Physiology & Behavior, Vol. 22, pp. 1141-1148. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Habituation of the Nictitating Membrane Reflex Response in the Intact Frog D E N N I S L. G L A N Z M A N A N D E U G E N E C. S C H M I D T
Department of Psychology, Arizona State University, Tempe, AZ 85281 (Received 2 D e c e m b e r 1978) GLANZMAN, D. L. AND E. C. SCHMIDT. Habituation of the nictitating membrane reflex response in the intact frog. PHYSIOL. BEHAV. 22(6) 1141-1148, 1979.--Habituation of the nictitating membrane reflex response was measured in the intact bullfrog. A stimulus map was created, and stimulating electrode pairs directly opposed across the eye proved to be the optimal loci for cutaneous electrical stimulation. The response was found to exhibit all of the parametric characteristics of habituation. To date, this represents what is perhaps the simplest intact vertebrate preparation demonstrating habituation. The response was also found to exhibit a surprising periodicity in response amplitude when unchanging stimuli were infrequently presented over long periods of time. The convenience, reliability and successful behavioral assessment strongly recommend this preparation as an ideal simple vertebrate model system for further examination of the neural basis of behavioral plasticity.
Habituation
Modelsystem
Nictitatingmembrane
HABITUATION, especially within the past decade, has come to be regarded as an ubiquitous example of behavioral and neural plasticity, encompassing phyla ranging from protozoans to man [1, 2, 12, 15, 17, 19, 21, 22, 30]. Dating from the earliest reports of"fatigue" of reflex responses [26], the emphasis has been on attempting to elucidate the mechanisms underlying the phenomenon. One very productive procedure for examination of the cellular basis of habituation has been the use of "model" systems [10, 11, 31], that is, the use of a reduced preparation such as spinal cat [9, 13, 14, 15, 16, 27, 28, 29] or the isolated spinal cord of the frog [7, 8, 10, 31] to examine habituation-like decrements in response amplitudes to repeated presentation of unchanging stimuli. Additionally, the use of "simple" systems has allowed the studies of these behaviors in less complex but intact animals such as the marine mollusk Aplysia [25], the praying mantis [3] and the frog [6,21]. It has also been convenient to combine these techniques, such as using a reduced preparation of a simple system, e.g., electrophysiological investigations of the isolated abdominal ganglion of Aplysia [4,19]. The ideal preparation, then, would be an intact, simple vertebrate system which would allow behavioral observations to be recorded without interference by surgical or pharmaceutical manipulation, yet would involve only a relatively discrete number of neuronal elements. We report here the development and utilization of such a preparation for the investigation of habituation: the nictitating membrane (NM) reflex response in the intact frog. This behavioral preparation closely conforms to our concept of an ideal system. Being a vertebrate but possessing no neocortex, the frog represents a convenient intermediate step between isolated neural systems and intact lower mammals at which the search for mechanisms underlying the simplest learning processes should prove fruitful. The neural circuitry of the NM response in the frog has
Dishabituation
Generalization
Bullfrog
been described; the III cranial nerve (occulomotor) has a termination in the retractor bulbi muscle, which retracts the bulb and "leads automatically to a movement of the 'nictitating m e m b r a n e ' . . , from the lower part of the cornea to the upper eyelid" [18]. Elevation of the bulb can be actively aided by the M. levator bulbi, innervated by the N. maxillaris branch of the V cranial nerve (trigeminal). Segments of this muscle also attach to the lowest portions of the NM which forms a lower eyelid; the segments have been separately identified as M. depressor membranae nictitantis [ 18]. Comparisons with other species indicate a lack of commonality of neural pathways underlying the response. For example, the NM reflex of the rabbit is produced in a similar fashion, save that the bulbus retractor muscle is innervated by the VI cranial nerve (abducens), not the III. The rabbit III nerve directly innervates muscle fibers in the NM proper which act to retract or return the NM to the lower eyelid [5]. Extension of the NM in the cat is mediated by the same nerve (VI) as in the rabbit, but retraction of the NM is actively mediated through autonomic activity [24]. A framework for the characterization of habituation has been established by Thompson and Spencer [30]. They outlined nine parametric characteristics of habituation, dishabituation and generalization of habituation, each of which has been applied here to assessment of habituation in the intact frog. EXPERIMENT i: STIMULUS MAP
Method All experiments were performed on bullfrogs (Rana catesbeiana) with body lengths ranging from 6 to 15 cm. Generally, animals with body lengths of 8 to 10 cm proved the most reliable and easiest to accommodate in our apparatus. Upon receipt from animal suppliers the frogs were
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/061141-08502.00/0
1142
housed either separately or in group cages, with tap water maintained at a depth of about 2 cm, the water being changed daily. Following at least 2 days to adapt to their new surroundings, animals were anesthetized by whole body immersion into a 1% solution of ethyl-m-aminobenzoate methane sulfonic acid (tricaine methanesulfonate, Sigma Chemical Co., No. 1626) in tap water, until all musculature was flaccid and no corneal reflexes could be elicited, usually requiring from 5 to 10 rain. Under anesthetic, a single silk suture (No. 6-t)) was tied into the nictitating membrane just beneath the thickened rim and a small loop (2 mm diameter) was left in the suture for attachment of the recording transducer during testing. In addition, eight stainless steel (SS) sutures (No. 30 SS wire) were implanted into the superficial skin of the periorbital region to serve as stimulating electrodes. These electrode loops were fluxed and touch soldered in place to prevent accidental removal, and any acid flux was washed from the skin. At least 2 days were allowed for complete recovery from suturing and anesthetic effects before testing was commenced in any paradigm. Apparatus. Measurement of the NM reflex response required a restraining chamber, electrode connections and a low mass transducer with a positive mechanical coupling to the NM proper. All studies were performed using the same apparatus. Each frog to be tested was held immobile without anesthetic in a plexiglas frame designed by Isidore Gormezano (personal communication). The restraining device consisted of a jaw pedestal, a head/neck clamp, a shallow trough to hold the animal's body and a V shaped leg clamp which was positioned between the animal's hind legs and secured firmly in place. Animals securely clamped into the apparatus accepted this form of restraint placidly for upwards of 2 hours without frequent or severe struggling. Tiny gold plated electrode hooks (E-Z Microhook, weighing less than I gram) were used to make electrical connections from the SS suture electrodes in the periorbital skin to a terminal strip firmly secured on the neck clamp. A second connection was then made from the terminal strip to the stimulus isolation units. This scheme allowed the microhook electrodes to be suspended immediately above the SS sutures, thus eliminating most of the strain on the implanted SS loops. Grass $8 and $88 stimulators were used to generate all electrical stimuli, and Grass stimulus isolators were used to provide isolation and constant current. In the first few tests of stimulus effectiveness, each stimulus consisted of a 15 msec train of pulses delivered a t 500/sec, totaling eight pulses of 0.10 msec duration each. These results were indistinguishable from experiments using single pulse stimuli, and the single pulse paradigm was used thereafter. The rectangular pulses ranged from 0.1 to 2.0 msec duration and 0.5 to 10.0 mA intensity. Responses were recorded by means of a rotary transducer, consisting of a light SS lever arm (about 10 cm) attached to the shaft of a microtorque potentiometer (Conrac Corp., Model No. 85153-2-250). The short end of the lever arm was counterbalanced with a small adjustable weight. The long end was in turn attached to a 10 cm length of No. 6-0 surgical silk, which was tied to a tiny SS hook. This hook was then looped through the ligature in the NM. The potentiometer was firmly affixed to the restraining apparatus below the animal's head so that an NM response would produce a deflection of the potentiometer wiper arm of about 2 to 3 degrees. The recording system was checked and found to be linear with accuracy to --*-0.3%. The potentiometer was
(iI,ANZMAN
A
(~
ANI) S(:HMII)'t
B
',
( ~
r
r:
~ c "2o g.o p
'1
0"~
i
~a~20 z
I
5
I 10
I t5
210
2 ,5
I In
Minutes
FIG. 1. Nictitating membrane reflex response topograph to mild (A) and to intense (B) stimulus. Note prolonged NM extension in (B). Calibration bars are I mm and 100 msec. Periodicity of frog nictitating membrane reflex response to mild electrical stimulation of superficial skin area of periorbital region {C). Stimuli were held constant at 2mA for 1 msec and delivered at 1 minute intervals. No other stimuli were presented during this period. Line labeled X is the mean response amplitude for the entire series of 32 stimulus presentations.
wired as a simple voltage divider, using two mercury bat, teries as a power source. Nictitating membrane extensions were read directly as a voltage change at the potentiometer wiper terminal. These DC signals were recorded oscill0graphically on a Grass Model 7 polygraph (filter settings DC to 35 Hz), visually displayed on Tektronix Model 565 or 502 oscilloscopes, and were also FM modulated (Vetter Model 2 or 2D FM adapters; frequency response DC to 300 Hz) and permanently stored on magnetic tape (Sony Model 366-4 and Teac Model A-2340-SX tape decks) for further analysis and back-up. The polygraph was calibrated so that 1 mm of NM deflection produced 5 mm of pen deflection on the hard copy records. All response amplitudes were measured by hand from polygraph records, which were either directly recorded "on-line" or made from playback of the magnetic tape, and tabulated for later analysis. Hard copy records were made at chart speeds of 60 or 100 mm/sec to provide an accurate record of NM response topographies. Response topography. The mean and standard deviation of response measures were for onset latency, 23.3_+3.03 msec; for peak latencies 90.23---15.95 msec. Total duration was measured in two ways. The wide distribution of response topographies led to a difficulty in establishing a measure of response duration, When higher stimulus intensities were used, the NM would often return to a position slightly elevated above pre-stimulation (baseline) levels, and in many instances remain at this elevated position for upwards of a few minutes. For this reason, both initial (onset or "reflex" component) and total duration were measured. The first measure was made from the response onset to the inflection point in the falling phase of the response trace (see Figs. 1A and 1B), and yielded a mean response duration of 113.57 _+ 18.75 msec. When the entire response was meas-
H A B I T U A T I O N IN T H E I N T A C T F R O G ured, an arbitary upper time limit of 500 msec was imposed. Any response not returning to baseline levels within this time period was then assigned a total duration value of 500 msec. The results of this determination yielded a total response duration of 424.64___ 126.00 msec. These measures were all taken during the " c o n t r o l " periods, in the absence of observable habituation. Procedure. A stimulus map was generated by using all permutations of sets of two of the electrodes about the eye. Eight SS electrodes had been implanted at approximately 45 ° intervals around one eye in each of the nine frogs used in this investigation. The purpose of the mapping study was to determine the optimum placement of electrodes for producing the maximal behavioral response amplitude (NM extension) to minimal intensity stimuli. Results The stimulus map demonstrated that the optimal electrode placement occurred when the widest periorbital field was stimulated, e.g., anterior-posterior, dorsal-ventral, etc. Stimulus polarity appeared to be non-critical. The most effective stimulus configuration occurred when an imaginary line connecting the two electrodes would pass through the center of the pupil. The lateral orbital ridge was the most convenient locus for implanting the posterior electrode loop. The mapping study also revealed a surprising variability in the excitability or responsiveness of the NM reflex during the course of a typical 2-hour recording session. Figure 1C demonstrates this variability. In this example the same electrode pair was used for each stimulus presentation, and the stimulus parameters were held constant (2.0 mA; 1.0 msec) as the NM reflex responses were measured at 1 minute intervals. The response variability was specifically measured in each of five different preparations, and exhibited an oscillating response amplitude with a period averaging about 5 minutes across all preparations. (Gormezano (personal communication) has observed a similar form of response variability in the NM reflex of the albino rabbit when the animal is subjected to air-puff stimulation of the cornea.) When the stimulus maps were made, this inherent variability in sensitivity was accommodated by comparing the response amplitudes for the various electrode pairs to the response for a standard pair (usually the anterior-posterior opposed pair). The standard and comparison electrode pairs were stimulated in alternation at one minute intervals. This procedure yielded a measure of the responsiveness or excitability of the NM response to the standard pair stimuli, and corrections for this change in response amplitude over time could be applied to the responses measured for the other electrode pairs. Ideally the response measure should be the area under the response amplitude---time trace (as in Fig. 1A), but this was often unsuitable for the reasons stated above (as in Fig. 1B). Instead of measuring and reporting the response amplitudes as areas, we have presented the response height (i.e., mm of peak NM extension above baseline) as our response measure. In a few records we have calculated a Pearson product-moment correlation coefficient between the response height and response amplitude (of those responses exhibiting an early onset component and then returning to baseline levels within 250 msec), and obtained a value of 0.934. Thus, under our conditions of recording, response height was found to be a reliable indicator of the reflex response amplitude.
1143 6
E E 4
3
. g = z
2
I
7-; Controls
'
5
Habituation
'0
I
Stimulus
'5
I
2
'o
215
Number
. 0. . 2.
30
4
Recovery Trials
FIG. 2. Habituation and spontaneous recovery of NM reflex responses to repeated stimulation in one preparation. Stimulus conditions: frequency, 1/sec: intensity, 1.0 mA" duration, a 15 msec train at 500/sec (for eight stimulus pulses of 0.1 msec duration each).
EXPERIMENT 2: HABITUATION Method The animals were prepared for the habituation studies using the same techniques as used in Experiment 1. However, usually only two SS electrode loops were implanted in the anterior-posterior orientation. Of the 17 bullfrogs prepared for these studies, two also received an additional pair of electrodes in the dorsal-ventral orientation for tests of generalization of habituation, and seven frogs also received an additional pair of electrodes about the contralateral eye for tests of dishabituation. In these cases the primary electrode pair received the stimuli labeled S1 (habituation stimuli) and the secondary electrode pair the stimuli labeled $2 (dishabituation or generalization stimuli). Recording and stimulation apparatus were the same as in Experiment 1. Stimulus parameters were adjusted for each animal on each day of testing to provide an initial NM deflection of approximately 3--4 mm, or about one half of the maximal possible excursion of the membrane. At the beginning of each testing session the animal was given a single subthreshold stimulus pulse, which was then repeated at 1 minute intervals. The intensity of the stimulus was increased between presentations until the desired response amplitude was obtained. When the stimuli were given at this low rate, no habituation was observed to occur. The procedure for presenting stimuli at the faster rates which yield habituation differed slightly with each of the tests appropriate to the many parametric characteristics of habituation. However, in general the procedure required presenting a series of 10 identical control stimuli at 1/min, followed by a series of identical stimuli delivered at a faster rate to provide habituation training. Recovery stimuli were then presented at infrequent intervals to test for the return of responsiveness without perpetuating the habituation effect. Response amplitudes were measured by hand from the polygraph records, and the responses to the control stimuli were averaged to yield a control level for each test. Responses to the habituation and recovery stimuli were expressed as a percentage of the control level. As in Experiment 1, the measure of response amplitude for the tests of
1144
(H~ANZMAN ANI) SCHMIlYl
__ 120
120
-
c
:
Habituation of the Nictitating Membrane Reflex Response in the Intact Frog D E N N I S L. G L A N Z M A N A N D E U G E N E C. S C H M I D T
Department of Psychology, Arizona State University, Tempe, AZ 85281 (Received 2 D e c e m b e r 1978) GLANZMAN, D. L. AND E. C. SCHMIDT. Habituation of the nictitating membrane reflex response in the intact frog. PHYSIOL. BEHAV. 22(6) 1141-1148, 1979.--Habituation of the nictitating membrane reflex response was measured in the intact bullfrog. A stimulus map was created, and stimulating electrode pairs directly opposed across the eye proved to be the optimal loci for cutaneous electrical stimulation. The response was found to exhibit all of the parametric characteristics of habituation. To date, this represents what is perhaps the simplest intact vertebrate preparation demonstrating habituation. The response was also found to exhibit a surprising periodicity in response amplitude when unchanging stimuli were infrequently presented over long periods of time. The convenience, reliability and successful behavioral assessment strongly recommend this preparation as an ideal simple vertebrate model system for further examination of the neural basis of behavioral plasticity.
Habituation
Modelsystem
Nictitatingmembrane
HABITUATION, especially within the past decade, has come to be regarded as an ubiquitous example of behavioral and neural plasticity, encompassing phyla ranging from protozoans to man [1, 2, 12, 15, 17, 19, 21, 22, 30]. Dating from the earliest reports of"fatigue" of reflex responses [26], the emphasis has been on attempting to elucidate the mechanisms underlying the phenomenon. One very productive procedure for examination of the cellular basis of habituation has been the use of "model" systems [10, 11, 31], that is, the use of a reduced preparation such as spinal cat [9, 13, 14, 15, 16, 27, 28, 29] or the isolated spinal cord of the frog [7, 8, 10, 31] to examine habituation-like decrements in response amplitudes to repeated presentation of unchanging stimuli. Additionally, the use of "simple" systems has allowed the studies of these behaviors in less complex but intact animals such as the marine mollusk Aplysia [25], the praying mantis [3] and the frog [6,21]. It has also been convenient to combine these techniques, such as using a reduced preparation of a simple system, e.g., electrophysiological investigations of the isolated abdominal ganglion of Aplysia [4,19]. The ideal preparation, then, would be an intact, simple vertebrate system which would allow behavioral observations to be recorded without interference by surgical or pharmaceutical manipulation, yet would involve only a relatively discrete number of neuronal elements. We report here the development and utilization of such a preparation for the investigation of habituation: the nictitating membrane (NM) reflex response in the intact frog. This behavioral preparation closely conforms to our concept of an ideal system. Being a vertebrate but possessing no neocortex, the frog represents a convenient intermediate step between isolated neural systems and intact lower mammals at which the search for mechanisms underlying the simplest learning processes should prove fruitful. The neural circuitry of the NM response in the frog has
Dishabituation
Generalization
Bullfrog
been described; the III cranial nerve (occulomotor) has a termination in the retractor bulbi muscle, which retracts the bulb and "leads automatically to a movement of the 'nictitating m e m b r a n e ' . . , from the lower part of the cornea to the upper eyelid" [18]. Elevation of the bulb can be actively aided by the M. levator bulbi, innervated by the N. maxillaris branch of the V cranial nerve (trigeminal). Segments of this muscle also attach to the lowest portions of the NM which forms a lower eyelid; the segments have been separately identified as M. depressor membranae nictitantis [ 18]. Comparisons with other species indicate a lack of commonality of neural pathways underlying the response. For example, the NM reflex of the rabbit is produced in a similar fashion, save that the bulbus retractor muscle is innervated by the VI cranial nerve (abducens), not the III. The rabbit III nerve directly innervates muscle fibers in the NM proper which act to retract or return the NM to the lower eyelid [5]. Extension of the NM in the cat is mediated by the same nerve (VI) as in the rabbit, but retraction of the NM is actively mediated through autonomic activity [24]. A framework for the characterization of habituation has been established by Thompson and Spencer [30]. They outlined nine parametric characteristics of habituation, dishabituation and generalization of habituation, each of which has been applied here to assessment of habituation in the intact frog. EXPERIMENT i: STIMULUS MAP
Method All experiments were performed on bullfrogs (Rana catesbeiana) with body lengths ranging from 6 to 15 cm. Generally, animals with body lengths of 8 to 10 cm proved the most reliable and easiest to accommodate in our apparatus. Upon receipt from animal suppliers the frogs were
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/061141-08502.00/0
1142
housed either separately or in group cages, with tap water maintained at a depth of about 2 cm, the water being changed daily. Following at least 2 days to adapt to their new surroundings, animals were anesthetized by whole body immersion into a 1% solution of ethyl-m-aminobenzoate methane sulfonic acid (tricaine methanesulfonate, Sigma Chemical Co., No. 1626) in tap water, until all musculature was flaccid and no corneal reflexes could be elicited, usually requiring from 5 to 10 rain. Under anesthetic, a single silk suture (No. 6-t)) was tied into the nictitating membrane just beneath the thickened rim and a small loop (2 mm diameter) was left in the suture for attachment of the recording transducer during testing. In addition, eight stainless steel (SS) sutures (No. 30 SS wire) were implanted into the superficial skin of the periorbital region to serve as stimulating electrodes. These electrode loops were fluxed and touch soldered in place to prevent accidental removal, and any acid flux was washed from the skin. At least 2 days were allowed for complete recovery from suturing and anesthetic effects before testing was commenced in any paradigm. Apparatus. Measurement of the NM reflex response required a restraining chamber, electrode connections and a low mass transducer with a positive mechanical coupling to the NM proper. All studies were performed using the same apparatus. Each frog to be tested was held immobile without anesthetic in a plexiglas frame designed by Isidore Gormezano (personal communication). The restraining device consisted of a jaw pedestal, a head/neck clamp, a shallow trough to hold the animal's body and a V shaped leg clamp which was positioned between the animal's hind legs and secured firmly in place. Animals securely clamped into the apparatus accepted this form of restraint placidly for upwards of 2 hours without frequent or severe struggling. Tiny gold plated electrode hooks (E-Z Microhook, weighing less than I gram) were used to make electrical connections from the SS suture electrodes in the periorbital skin to a terminal strip firmly secured on the neck clamp. A second connection was then made from the terminal strip to the stimulus isolation units. This scheme allowed the microhook electrodes to be suspended immediately above the SS sutures, thus eliminating most of the strain on the implanted SS loops. Grass $8 and $88 stimulators were used to generate all electrical stimuli, and Grass stimulus isolators were used to provide isolation and constant current. In the first few tests of stimulus effectiveness, each stimulus consisted of a 15 msec train of pulses delivered a t 500/sec, totaling eight pulses of 0.10 msec duration each. These results were indistinguishable from experiments using single pulse stimuli, and the single pulse paradigm was used thereafter. The rectangular pulses ranged from 0.1 to 2.0 msec duration and 0.5 to 10.0 mA intensity. Responses were recorded by means of a rotary transducer, consisting of a light SS lever arm (about 10 cm) attached to the shaft of a microtorque potentiometer (Conrac Corp., Model No. 85153-2-250). The short end of the lever arm was counterbalanced with a small adjustable weight. The long end was in turn attached to a 10 cm length of No. 6-0 surgical silk, which was tied to a tiny SS hook. This hook was then looped through the ligature in the NM. The potentiometer was firmly affixed to the restraining apparatus below the animal's head so that an NM response would produce a deflection of the potentiometer wiper arm of about 2 to 3 degrees. The recording system was checked and found to be linear with accuracy to --*-0.3%. The potentiometer was
(iI,ANZMAN
A
(~
ANI) S(:HMII)'t
B
',
( ~
r
r:
~ c "2o g.o p
'1
0"~
i
~a~20 z
I
5
I 10
I t5
210
2 ,5
I In
Minutes
FIG. 1. Nictitating membrane reflex response topograph to mild (A) and to intense (B) stimulus. Note prolonged NM extension in (B). Calibration bars are I mm and 100 msec. Periodicity of frog nictitating membrane reflex response to mild electrical stimulation of superficial skin area of periorbital region {C). Stimuli were held constant at 2mA for 1 msec and delivered at 1 minute intervals. No other stimuli were presented during this period. Line labeled X is the mean response amplitude for the entire series of 32 stimulus presentations.
wired as a simple voltage divider, using two mercury bat, teries as a power source. Nictitating membrane extensions were read directly as a voltage change at the potentiometer wiper terminal. These DC signals were recorded oscill0graphically on a Grass Model 7 polygraph (filter settings DC to 35 Hz), visually displayed on Tektronix Model 565 or 502 oscilloscopes, and were also FM modulated (Vetter Model 2 or 2D FM adapters; frequency response DC to 300 Hz) and permanently stored on magnetic tape (Sony Model 366-4 and Teac Model A-2340-SX tape decks) for further analysis and back-up. The polygraph was calibrated so that 1 mm of NM deflection produced 5 mm of pen deflection on the hard copy records. All response amplitudes were measured by hand from polygraph records, which were either directly recorded "on-line" or made from playback of the magnetic tape, and tabulated for later analysis. Hard copy records were made at chart speeds of 60 or 100 mm/sec to provide an accurate record of NM response topographies. Response topography. The mean and standard deviation of response measures were for onset latency, 23.3_+3.03 msec; for peak latencies 90.23---15.95 msec. Total duration was measured in two ways. The wide distribution of response topographies led to a difficulty in establishing a measure of response duration, When higher stimulus intensities were used, the NM would often return to a position slightly elevated above pre-stimulation (baseline) levels, and in many instances remain at this elevated position for upwards of a few minutes. For this reason, both initial (onset or "reflex" component) and total duration were measured. The first measure was made from the response onset to the inflection point in the falling phase of the response trace (see Figs. 1A and 1B), and yielded a mean response duration of 113.57 _+ 18.75 msec. When the entire response was meas-
H A B I T U A T I O N IN T H E I N T A C T F R O G ured, an arbitary upper time limit of 500 msec was imposed. Any response not returning to baseline levels within this time period was then assigned a total duration value of 500 msec. The results of this determination yielded a total response duration of 424.64___ 126.00 msec. These measures were all taken during the " c o n t r o l " periods, in the absence of observable habituation. Procedure. A stimulus map was generated by using all permutations of sets of two of the electrodes about the eye. Eight SS electrodes had been implanted at approximately 45 ° intervals around one eye in each of the nine frogs used in this investigation. The purpose of the mapping study was to determine the optimum placement of electrodes for producing the maximal behavioral response amplitude (NM extension) to minimal intensity stimuli. Results The stimulus map demonstrated that the optimal electrode placement occurred when the widest periorbital field was stimulated, e.g., anterior-posterior, dorsal-ventral, etc. Stimulus polarity appeared to be non-critical. The most effective stimulus configuration occurred when an imaginary line connecting the two electrodes would pass through the center of the pupil. The lateral orbital ridge was the most convenient locus for implanting the posterior electrode loop. The mapping study also revealed a surprising variability in the excitability or responsiveness of the NM reflex during the course of a typical 2-hour recording session. Figure 1C demonstrates this variability. In this example the same electrode pair was used for each stimulus presentation, and the stimulus parameters were held constant (2.0 mA; 1.0 msec) as the NM reflex responses were measured at 1 minute intervals. The response variability was specifically measured in each of five different preparations, and exhibited an oscillating response amplitude with a period averaging about 5 minutes across all preparations. (Gormezano (personal communication) has observed a similar form of response variability in the NM reflex of the albino rabbit when the animal is subjected to air-puff stimulation of the cornea.) When the stimulus maps were made, this inherent variability in sensitivity was accommodated by comparing the response amplitudes for the various electrode pairs to the response for a standard pair (usually the anterior-posterior opposed pair). The standard and comparison electrode pairs were stimulated in alternation at one minute intervals. This procedure yielded a measure of the responsiveness or excitability of the NM response to the standard pair stimuli, and corrections for this change in response amplitude over time could be applied to the responses measured for the other electrode pairs. Ideally the response measure should be the area under the response amplitude---time trace (as in Fig. 1A), but this was often unsuitable for the reasons stated above (as in Fig. 1B). Instead of measuring and reporting the response amplitudes as areas, we have presented the response height (i.e., mm of peak NM extension above baseline) as our response measure. In a few records we have calculated a Pearson product-moment correlation coefficient between the response height and response amplitude (of those responses exhibiting an early onset component and then returning to baseline levels within 250 msec), and obtained a value of 0.934. Thus, under our conditions of recording, response height was found to be a reliable indicator of the reflex response amplitude.
1143 6
E E 4
3
. g = z
2
I
7-; Controls
'
5
Habituation
'0
I
Stimulus
'5
I
2
'o
215
Number
. 0. . 2.
30
4
Recovery Trials
FIG. 2. Habituation and spontaneous recovery of NM reflex responses to repeated stimulation in one preparation. Stimulus conditions: frequency, 1/sec: intensity, 1.0 mA" duration, a 15 msec train at 500/sec (for eight stimulus pulses of 0.1 msec duration each).
EXPERIMENT 2: HABITUATION Method The animals were prepared for the habituation studies using the same techniques as used in Experiment 1. However, usually only two SS electrode loops were implanted in the anterior-posterior orientation. Of the 17 bullfrogs prepared for these studies, two also received an additional pair of electrodes in the dorsal-ventral orientation for tests of generalization of habituation, and seven frogs also received an additional pair of electrodes about the contralateral eye for tests of dishabituation. In these cases the primary electrode pair received the stimuli labeled S1 (habituation stimuli) and the secondary electrode pair the stimuli labeled $2 (dishabituation or generalization stimuli). Recording and stimulation apparatus were the same as in Experiment 1. Stimulus parameters were adjusted for each animal on each day of testing to provide an initial NM deflection of approximately 3--4 mm, or about one half of the maximal possible excursion of the membrane. At the beginning of each testing session the animal was given a single subthreshold stimulus pulse, which was then repeated at 1 minute intervals. The intensity of the stimulus was increased between presentations until the desired response amplitude was obtained. When the stimuli were given at this low rate, no habituation was observed to occur. The procedure for presenting stimuli at the faster rates which yield habituation differed slightly with each of the tests appropriate to the many parametric characteristics of habituation. However, in general the procedure required presenting a series of 10 identical control stimuli at 1/min, followed by a series of identical stimuli delivered at a faster rate to provide habituation training. Recovery stimuli were then presented at infrequent intervals to test for the return of responsiveness without perpetuating the habituation effect. Response amplitudes were measured by hand from the polygraph records, and the responses to the control stimuli were averaged to yield a control level for each test. Responses to the habituation and recovery stimuli were expressed as a percentage of the control level. As in Experiment 1, the measure of response amplitude for the tests of
1144
(H~ANZMAN ANI) SCHMIlYl
__ 120
120
-
c
:
