERG by 90% or more blocked phase shifting whereas solutions which decreased the ERG by less than 74% had no effect on phase shifting by light. The results from these studies are consistent with two pathways for the flow of phase shifting information to the circadian oscillator. The circadian oscillator may be associated with receptor cells and the entrainment pathway would include a step involving the photoreceptor potential. Alternatively, the circadian oscillator may be associated with secondary cells and receive entrainment information via the photoreceptor potential and passive spread of current through a gap junction. Higher order cells than second-order ones are probably not involved in the entrainment pathway. INTRODUCTION
The circadian rhythm of spontaneous nerve activity from the isolated eye of Aplysia californica can be photoentrained in vitro (Eskin, 1971). Therefore, 273 01977 by John Wiley & Sons, Inc.
274
ESKIN
the mechanisms responsible for photo-entrainment can be studied in the isolated eye. Several general steps must be involved in the entrainment process as entrainment information flows from the environment to the oscillator mechanism responsible for the eye rhythm. The environmental information must first be absorbed by a photopigment. The information is then transduced and coded into a form which can be propagated to the circadian oscillator. After propagation, the information must be decoded by the oscillator resulting in a phase shift of the rhythm. Different processes such as action potentials or synaptic transmission could be involved in the flow of information along this pathway. The two possible general locations for the circadian oscillator in the eye are the photoreceptor cells and nonphotoreceptor second-lor higher order neurons (assuming that support or glial cells are not involved). If the circadian oscillator is associated with the receptors, the flow of phase shifting information to the oscillator may or may not involve an electrical membrane event. If second- or higher order neurons are involved, the phase shifting information may be propagated electronically (passively) or via action potentials and by either secretory or electrical junctions between cells. An attempt has been made to identify the physiological processes involved in entrainment of the rhythm from the eye by determining whether pharmacological agents or ion substitutions could block phase shifts produced by single light pulses. Knowing which physiological processes are involved in entrainment should help define the morphological pathway traveled by entrainment information. The present results indicate that for phase shifting by light a membrane event is required, action potentials are not required, and chemical synaptic transmission and neurosecretion are not required. Several locations of the oscillator are consistent with these results. The oscillator may be associated with receptor cells and require an electrical membrane event for entrainment. Alternatively, the oscillator may be associated with second-order cells. In this case, the entrainment information would propagate electrotonically and be transmitted between cells by electrical junctions. MATERIALS AND METHODS Aplysia californica were obtained from Pacific Biomarine (Venice, California) and maintained on a 12:12 light-dark cycle in aquaria filled with Instant Ocean Sea Water a t 15°C. The eyes with optic nerves attached were removed within two hours before the onset of dark and placed in 20 ml Nalgene beakers containing two polyethylene tubes embedded in 10 ml of Sylgard (Dow Corning). The optic nerve was pulled into one of these tubes (PE 20) using suction. The suction was released and a platinum wire (platinum-blacked) was lowered into the tube to record the optic nerve impulses. ERGS were recorded using suction electrodes attached to the surface of the eye in the vicinity of the cornea. The impulses and ERGS were amplified and recorded using a Grass polygraph. The Nalgene beakers were partially submerged in a temperature regulated bath which maintained the eyes within a range of 0.4'C throughout an experiment. The average temperature of different experiments varied from 14.5 to 15.5OC. The beakers containing the eyes were housed in light-tight boxes. The light pulses were produced by a Bausch and Lomh microscope illuminator (6.5 V) centered 20 in. above the eyes. The lucite bath containing the beakers was wrapped with aluminum foil to reflect the light back toward the eyes. The temperature in the solutions surrounding the eyes increased by less than 1°C during the light pulses.
ENTRAINMENT MECHANISMS OF APLYSIA EYE RHYTHM 275 An attempt to determine the physiological processes involved in phase shifting by light pulses was made by examining which processes were necessary for such a phase shift to occur. Experimental solutions which interfere with physiological processes were administered to the eye one-half hour before the eye was exposed to light. The eye was then exposed to light for six hours a t CT 18-24 (CT 00 is the last dawn seen by the intact animal) and returned to constant dark for the remainder of the experiment (3 days). Within ten minutes after the end of the light pulse the experimental solution was withdrawn and the eyes were rinsed, and exposed once again to the control solution. The experimental solutions had to satisfy certain criteria for use in these experiments. Any effects of the treatments on impulse activity from the eye had to he reversible. In addition, the experimental solutions themselves could not produce phase shifts in the rhythm when presented in the absence of light. If any solution produced phase shifts, the interpretation of results regarding its ability to interfere with the phase shift produced by light would he difficult. Thus, before each of the different attempts to block phase shifting by light, a group of eyes was exposed to the different treatments for 6.5 hrs a t C T 17.5-24 to determine if the solution itself caused phase shifts. The experiments were performed by administering the experimental solution to one eye while the other eye from the same animal served as the “dark” control in the experiments with no light, and the “light” control in the experiments with light pulses. The phase shifts (Ab) produced by exposing eyes only to the experimental solutions were determined by the difference in phase (defined as the peak of the rhythms) between the experimental and control eyes (positive differences are advances and negative differences are delays). The blocking effect of a solution on the phase shift produced by light was determined by the difference ( D )in phase between the light control eye and the eye receiving the experimental solution plus light. (Positive differences indicate that the experimental eye was phase shifted by the light more than the control eye and negative differences indicate that the experimental solution had a blocking effect on phase shifting by light.) All Ag’s and D’s in the text were calculated using data obtained during the third cycle of the rhythm after the experimental treatment (see Fig. 2). The control solution (ASW) contained Instant Ocean Sea Water, 30 mM HEPES buffer, and 100 units/ml of penicillin and 100 mcg/ml of streptomycin. The experimental solutions and ion substitutions also contained this same amount of buffer and antibiotic. The pH of all solutions was 7.85 (adjusted using Ca (0H)z for the LoNa-Mannitol solutions and NaOH for all other solutions) and all solutions were filtered (Millipore, 0.22 pm) before their use. The drags were added directly to ASW. The following were obtained from Sigma: Procaine-HCL, Mephenesin, Glycine, yamino-n-butyric acid (GABA), L-glutamic acid (monosodium salt), Tetrodotoxin ( T T X ) ,Acetylcholine chloride, choline chloride, 5 Hydroxytryptamine creatinine sulfate (Serotonin), creatinine sulfate, DL-octopamine hydrochloride, L-aspartic acid, 3-Hydroxytyramine-HCL (Dopamine), Ethyleneglycol his(@-Aminoethylether)N,N’-tetraacetic acid (EGTA),Histamine (free base), and L-Ascorbic acid. The basic solutions with their ion substitutions are listed in the table. The low sodium solutions had sodium reduced from 463 mM to either 70 (LoNa-70 mM), or 7 mM (LoNa-7 mM). Tris (pH 7.85 at 15”C),choline, mannitol (D-mannitol), cesium, and lithium were used as substitutes for Na+ or NaCl. The amount of mannitol required to maintain the LoNa (7 mM-Mannitol solution isotonic with ASW was determined using an osmometer (Advanced Instruments). No Hepes buffer was added to the solutions containing Tris. Mg2+ was increased 2.5 times an&Ca2+decreased by lo2 times in the HiMg LoCa solution. The molarity of this solution was normalized by reducing NaCl. Ca2+was buffered with EGTA in some of the reduced Ca2+solutions and the concentration of Ca2+ was calculated after Portzehl, Caldwell, and Ruegg (1964). The solutions had various amounts of EGTA, Ca2+and Na+, the concentrations of the other ions remaining constant. The recipe for EGTA M ) is given in the table. The amounts of Ca2+ and EGTA added, respectively, I (Ca2+= 1.3 X to the other solutions were: EGTA I1 (Ca2+ = 6.4 X 10W7M ) - 5,6 mM; EGI’A I11 (Ca2+ = 1.4 X M ) - 10, 10.05 mM: EGTA IV (Ca2+ = 4.5 X lo-* M ) - 10.5,10.05 mM. In the high Ca2+ solution (HiCa),the Ca2+ concentration was increased 6 times and Na+ was reduced an appropriate amount. Na acetate and Na propionate were substituted for NaCl in the chloride substitution experiments. Solutions were made hypotonic by diluting ASW with an appropriate amount of deionized water and were made hypertonic by adding 0.5 M mannitol to ASW. The solutions surrounding the eyes were completely exchanged by coupling a polyethylene tube, with an attached 10 ml syringe, outside of the box, to the other polyethylene tube (PE 150) embedded
276
ESKIN
Fig. 1. An advance phase shift produced by a single 6-hr light pulse. The optic nerve impulse rhythms in all figures are from two isolated eyes, both from the same animal. One eye, the control, was in constant darkness throughout the experiment. The other eye was exposed to 6 hours of light during the time shown by the closed bar a t the bottom of the graph. Before and after the light pulse, this eye was in constant darkness. The open bars a t the bottom of the graphs represent the projected light portion (12 hours) of the light-dark cycle used to entrain the intact animal. All of the eyes were dissected and then placed in light-tight boxes a t the end of a light period. The data from this time to the projected onset of light shown in the figures were recorded, but they are not shown in any of the figures. in the Sylgard in the Nalgene beaker. The experimental eyes were rinsed twice (10 ml each time) with the experimental solutions before remaining in these solutions. At the end of treatments, the experimental solutions were withdrawn and the eyes were rinsed 6 to 9 times (10 ml each time) with filtered Instant Ocean Sea Water before being returned to ASW. The control eyes were rinsed one time with ASW at the beginning of treatments and were rinsed one time each with filtered Instant Ocean Sea Water and ASW a t the end of the treatments.
RESULTS
Phase s h i f t by light Eyes were exposed to single 6-hr light pulses at different phases of the rhythm during the first 24 hours of isolation to determine at which phase the largest phase shift would occur. The largest phase shifts were produced by light during C T 18-24. All experiments were performed a t this phase. At this phase, the light produced a 3.21 f 0.31-hr advance phase shift on the second day after the light pulse and a 3.28 f 0.45-hr phase shift on the third day after the light pulse (Fig. 1). (All f values are 95% confidence intervals). These average phase shifts were obtained by subtracting the mean phase of the “light” controls ( N = 36) from the mean phase of the “dark” controls ( N = 40) used in the experiments presented below.
Treatments which a f f e c t membrane potential changes
A variety of membrane conductance or potential changes might be involved in carrying the phase shifting information to the circadian oscillator. The amplitudes of these potential changes are a function of the ionic gradients for Na+,
ENTRAINMENT MECHANISMS OF APLYSIA EYE RHYTHM 277
K+, C1-, and Ca2+depending upon the specific currents involved in producing a particular potential change. The Aplysia photoreceptor potentials might vary with the concentration of Na+
