The isolated circumesophageal nervous system is placed, immersed in a few drops of seawater, on ... A ring of Vaseline (approximately 0.4 cm high and 3 cm in.
Neural Modification by Paired Sensory Stimuli D A N I E L L. A L K O N F r o m t h e Section o n N e u r a l Systems, L a b o r a t o r y o f Biophysics, National Institute o f N e u r o logical a n d C o m m u n i c a t i v e Disorders a n d Stroke, National Institutes o f H e a l t h , B e t h e s d a , M a r y l a n d 20014, a n d t h e M a r i n e Biological Laboratory, W o o d s Hole, Massachusetts 02543
AB ST R AC T With repetitive stimulation of two sensory pathways which are intact within the isolated nervous system of Hermissenda, features of a cellular conditioning paradigm were identified. Type A photoreceptors, unlike type B photoreceptors, produce fewer impulses in response to light following temporally specific pairing of light stimuli with rotation stimuli. Type A photoreceptor impulse waveforms are also specifically changed by such stimulus regimens. These findings can be explained, at least in part, by increased inhibition of type A cells by type B cells after stimulus pairing. INTRODUCTION
E x p o s u r e to 3 h o f light stimuli associated with 3 h o f rotation stimuli decreases or eliminates for some hours the m o v e m e n t o f the n u d i b r a n c h mollusc, Hermissenda crassicornis toward a light source (Alkon, 1974b). Excitation o f hair cells by light, normally caused by type A p h o t o r e c e p t o r impulses, is r e d u c e d in animals exposed to such a r e g i m e n (Alkon, 1973b, 1975a, b). In the work p r e s e n t e d here, the light response o f type A p h o t o r e c e p t o r s in isolated nervous system preparations is shown to be c h a n g e d by temporally specific pairing o f light and rotation. Responses o f type B p h o t o r e c e p t o r s , however, are c h a n g e d slightly, if at all, by such a stimulus regimen. This a p p r o a c h was made possible by a technique which permits intracellular recording while delivering both visual and statocyst stimuli to the isolated nervous system ofHermissenda (Alkon, 1975c; Alkon, 1976). With this technique, type A p h o t o r e c e p t o r , but not type B p h o t o r e c e p t o r , impulse activity d u r i n g steady-state light responses is abolished by rotation o f the nervous system with a specific orientation with respect to the center of the rotating table. This response o f type A p h o t o r e c e p t o r s was d e m o n s t r a t e d to arise f r o m synaptic inhibition by hair cells. T h e change in type A p h o t o r e c e p t o r light responses which will be shown to result f r o m stimulus regimens of light and rotation is a persistent reduction o f impulse activity as well as a modification of the impulse amplitude and wavef o r m . T h e g e n e r a t o r response to light is not specifically c h a n g e d by the same stimulus regimen. T h e observed changes can account, at least in part, for the previously observed neural correlates o f associative training o f intact Hermissenda. THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 68, 1976 " p a g e s 341-358
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T H E JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 6 8 ' 1 9 7 6 METHODS
Preparation Hermissenda were provided by Dr. Rimmon Fay of the Pacific Biomarine Supply Co. (Venice, Calif.) and Mr. Michael Morris of the Peninsula Marine Biological Supply Co. (Monterey, Calif.). Animals were maintained with 61/2 h of daily light (cf. Alkon, 1974b) in an "Instant Ocean" aquarium at 13"C. T h e circumesophageal nervous system of Hermisenda was dissected and isolated as previously described (cf. Alkon, 1973a; Alkon and Bak, 1973; Alkon, 1974a). T y p e A photoreceptors (Alkon, 1973a) were penetrated by placement of the electrode tip in the ventral portion of the eye. T h a t type A photoreceptor in the lateral portion of the eye was used. A silent nonspiking, unresponsive cell, presumably a pigment cell, was almost always p e n e t r a t e d before impalement of the type A photoreceptor. T y p e B photoreceptors were penetrated by placement of the electrode tip in the dorsal posterior portion of the eye. Occasionally a silent cell was encountered before a successful type B impalement was made. Unless otherwise indicated all photoreceptors were darkadapted at least 15 rain before light stimuli were given.
Rotation of Circumesophageal Nervous System T h e isolated circumesophageal nervous system is placed, immersed in a few drops of seawater, on a conventional microscope glass slide. Two strips o f Vaseline, 1 cm long, are placed immediately above and below the preparation. Stainless steel pins are then laid across the connectives of the nervous system. T h e pins are in direct contact with the surface of the glass slide and are held this way because the ends of the pins are i m b e d d e d in the Vaseline strips. A ring of Vaseline (approximately 0.4 cm high and 3 cm in diameter) is then spread a r o u n d the p r e p a r a t i o n pinned as described between the two Vaseline strips. A Lucite disc, 0.2 m m thick and 3.5 cm in diameter, is then placed on top of the Vaseline ring after it has been filled with seawater. Numerous holes (1.1 mm in diameter) permit passage of microelectrodes. T h e advantages of this means of mounting for rotation are discussed elsewhere (Alkon, 1975c). T h e p r e p a r a t i o n , ventral side downward a n d m o u n t e d as described above, was placed on the turntable. T h e central axis of the cephalic end of the circumesophageal nervous system pointed toward the center o f rotation. Unless otherwise specified, rotation of the preparation (13 cm from the center of rotation) was effected by a G a r r a r d turntable (Garrard Co., Swindon-Wiltshire, England, model Zero 92) which had been reinforced by a 0.5-in. aluminum annulus (Fig. 2). All the necessities for intracellular recording were m o u n t e d on the turntable including a Lucite stage, a Prior micromanipulator, and an amplifier. Electrical contacts for recording were made via six c o p p e r - c o p p e r slip rings (Airflyte, Bayonne, N. J.). T h e velocity of rotation was monitored by measuring the current induced in a stationary coil by a magnet fixed to the rotating table. (This is possible because the current induced is directly proportional to the velocity which which the magnet moves past the coil.) Rotation was begun when 70% of the ll0-V electricity source was delivered to the turntable (in an "on" position) via a transformer.
Illumination Unless otherwise specified, light was provided by 10 G. E. lamps (Type CM 332, 0.2 a m p , 6 volt, 1 cd each) spaced evenly on a circle whose center was the center o f rotation, 14 cm diameter and 45 cm immediately above the preparation.
DANIZL L. ALgON Neural Modification by Paired Sensory Stimuli
343
Intracellular Recording As in previous studies, the circumesophageal nervous system was incubated in a solution o f a digestive enzyme prior to intracellular recording. In this study a somewhat milder treatment was used: 17 rain o f incubation in collagenase (type 1, Sigma Chemical Co., St. Louis, Mo.) solution (0.5-1.0 mg/cc). Intracellular recordings were made with glass micropipettes filled with 4 M potassium acetate (resistance of 60-100 MI~). T h e electrode was connected via a silver wire to the input stage of the high impedance amplifier. T h e reference electrode was a chloride/ silver wire. A Wheatstone bridge circuit was used to pass current through the recording electrode. C u r r e n t was monitored by recording the potential d r o p across a 10 MI'~ resistor in series w i t h the electrode. All experiments were p e r f o r m e d at r o o m t e m p e r a t u r e (approximately 22°C). Details concerning the amplifier used for potential recordings are given in Alkon (1975c). INITIAL TEST PERIOD ¢:3 C3 ~
SECOND TEST PERIOD
STIMULUS REGIMEN
C3 C31:3 C31:31~
LIGHT ALONE ROTATION ALONE :
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LIGHT AND ROTATION l
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Rotation Stimulus t 4 rain
FIGURE 1. Cell treatment. Initial a n d second test periods include four to eight light steps, each 60 s, at 35 s intervals. Stimulus regimens, each 30 min, consist of light stimuli alone, rotation stimuli alone, or light with rotation stimuli. Other stimulus regimens used are not r e p r e s e n t e d .
Cell Treatment Photoreceptors were exposed to three distinct treatment intervals (Fig. 1): an initial test period, a stimulus regimen, and a second test period. Unless otherwise specified (see T e m p o r a l Specificity u n d e r Results) for the initial test period, lateral type A and type B photoreceptors were d a r k - a d a p t e d for 15 rain before r e p e a t e d 60 s light steps at 35 s intervals were begun. Most cells r e s p o n d e d with an approximately constant n u m b e r of impulses after the fourth to eighth-light step (Fig. 2). Occasionally a few additional light steps were necessary before such a steady state was reached. T h e cells were exposed to between four a n d ten steps of light (at 35 s intervals) before beginning the stimulus regimen. A few photoreceptors were discarded which: (1) never achieved a steady state o f impulse response; i.e., progressively fewer impulses occurred d u r i n g the light response a n d / o r r e s p o n d e d with progressively smaller generator potential amplitudes; (2) re-
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THE
JOURNAL
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GENERAL
PHYSIOLOGY
• VOLUME
68
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1976
sponded with less than sixty impulses d u r i n g the light response and a small generator potential. The amplitude of the initial response of the photoreceptors following dark adaptation (using 1 cd lights arranged as described u n d e r Methods) ranged between 30 and 60 mV. This range approaches the m a x i m u m response range previously observed for photoreceptors (Alkon, 1973a, Alkon and Fuortes, 1972) using a quartz-iodide light source delivering 2 × 105 ergs cm -2 s-I to the eyes. The initial test period was followed by one of four possible physiologic stimulus regimens (Fig. 1): light stimuli alone, rotation stimuli alone, light with rotation stimuli, or a period of light stimuli followed by a period of rotation stimuli. T h e first three regimens lasted for 30 min. T h e last regimen consisted of a 30 min period of light stimuli followed by a 30 min period of rotation stimuli. c
A
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FIGURE 2. Responses to light stimuli alone. A, initial response of a type A photoreceptor to a 60-s light stimulus. B, response of photoreceptor to fourth light step delivered at 35 s intervals. C, response to light step after 30 min of light steps at 35 s intervals. Note the hyperpolarization following the response to the first light step. Note also that the impulse activity in response to the fourth light step is unchanged after 30 rain, although the amplitude of the generator potential has increased. The initial test period was also followed in other experiments, by one of two stimulus regimens: hyperpolarizing current pulses at 95 s intervals, or light stimuli with current pulses. These two regimens lasted for 20 min. Additional details concerning these and other stimulus regimens used will be included u n d e r the Results section. The initial test period and stimulus regimen were followwed by a second test period. This consisted of delivering four or more light stimuli of exactly the same intensity duration and frequency used for the initial test period.
Analysis of Stimulus Regimen Effects In order to evaluate quantitatively the effects of the stimulus regimens just described, impulse frequency ratios were determined for each cell. T h e denominator of each ratio was given by the n u m b e r of photoreceptor impulses occurring in response to the last 10 s
DANIEL L. ALKOI,J
Neural Mod~'ication by Paired Sensory Stimuli
345
of the light stimulus during the initial test period. This number was the average of two or more, obtained when the photoreceptor responded with an approximately constant number of impulses to each light step. The numerator of each ratio was given by the number of photoreceptor impulses obtained in exactly the same way, but after a stimulus regimen; i.e., during the second test period. Thus, a ratio measures the change in firing frequency of a photoreceptor in response to light during,the two test periods, produced by a stimulus regimen. The ratios, for example, under R + L of Table I compare firing frequencies during photoreceptbr responses before and after 30 rain of light and rotation stimuli. The ratios under L measure the change in firing frequency of photoreceptors produced by 30 min of light stimuli alone. The ratios under R + D measure the effect on photoreceptor responses of 30 min of rotation stimuli alone. RESULTS
E x p e r i m e n t s were p e r f o r m e d to e x a m i n e the effect on p h o t o r e c e p t o r light responses of the physiologic stimulus r e g i m e n s described above. O t h e r experim e n t s involved intracellular injection o f currents.
Responses to Light Stimuli Alone T h e initial r e s p o n s e o f the type A p h o t o r e c e p t o r to a 60 s light stimulus is a depolarizing g e n e r a t o r potential (30-60 mV) with m a n y s u p e r i m p o s e d impulses (Fig. 2A). I m m e d i a t e l y following the light step, the cell h y p e r p o l a r i z e s (5-10 mV) a n d the m e m b r a n e potential does not r e t u r n to the resting level for 40 to 60 s. T h e next light step begins a f t e r 35 s, b e f o r e the resting level has b e e n achieved. With successive light steps a new steady-state level o f m e m b r a n e potential ( d u r i n g the d a r k intervals) is r e a c h e d , 10-15 m V negative with l:espect to the resting level. A f t e r f o u r to eight light steps, i.e. the n u m b e r o f steps in the initial test p e r i o d , the n u m b e r o f impulses o c c u r r i n g d u r i n g the r~sponse o f the type A p h o t o r e c e p t o r to each light step is a p p r o x i m a t e l y const~itit (Fig. 2 B). Following the initial test p e r i o d , the light stimuli are c o n t i n u e d for 30 min; i.e., t h r o u g h o u t the stimulus r e g i m e n . T h e n u m b e r o f impulses o c c u r r i n g d u r i n g the r e s p o n s e o f the type A p h o t o r e c e p t o r to each s u b s e q u e n t light step following this stimulus r e g i m e n , d u r i n g the second test p e r i o d , was not significantly d i f f e r e n t f r o m the n u m b e r o f impulses o c c u r r i n g d u r i n g the initial test period (Fig. 2 C, L, Tables I a n d II). T h e a m p l i t u d e o f the g e n e r a t o r potential, however, gradually increased o v e r 20 to 30 min b e f o r e it also r e a c h e d a constant value (Fig. 2 A - C ) . T h e effect o f light stimuli on type B p h o t o r e c e p t o r s was similar with the exception that t h e h y p e r p o l a r i z a t i o n following the d e p o l a r i z i n g g e n e r a t o r potential was usually not present.
Responses to Rotation Stimuli Alone Following the initial test p e r i o d , the cells are e x p o s e d to continual darkness a n d rotation stimuli for 30 min. T h e onset o f each rotation stimulus o c c u r r e d at 95 s intervals. M a x i m u m r p m was achieved within 15 s a n d lasted for 18 s. T h e initial r e s p o n s e o f the type A p h o t o r e c e p t o r to rotation in d a r k n e s s is a h y p e r p o ! a r i z a tion of 2-8 mV (Fig. 3 A). T h i s h y p e r p o l a r i z a t i o n d e c r e a s e d slightly with successive rotation stimuli (Fig. 3 B, C) b e f o r e r e a c h i n g an a p p r o x i m a t e l y constant
1.575
R]L
Type A ratios--20 min regimen R'+L' (out-of-phase)
Type B ratios--30 rain regimen R+L
Cut N R +L
0.814 1.21 0.67
R+D
0.423 0.628 0.66
0.78
0.78
0.57
0.9 1.07 0.847
1.970
0.830
0.966 0.612 1.44 1.15 0.95
0.113 0.239 0.0 0.290 0.018 0.156
Ratios
L
Type A ratios--30 rain regimen R+L
Grou p
3
3
3
3
3
5
6
N
0.5703
0.710
0.9390
1.4583
0.8980
1.0236
0.136
Mean
I
0.1286
0.12124
0.1165
0.5789
0.27963
0.3031
0.1t63
SD
RESPONSE
TABLE PHOTORECEPTOR
0.07424
0.07
0.06726
0.3342
0.16144
0.1356
0.0475
SEM
RATIOS
-5.788
-4.143
-0.9069
1.370
-0.6318
0.174
-18.19
t
2
2
2
2
2
4
5
df
0.02
