Physiology & Behavior, Voi. 23, pp. 299-308. Pergamon Press and Brain Research Pubi., 1979. Printed in the U.S.A.
Efferent Neuronal Control of the Nictitating Membrane Response in Rabbit (Oryctolagus cuniculus): A Reexamination I G E R A L D M. P O W E L L , N E I L E. B E R T H I E R A N D J O H N W. M O O R E
Department o f Psychology, Middlesex House University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 29 S e p t e m b e r 1978 POWELL, G. M., N. E. BERTHIER AND J. W. MOORE. Efferent neuronal control of the nictitating membrane response in rabbit (Oryctolagus cuniculus): A reexamination. PHYSIOL. BEHAV. 2,3(2) 299--308, 1979.--Four Albino rabbits acquired a classically conditioned nictitating membrane response (NMR) to light, tone, and back shock conditioned stimuli using a paraorbital shock applied to the right eye as the unconditioned stimulus. Electrocoagulation lesions were made in the ipsilateral abducens nucleus of the pons in two animals, and the pontine reticular formation in two animals. Reinstitufion of conditioning following surgery (retention test) indicated that the lesions failed to eliminate conditioned or unconditioned responses. This result is surprising in light of recent evidence that efferent control of the rabbit NMR derives solely from motoneurons of the ipsilateral abducens nucleus. Alternative hypotheses are considered. Nictitating membrane response
Abducens nucleus
C L A S S I C A L (Pavlovian) conditioning of the rabbit nictitating membrane response (NMR) offers the investigator concerned with the neural basis of associative learning in mammals a preparation with several attractive features [3, 15, 17]. High on the list is the evident simplicity of neural efferent control of the response. Motoneurons responsible for the NMR are thought to be located, together with those innervating the lateral rectus muscle, in the ipsilateral abducens nucleus of the pons [3, 4, 17, 18]. The electrophysiological activity of motoneurons in the abducens nucleus is mirrored with high fidelity by the peripheral response [4, 9, 17]: "The behavioral response [provides] a 'label' to be compared with activity in higher brain centers ([3], p. 324)." In short, the preparation seems to offer a system that simplifies the task of tracing the neural circuits involved in conditioning from the motoneurons of the "final common path" to other regions of the brain. The NMR occurs when the membrane sweeps across the eye, in a nasal to temporal direction, as part of an integrated pattern of defensive reactions that include the eyeblink and eyeball retraction. Reflexive extension of the NMR is typically elicited by noxious stimulation of the cornea by air puffs or electric shock applied paraorbitally. These unconditioned stimuli (USs) are sufficient to reinforce a conditioned anticipatory extension of the membrane (CR) to visual, auditory, or tactile conditioned stimufi (CSs). While a small strip of muscle innervated by motoneurons in the oculomotor complex subserves retraction of the membrane, extension is a passive consequence of eyeball retrac-
Rabbit
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tion [4]. In the most thorough investigation of efferent control of the NMR in rabbit to date, Cegavske, Thompson, Patterson and Gormezano [4] reported that: (a) Electrical stimulation of the abducens nucleus elicited a normal appearing NMR, but stimulation of the oculomotor, trochlear, facial nuclei or superior cervical ganglion did not; (b) Stimulation of the distal end of the transected sixth cranial nerve produced a normal appearing NMR, but stimulation of the third, fourth, or seventh nerve did not; (c) Destruction of the sixth nerve in one animal by an electrolytic lesion eliminated the NMR; (d) Corneal stimulation elicited electrophysiological activity from neurons in the abducens nucleus. Mis [12] has provided additional relevant evidence by demonstrating that both the conditioned and unconditioned NMR are unaffected by transection of all extrinsic eye muscles (four rectus muscles and two oblique muscles) excepting the retractor bulbi muscles. Clearly, the retractor bulbi muscles are sufficient for eyeball retraction and the NMR. The present investigation sought to further explore the neural control of the conditioned and unconditioned NMR in rabbits by making lesions in the brain stem in the vicinity of the abducens nucleus. Two considerations motivated this research: First, the lesion data presented by Cegavske et al. [4] are sparse and problematic in that one reported instance of response disruption involved six lesions in unspecified loci as well as destruction of the sixth nerve. Some combination of these multiple lesions might have been responsible for the disruption attributed to destruction of the sixth nerve. Secondly, indirect evidence from a variety of sources
1This research was supported by National Science Foundation Grant BNS 77-14871 to the third author.
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FIG. 1. Reconstruction of lesions for Animals 14 and 15 (top row) and Animals 13and 16 (bottom row). Section numbers are from Messen and Olszewsky [8], moving caudally from left to right. The genu of the facial nerve is labeled G in sections No. 450 and No. 424 of the upper row. The abdueens nucleus is labeled N in section No. 450.
suggests that the locus of associative connections responsible for acquisition and performance of the conditioned NMR might lie in the reticular formation ventral to the abducens nucleus [14]. Given the evident compact and discrete nature of the neural systems providing efferent control of the NMR, we reasoned that the volume of tissue subserving the critical linkages of conditioning might be equally compact and located near to, but not within, the abducens nucleus. We hoped that a suitable lesion would disrupt a previously established CR to a variety of CSs while leaving intact the UR to paraorbital shock. Instead, we found that lesions of the abducens nucleus and reticular formation had little or no effect on either the conditioned or unconditioned NMR. METHOD
Animals Four experimentally naive albino rabbits, weighing 2.63,0 kgand approximately 100 days old at the beginning of the experiment, were maintained on ad lib food and water.
Surgery Animals were anesthetized with 12 mg/k8 Thorazine (IM) followed by 20-25 mg/kg Nembutal (IV) prior to placement into a Kopf model 900 stereotaxic instrument equipped with a rabbit head holder. Following an injection of Xylocaine to
the scalp, a 4-5 cm midline incision was made and the skull prepared for stereotaxic positioning of the lesioning electrode. A hole was drilled 16.5 mm posterior to bregma and 1.5 mm right lateral to the midiine. Lambda was set to be 1.5 mm lower than bregma. Enough skull was removed with ronjeurs to permit an incision to be made in the underlying dura mater. Lesioning electrodes were constructed of nichrome wire (diameter 0.25 mm; length 2.2 cm) insulated with three coats of DuPont enamel with each coat-baked for 30 minutes at 165"C. Tip exposure was 1.5 ram. The lesioning electrode was advanced to a depth of 15.5-17.5 mm from the dorsal surface of the brain, and an electrocoagulation lesion was made by increasing the output of a Grass Model LM4 radio frequency lesion maker from 0 to 20 mA over a period of 30 seconds. Time at peak current was varied to control the size of the lesion. Animals 13 and 16 received lesions of the right abducens nucleus, Animal 14 received a large lesion of the fight reticular formation, and Animal 15 received a small lesion in the reticular formation (see Fig. 1). Following retraction of the electrode, the exposed brain area was packed w/th absorbable gelat/n sponge, and Neosporin (polymyxin B-Bacitracin-neomycin) was applied liberally before the incision was closed.
Apparatus The four rabbits were trained and tested concurrently in
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unsystematic order with a constant intertrial interval of 30 seconds. The CS--US interval was 300 msec for each CS, the CS and US terminated together 350 msec after CS onset.
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Immediately following the retention test, the animals were perfused transcardially from Nembutal anesthesia with isotonic saline followed by 10% Formalin. Brains were stored in Formalin for 19 days followed by 3 days in 30% sucrose-formalin. Frozen coronal sections (40/z) were taken through the lesion area. Every fourth section was mounted on glass and stained with 0.5%-cresyl violet.
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FIG. 2. Tracing of responses from Trials 27, 30 and 43 of the last acquisition session and from Trials 27, 30 and 43 of the retention test. Horizontal calibration bars correspond to the CS-US interval (300 reset) and 1.5 era. The vertical bar corresponds to 1 cm. The numbers 13-16 are animal numbers, and the letters T, L, and B stand for tone, light and back shock CSs, respectively.
four ventilated, fireproofed, and sound attenuated file cabinet drawers as described in previous published reports from our laboratory (e.g. [15]). Each rabbit was restrained in a Plexiglas box like those described by Gormezano [6]. A minitorque potentiometer, mechanically linked by a nylon suture in the right NM, provided a DC signal which was recorded on a four-channel Grass polygraph. The potentiometer was calibrated such that 0.5 mm of NM extension was reflected in a 1.0 mm positive deflection of the recording pen. A CR'was defined as a positive pen deflection which w.as at least 1.0 mm. The larger pen deflections illustrated in Fig. 2 represent complete extensions of the NM. Responses of smaller amplitude in normal unoperated animals typically come about when the membrane assumes a partially extended position between trials. The CSs consisted of (a) illumination of two 6 W (4.5 V) incandescent lamps located behind white plastic translucent screens, (b) a 1200 Hz tone of 85 dB (re: 20 uN/mZ), and (c) a mild 8 V AC electric shock applied via safety pin electrodes implanted chronically in the skin of the back above the scapulae. The US was a 2 mA AC electrode shock of 50 msec duration delivered via stainless steel suture (Clay Adams 9 ram) placed approximately 3 mm inferior to the right eye. Procedures The animals were sutured and adapted to restraint and the experimental enclosures for 45 minutes. Acquisition training began the next day and continued for a total of five successive daily sessions. On the day following the last acquisition session, the animals were lesioned as described above. After five days of recovery, they were given a sixth session identical to the five acquisition sessions. This last session served as the retention test, and all trials were reinforced by presentation of the US. Each of the three CSs, lights, tone, and back shock, was paired with the US 30 times daily for a grand total of 90 trials per day. The three types of trials were presented in a fixed
Figure 1 depicts reconstructions of the lesions for each animal on plates traced from the Messen and Olszewsky [11] atlas of the rabbit rhombencephalon. The numbers in the lower fight comer of each tracing correspond to the section number in the lower right comer of photographs in the atlas. Figure 2 shows tracings of the polygraph record for each animal and type of CS, both before (acquisition) and after lesioning (retention). The histological and behavioral evidence are considered in greater detail for each animal individually. Animal 15 Figure 1 shows that Animal 15 sustained a very small lesion of the reticular formation that spared both the abducens nerve and nucleus. Figure 2 shows that CR amplitude, but not UR amplitude, decreased after lesioning. Measures of response topography for the session before surgery and the retention sessions were taken. Trials not containing a CR were excluded and not replaced. The mean CR amplitude before lesions was 9.9 mm (Standard deviation (SD)=4.9, range (R)=l-16) while after lesioning the mean CR amplitude was 2.7 mm (SD= 1.82, R= 1-10). The mean UR amplitude before surgery was 13.3 mm (SD=4.5, R=8-25), and after lesioning it was 20.4 mm (SD=ll.6, R=2-42). The average CR latency was unaffected by the lesion. The measures before and after surgery were 201.6 msec (SD=37.1) and 206 msec (SD=49.27), respectively. CRs occurred on 77% of the trials before lesioning and on 87% after lesioning. Animal 14 Figure 1 indicates that Animal 14 suffered an extensive lesion of the pontine reticular formation (nucleus reticularis pontis caudalis; nucleus reticularis parvocellularis) ranging from the level of the trigeminal nerve (No. 534) and extending caudally to the level of the nucleus of the facial nerve (No. 424). Figure 2 suggests that the lesion failed to disrupt either the conditioned or unconditioned components of the response. This impression was confirmed by measures of response topography. Amplitude of the CR averaged 5.8 mm (SD=I.6, R = I - l l ) on the session before surgery. The average CR amplitude following lesioning was 4.0 mm (SD= 1.0, R= 1-6). Before surgery, the latency of CRs averaged 190.4 msec (SD=40). After lesioning, the latency of CRs averaged 186.7 msec (SD=50). Conditioned responses occurred on 90% of all trials before surgery and 98% of all trials on the retention test. The average UR amplitude before lesioning
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bl(_i. 3. Photomicrograph of lesion in Animal 14 at the level of section No. 450 (see Fig. 1). The calibration b a r = 5 0 0 #. The arrow indicates the rootlets of the a b d u c e n s nerve (labeled nl.
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FIG. 4. Photomicrograph of right abducens nucleus of Animal 14 at the level of section No. 450 [8]. Calibration bar= 100 g..
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FIG. 5. Photomicrograph of area demarcated by the rectangle in Fig. 4. Calibration bar=50/z. Explanation in text.
was 9.3 mm (SD=3.4, R=4-19). After lesioning, the mean UR amplitude was 10.9 mm (SD= 10, R=4-47). Although the lesion in Animal 14 was relatively large, it spared a portion of the reticular formation immediately ventral to the abducens nucleus (No. 450). An estimate o f the damage to the reticular formation at the level of the abducens nucleus (No. 450) indicated that approximately 34% of the area was spared. Consequently, this case cannot be taken as disconfirmation of the hypothesis that the critical events in conditioning the N M R occur in the reticular formation. Microscopic examination o f the section corresl~0nding tO No. 450 indicated that the lesion in Animal 14 impinged on rootlets of the sixth nerve (Fig, 3). The arrow in the lower portion of Fig. 3 marks the location of these fibers ventral to the lesion. Figure 4 is a photograph o f thd ipsilateral abdut e n s nucleus on the same section. The pale appearance of cells in the lateral zone of the nucleus (right hand portion of Fig. 4) suggests a chromatolytic reaction to the fiber damage
shown in Fig. 3. Figure 5 is a photograph taken at higher power of the area demarcated by the rectangle in Fig. 4. Figure 5 shows at least three neurons at various stages of chromatolysis (labeled C) and one normal appearing neuron (labeled N). (The evidence of chromatotysis in response to partial transection of the abducens nerve is interesting in light of a recently reported failure to observe chromatolysis of abducens motoneurons following axotomy of the sixth nerve in cat [5]. Since the abducens nucleus presumably provides the last efferent link in the neural circuit controlling the NMR, the failure of the lesion to disrupt the NMR is surprising, Perhaps sparing of a healthy subpopulation o f motoneurons in the abducens nucleus was sufficient to maintain the NMR. Animal 13
Not so readily explained is Animal 13 which suffered virtually complete destruction of the ipsilateral abducens nu-
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FIG. 6. Montage of photomicrographs of the fight and left abducens nucleus (N) of Animal 13 through its rostral-caudal extent (A-C). Calibration bar=500/~. Explanation in text.
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FIG. 7. Montage of photomicrographs of the right and left abducens nucleus (N) of Animal 16 through its rostral-caudal cxten~ (A-C). Calibration bar=500/x. Explanation in text.
EFFERENT CONTROL OF NICTITATING MEMBRANE RESPONSE cleus (see Fig. 1) yet survived surgery with its NMR intact (see Fig. 2). Photographic reconstructions (Fig. 6) show the rostral-caudal extent of damage to the abducens nucleus on the right of panels A-C, as contrasted with the normal nucleus (labeled N) seen most readily on the left of panel A. Microscopic inspection suggested sparing of some motoneurons located immediately to the right and slightly ventral to the genu (Fig. 6, panel B). In cell counts made of the ipsilateral abducens nucleus and the intact contralateral abducens nucleus, it was estimated that approximately 91% of the neurons of the ipsilateral abducens nucleus were destroyed. Animal 13 gave an average of 93% CRs before surgery and 100% during the retention test. Measures of response topography failed to uncover a significant effect of the lesions. Amplitude of the CR averaged 8.7 mm (SD=3.0, R=l-13) before surgery and 10.9 mm (SD=3.5, R=2-15) on the retention test. Latency of the CR averaged 156 msec (SD=48) before surgery and 162.0 msec (SD=48) during retention. Average UR amplitude before surgery was 10.9 mm (SD=3.2, R=I-13) and 13.6 mm (SD=I.8, R=9-18) on the retention test. A n i m a l 16
Animal 16 sustained a total ablation of the ipsilateral abducens nucleus (see Figs. 1 and 7), yet the conditioned and unconditioned responses were not eliminated. Figure 2 suggests a reduction of the amplitude of the response, and this tended to be confirmed by comparing the average amplitude from the session immediately before surgery with those on the retention test. The CR amplitude averaged 6.4 mm (SD=3.7, R=l-15) before surgery and 1.4 mm (SD=0.6, R = 1-4) after surgery. Latency of CRs, however, was unaffected by the lesion, averaging 207 msec (SD=38) before surgery and 170 msec (SD=69) during the retention test. Before surgery UR amplitude averaged 12.9 mm (SD=4.7, R=8-21) and after surgery it averaged 4.4 mm (SD=I.4, R = 1-8). Although the unconditioned NMR was apparent on 100% of trials during the retention test, CRs occurred on 75% of trials compared to 87% before surgery. This decrease was due largely to the backshock trials which, unlike tone or light trials, yielded only 60% of CRs after surgery compared to 93% before surgery. Although the average UR amplitude in Animal 16 after surgery (4.4 mm) was only 34% of the average UR amplitude for this animal before surgery (12.9 mm), an average UR amplitude of this extent suggests that some eyeball retraction had occurred. Given that the UR amplitude of the response ranged as high as 8 mm, it seems unlikely that NM extension was due solely to the effects of eyelid closure. (Cegavske et al. [4] found that the mean amplitude of the unconditioned response following section of the sixth nerve was 1.2 mm, compared with 19.0 mm before transection. The authors attributed this small response to the animals' attempt to close their eyes against the eyelid retractors employed in their study.) GENERAL DISCUSSION
Motoneurons of the abducens nucleus evidently do not provide the only efferent control of the rabbit NMR. Ninety-one percent destruction of the abducens nucleus in one case and complete destruction in another case failed to eliminate the conditioned or unconditioned responses (Animals 13 and 16). How can these findings be reconciled with
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the evidence cited in the introduction implicating abducens motoneurons in the NMR? One possibility is that retractor bulbi muscles are innervated by the contralateral (spared) abducens nucleus. Another possibility is that other extraocular muscles in addition to the retractor bulbi muscles participate in the NMR. Finally, the motoneurons innervating the retractor bulbi muscles may not originate in the abducens nucleus but in some other nuclear group. Crossed efferent control might be ruled out as an explanation because anatomists have not reported contralateral projections of the abducens nerve. This is consistent with our observations that stimulation of the abducens nucleus (e.g., in Animals 13 and 16 prior to lesioning) evokes retraction of the ipsilateral eyeball but no reaction in the contralateral eyeball. Far more likely is the possibility that other extraocular muscles in addition to the retractor bulbi muscles participate in eyeball retraction. Thus, while the retractor bulbi muscles may be sufficient to retract the eyeball and produce NM extension [12], these muscles may not be necessary, at least when paraorbital shock is employed as the eliciting stimulus. Lorente de N6 [10] stated that the rabbit NMR is produced by "simultaneous contraction of the retractor bulbi and the six extrinsic ocular muscles ([10], p. 704)." This contention is supported by evidence that peripheral trigeminal stimulation elicited contraction of all six extrinsic muscles. Contraction of the four rectus muscles was more pronounced than contraction of the oblique muscles ([10], p. 706, Fig. 1). The negligible role of oculomotor and trochlear motoneurons in eyeball retractions reported by Cegavske et al. [4] might reasonably be attributed to the use of a corneal airpuff as the eliciting stimulus in their study in contrast to the use of electrical shock by Lorente de N6 [10] and paraorbital shock in the present investigation. The possibility that the retractor bulbi muscles are not innervated by motoneurons of the abducens nucleus seems plausible in light of the recent HRP-studies with cats that report the retractor bulbi muscles to be innervated by the accessory abducens nucleus [2] which lies dorsal to the superior olive [7, 8, 16]. However, investigators differ as to whether other nuclei besides the accessory abducens innervate the retractor bulbi muscles. Spencer [16] concluded that the retractor bulbi is innervated by the abducens, accessory abducens, and oculomotor nuclei. Grant et al. [7] and Guegan, Geritaud and Horcholle-Bossavit [8] contend that the retractor bulbi is innervated solely by the accessory abducens nucleus. In all cases reported in the present experiment the accessory ahducens nucleus was spared. Further research in our laboratory is proceeding on the assumption that as many as five distinct nuclear groups may be involved in eyeball retraction and the NMR: (a) the ipsilateral abducens nucleus innervating the lateral rectus muscle, (b) the ipsilateral oculomotor nucleus innervating the medial and inferior rectus muscles and the inferior oblique, (c) the contralateral oculomotor nucleus innervating the superior rectus muscle, (d) the trochlear nucleus innervating the superior oblique, and (e) the accessory abducens nucleus, should its function in rabbit be the same as in cat, innervating the retractor bulbi muscles. This model of efferent control of the rabbit NMR can be reconciled with the available evidence. Cegavske et al. [4] reported that stimulation of the oculomotor nucleus produced retraction of the NM rather than extension. This finding does not rule out the possibility that extrinsic muscles
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innervated by the third nerve participate in eyeball retraction when elicited by noxious peripheral stimulation [10]. Stimulation of the area of sixth nucleus [4, 13, 17, 18] could conceivably produce eyeball retraction through either (a) spread of current to the sensory trigeminal nucleus or (b) direct stimulation of the sixth nerve containing axons from an accessory abducens nucleus projecting to the retractor bulbi muscles. Finally, the widely reported observation of elec-
trophysiological unit activity (multiple unit activity) from abducens motoneurons correlated with the conditioned and unconditioned NMR [4, 9, 17] would be consistent with the notion that the lateral rectus muscle, as well as the retractor bulbi muscles, participates in eyeball retraction. Alternatively, these studies might have been recording action potentials from axons of an accessory abducens nucleus which, in the cat, course through the abducens nucleus [7].
REFERENCES 1. Akagi, Y. The localization of the motor neurons innervating the extraocular muscles in the oculomotor nuclei of the cat and rabbit, using horseradish peroxidase. J. comp. Neurol. 181: 745-762, 1978. 2. Ariens Kappers, C. U., G. C. Huber and E. C. Crosby. The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. Volume One. New York: Harrier, 1936. 3. Berger, T. W. and R. F. Thompson. Neuronal plasticity in the limbic system during classical conditioningof the rabbit nictitating membrane response. I. The hippocampus. Brain Res. 145: 323--346, 1978. 4. Cegavske, C. F., R. F. Thompson, M. M. Patterson and I. Gormezano. Mechanisms of efferent neuronal control of the reflex nictitating membrane response in rabbit (Oryctolagus cuniculus). J. comp. physiol. Psychol. 90: 411-423, 1976. 5. Delgado-Garcia, J. M., R. Baker, K. Alley and R. McCrea. Axotomy and physiology of axotomized cat abducens motoneurons. Society for Neuroscience Abstracts 4: 168, 1978. 6. Gormezano, I. Classical conditioning. In: Experimental Methods and Instrumentation in Psychology, edited by J. B. Sidowski. New York:. McGraw-Hill, 1966. 7. Grant, K., P. Gutritaud, G. Horcholle-Bossavit and S. Ty~Dumont. Anatomical and electrophysiological identification of motoneurons supplying the cat retractor bulbi muscle. Expl Brain Res. 34: 541-550,_1979. 8. Gutgan, M., J-P. Gutritaud and G. Horcholte-Bossavit. Localisation des motoneurons du muscle retractor bulbi par transport rttrograde de peroxydase exogtne chez le chat. C. R. Acad. Sci. Paris (D). 286: 1355-1357, 1978. 9. Harrison, T. A. and C. F. Cegavske. Neural activity recorded in the abducens and oculomotor nuclei during nictitating membrane conditioning in the rabbit. Society for Neuroscience Abstracts 4: 259, 1978.
10. Lorente de Nt, R. The interaction of the corneal reflex and vestibular nystagmus. Am. J. Physiol. 103: 704-711, 1933. 11. Messen, H. and J. A. Olszewsky. A Cytoarchitectonic Atlas of the Rhombencephalon of the Rabbit. Basal: Karger, 1949. 12. Mis, F. W. A midbrain-brainstem circuit for conditioned inhibition of the nictitating membrane response in the rabbit (Oryctolagus cuniculus). J. comp. physiol. Psychol. 91: 975-988, 1977. 13. Mis, F. W., I. Gormezano, D. Rosewall and J. A. Harvery. Electrical stimulation of the abducens nucleus in classical conditioning of the rabbit's nictitating membrane response. Society for Neuroscience Abstracts 4: 261, 1978. 14. Moore, J. W. Brain processes and conditioning. In: Mechanisms of Learning and Motivation. A Memorial Volume to Jerzy Konorski, edited by A. Dickinson and R. A Boakes. Hillsdale, NJ: Erlbaum, 1979. 15. Moore, J. W., H. G. Marchant III, J. B. Norman and S. E. Kwaterski. Electrical brain stimulation as a Pavlovian conditioned inhibitor, Physiol, Behav. 10: 581-587, 1973. 16. Spencer, R. F. Identification and localization of motoneurons innervatingthe cat retractor bulbi muscle. Society for Neuroscience Abstracts 4: 168, 1978. 17. Thompson, R. F. The search for the engram. Am. Psychologist 31: 209-227, 1976. 18. Young, R. A., C. F. Cegavske and R. F. Thompson. Toneinduced changes in excitability of abdueens motoneurons and the reflex path of nictitating membrane response in rabbit (Oryctolagus cuniculus). J. comp. physiol. Psychol. 90: 424-434, 1976.
Efferent Neuronal Control of the Nictitating Membrane Response in Rabbit (Oryctolagus cuniculus): A Reexamination I G E R A L D M. P O W E L L , N E I L E. B E R T H I E R A N D J O H N W. M O O R E
Department o f Psychology, Middlesex House University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 29 S e p t e m b e r 1978 POWELL, G. M., N. E. BERTHIER AND J. W. MOORE. Efferent neuronal control of the nictitating membrane response in rabbit (Oryctolagus cuniculus): A reexamination. PHYSIOL. BEHAV. 2,3(2) 299--308, 1979.--Four Albino rabbits acquired a classically conditioned nictitating membrane response (NMR) to light, tone, and back shock conditioned stimuli using a paraorbital shock applied to the right eye as the unconditioned stimulus. Electrocoagulation lesions were made in the ipsilateral abducens nucleus of the pons in two animals, and the pontine reticular formation in two animals. Reinstitufion of conditioning following surgery (retention test) indicated that the lesions failed to eliminate conditioned or unconditioned responses. This result is surprising in light of recent evidence that efferent control of the rabbit NMR derives solely from motoneurons of the ipsilateral abducens nucleus. Alternative hypotheses are considered. Nictitating membrane response
Abducens nucleus
C L A S S I C A L (Pavlovian) conditioning of the rabbit nictitating membrane response (NMR) offers the investigator concerned with the neural basis of associative learning in mammals a preparation with several attractive features [3, 15, 17]. High on the list is the evident simplicity of neural efferent control of the response. Motoneurons responsible for the NMR are thought to be located, together with those innervating the lateral rectus muscle, in the ipsilateral abducens nucleus of the pons [3, 4, 17, 18]. The electrophysiological activity of motoneurons in the abducens nucleus is mirrored with high fidelity by the peripheral response [4, 9, 17]: "The behavioral response [provides] a 'label' to be compared with activity in higher brain centers ([3], p. 324)." In short, the preparation seems to offer a system that simplifies the task of tracing the neural circuits involved in conditioning from the motoneurons of the "final common path" to other regions of the brain. The NMR occurs when the membrane sweeps across the eye, in a nasal to temporal direction, as part of an integrated pattern of defensive reactions that include the eyeblink and eyeball retraction. Reflexive extension of the NMR is typically elicited by noxious stimulation of the cornea by air puffs or electric shock applied paraorbitally. These unconditioned stimuli (USs) are sufficient to reinforce a conditioned anticipatory extension of the membrane (CR) to visual, auditory, or tactile conditioned stimufi (CSs). While a small strip of muscle innervated by motoneurons in the oculomotor complex subserves retraction of the membrane, extension is a passive consequence of eyeball retrac-
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tion [4]. In the most thorough investigation of efferent control of the NMR in rabbit to date, Cegavske, Thompson, Patterson and Gormezano [4] reported that: (a) Electrical stimulation of the abducens nucleus elicited a normal appearing NMR, but stimulation of the oculomotor, trochlear, facial nuclei or superior cervical ganglion did not; (b) Stimulation of the distal end of the transected sixth cranial nerve produced a normal appearing NMR, but stimulation of the third, fourth, or seventh nerve did not; (c) Destruction of the sixth nerve in one animal by an electrolytic lesion eliminated the NMR; (d) Corneal stimulation elicited electrophysiological activity from neurons in the abducens nucleus. Mis [12] has provided additional relevant evidence by demonstrating that both the conditioned and unconditioned NMR are unaffected by transection of all extrinsic eye muscles (four rectus muscles and two oblique muscles) excepting the retractor bulbi muscles. Clearly, the retractor bulbi muscles are sufficient for eyeball retraction and the NMR. The present investigation sought to further explore the neural control of the conditioned and unconditioned NMR in rabbits by making lesions in the brain stem in the vicinity of the abducens nucleus. Two considerations motivated this research: First, the lesion data presented by Cegavske et al. [4] are sparse and problematic in that one reported instance of response disruption involved six lesions in unspecified loci as well as destruction of the sixth nerve. Some combination of these multiple lesions might have been responsible for the disruption attributed to destruction of the sixth nerve. Secondly, indirect evidence from a variety of sources
1This research was supported by National Science Foundation Grant BNS 77-14871 to the third author.
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FIG. 1. Reconstruction of lesions for Animals 14 and 15 (top row) and Animals 13and 16 (bottom row). Section numbers are from Messen and Olszewsky [8], moving caudally from left to right. The genu of the facial nerve is labeled G in sections No. 450 and No. 424 of the upper row. The abdueens nucleus is labeled N in section No. 450.
suggests that the locus of associative connections responsible for acquisition and performance of the conditioned NMR might lie in the reticular formation ventral to the abducens nucleus [14]. Given the evident compact and discrete nature of the neural systems providing efferent control of the NMR, we reasoned that the volume of tissue subserving the critical linkages of conditioning might be equally compact and located near to, but not within, the abducens nucleus. We hoped that a suitable lesion would disrupt a previously established CR to a variety of CSs while leaving intact the UR to paraorbital shock. Instead, we found that lesions of the abducens nucleus and reticular formation had little or no effect on either the conditioned or unconditioned NMR. METHOD
Animals Four experimentally naive albino rabbits, weighing 2.63,0 kgand approximately 100 days old at the beginning of the experiment, were maintained on ad lib food and water.
Surgery Animals were anesthetized with 12 mg/k8 Thorazine (IM) followed by 20-25 mg/kg Nembutal (IV) prior to placement into a Kopf model 900 stereotaxic instrument equipped with a rabbit head holder. Following an injection of Xylocaine to
the scalp, a 4-5 cm midline incision was made and the skull prepared for stereotaxic positioning of the lesioning electrode. A hole was drilled 16.5 mm posterior to bregma and 1.5 mm right lateral to the midiine. Lambda was set to be 1.5 mm lower than bregma. Enough skull was removed with ronjeurs to permit an incision to be made in the underlying dura mater. Lesioning electrodes were constructed of nichrome wire (diameter 0.25 mm; length 2.2 cm) insulated with three coats of DuPont enamel with each coat-baked for 30 minutes at 165"C. Tip exposure was 1.5 ram. The lesioning electrode was advanced to a depth of 15.5-17.5 mm from the dorsal surface of the brain, and an electrocoagulation lesion was made by increasing the output of a Grass Model LM4 radio frequency lesion maker from 0 to 20 mA over a period of 30 seconds. Time at peak current was varied to control the size of the lesion. Animals 13 and 16 received lesions of the right abducens nucleus, Animal 14 received a large lesion of the fight reticular formation, and Animal 15 received a small lesion in the reticular formation (see Fig. 1). Following retraction of the electrode, the exposed brain area was packed w/th absorbable gelat/n sponge, and Neosporin (polymyxin B-Bacitracin-neomycin) was applied liberally before the incision was closed.
Apparatus The four rabbits were trained and tested concurrently in
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unsystematic order with a constant intertrial interval of 30 seconds. The CS--US interval was 300 msec for each CS, the CS and US terminated together 350 msec after CS onset.
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Immediately following the retention test, the animals were perfused transcardially from Nembutal anesthesia with isotonic saline followed by 10% Formalin. Brains were stored in Formalin for 19 days followed by 3 days in 30% sucrose-formalin. Frozen coronal sections (40/z) were taken through the lesion area. Every fourth section was mounted on glass and stained with 0.5%-cresyl violet.
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FIG. 2. Tracing of responses from Trials 27, 30 and 43 of the last acquisition session and from Trials 27, 30 and 43 of the retention test. Horizontal calibration bars correspond to the CS-US interval (300 reset) and 1.5 era. The vertical bar corresponds to 1 cm. The numbers 13-16 are animal numbers, and the letters T, L, and B stand for tone, light and back shock CSs, respectively.
four ventilated, fireproofed, and sound attenuated file cabinet drawers as described in previous published reports from our laboratory (e.g. [15]). Each rabbit was restrained in a Plexiglas box like those described by Gormezano [6]. A minitorque potentiometer, mechanically linked by a nylon suture in the right NM, provided a DC signal which was recorded on a four-channel Grass polygraph. The potentiometer was calibrated such that 0.5 mm of NM extension was reflected in a 1.0 mm positive deflection of the recording pen. A CR'was defined as a positive pen deflection which w.as at least 1.0 mm. The larger pen deflections illustrated in Fig. 2 represent complete extensions of the NM. Responses of smaller amplitude in normal unoperated animals typically come about when the membrane assumes a partially extended position between trials. The CSs consisted of (a) illumination of two 6 W (4.5 V) incandescent lamps located behind white plastic translucent screens, (b) a 1200 Hz tone of 85 dB (re: 20 uN/mZ), and (c) a mild 8 V AC electric shock applied via safety pin electrodes implanted chronically in the skin of the back above the scapulae. The US was a 2 mA AC electrode shock of 50 msec duration delivered via stainless steel suture (Clay Adams 9 ram) placed approximately 3 mm inferior to the right eye. Procedures The animals were sutured and adapted to restraint and the experimental enclosures for 45 minutes. Acquisition training began the next day and continued for a total of five successive daily sessions. On the day following the last acquisition session, the animals were lesioned as described above. After five days of recovery, they were given a sixth session identical to the five acquisition sessions. This last session served as the retention test, and all trials were reinforced by presentation of the US. Each of the three CSs, lights, tone, and back shock, was paired with the US 30 times daily for a grand total of 90 trials per day. The three types of trials were presented in a fixed
Figure 1 depicts reconstructions of the lesions for each animal on plates traced from the Messen and Olszewsky [11] atlas of the rabbit rhombencephalon. The numbers in the lower fight comer of each tracing correspond to the section number in the lower right comer of photographs in the atlas. Figure 2 shows tracings of the polygraph record for each animal and type of CS, both before (acquisition) and after lesioning (retention). The histological and behavioral evidence are considered in greater detail for each animal individually. Animal 15 Figure 1 shows that Animal 15 sustained a very small lesion of the reticular formation that spared both the abducens nerve and nucleus. Figure 2 shows that CR amplitude, but not UR amplitude, decreased after lesioning. Measures of response topography for the session before surgery and the retention sessions were taken. Trials not containing a CR were excluded and not replaced. The mean CR amplitude before lesions was 9.9 mm (Standard deviation (SD)=4.9, range (R)=l-16) while after lesioning the mean CR amplitude was 2.7 mm (SD= 1.82, R= 1-10). The mean UR amplitude before surgery was 13.3 mm (SD=4.5, R=8-25), and after lesioning it was 20.4 mm (SD=ll.6, R=2-42). The average CR latency was unaffected by the lesion. The measures before and after surgery were 201.6 msec (SD=37.1) and 206 msec (SD=49.27), respectively. CRs occurred on 77% of the trials before lesioning and on 87% after lesioning. Animal 14 Figure 1 indicates that Animal 14 suffered an extensive lesion of the pontine reticular formation (nucleus reticularis pontis caudalis; nucleus reticularis parvocellularis) ranging from the level of the trigeminal nerve (No. 534) and extending caudally to the level of the nucleus of the facial nerve (No. 424). Figure 2 suggests that the lesion failed to disrupt either the conditioned or unconditioned components of the response. This impression was confirmed by measures of response topography. Amplitude of the CR averaged 5.8 mm (SD=I.6, R = I - l l ) on the session before surgery. The average CR amplitude following lesioning was 4.0 mm (SD= 1.0, R= 1-6). Before surgery, the latency of CRs averaged 190.4 msec (SD=40). After lesioning, the latency of CRs averaged 186.7 msec (SD=50). Conditioned responses occurred on 90% of all trials before surgery and 98% of all trials on the retention test. The average UR amplitude before lesioning
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bl(_i. 3. Photomicrograph of lesion in Animal 14 at the level of section No. 450 (see Fig. 1). The calibration b a r = 5 0 0 #. The arrow indicates the rootlets of the a b d u c e n s nerve (labeled nl.
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FIG. 4. Photomicrograph of right abducens nucleus of Animal 14 at the level of section No. 450 [8]. Calibration bar= 100 g..
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FIG. 5. Photomicrograph of area demarcated by the rectangle in Fig. 4. Calibration bar=50/z. Explanation in text.
was 9.3 mm (SD=3.4, R=4-19). After lesioning, the mean UR amplitude was 10.9 mm (SD= 10, R=4-47). Although the lesion in Animal 14 was relatively large, it spared a portion of the reticular formation immediately ventral to the abducens nucleus (No. 450). An estimate o f the damage to the reticular formation at the level of the abducens nucleus (No. 450) indicated that approximately 34% of the area was spared. Consequently, this case cannot be taken as disconfirmation of the hypothesis that the critical events in conditioning the N M R occur in the reticular formation. Microscopic examination o f the section corresl~0nding tO No. 450 indicated that the lesion in Animal 14 impinged on rootlets of the sixth nerve (Fig, 3). The arrow in the lower portion of Fig. 3 marks the location of these fibers ventral to the lesion. Figure 4 is a photograph o f thd ipsilateral abdut e n s nucleus on the same section. The pale appearance of cells in the lateral zone of the nucleus (right hand portion of Fig. 4) suggests a chromatolytic reaction to the fiber damage
shown in Fig. 3. Figure 5 is a photograph taken at higher power of the area demarcated by the rectangle in Fig. 4. Figure 5 shows at least three neurons at various stages of chromatolysis (labeled C) and one normal appearing neuron (labeled N). (The evidence of chromatotysis in response to partial transection of the abducens nerve is interesting in light of a recently reported failure to observe chromatolysis of abducens motoneurons following axotomy of the sixth nerve in cat [5]. Since the abducens nucleus presumably provides the last efferent link in the neural circuit controlling the NMR, the failure of the lesion to disrupt the NMR is surprising, Perhaps sparing of a healthy subpopulation o f motoneurons in the abducens nucleus was sufficient to maintain the NMR. Animal 13
Not so readily explained is Animal 13 which suffered virtually complete destruction of the ipsilateral abducens nu-
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FIG. 6. Montage of photomicrographs of the fight and left abducens nucleus (N) of Animal 13 through its rostral-caudal extent (A-C). Calibration bar=500/~. Explanation in text.
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FIG. 7. Montage of photomicrographs of the right and left abducens nucleus (N) of Animal 16 through its rostral-caudal cxten~ (A-C). Calibration bar=500/x. Explanation in text.
EFFERENT CONTROL OF NICTITATING MEMBRANE RESPONSE cleus (see Fig. 1) yet survived surgery with its NMR intact (see Fig. 2). Photographic reconstructions (Fig. 6) show the rostral-caudal extent of damage to the abducens nucleus on the right of panels A-C, as contrasted with the normal nucleus (labeled N) seen most readily on the left of panel A. Microscopic inspection suggested sparing of some motoneurons located immediately to the right and slightly ventral to the genu (Fig. 6, panel B). In cell counts made of the ipsilateral abducens nucleus and the intact contralateral abducens nucleus, it was estimated that approximately 91% of the neurons of the ipsilateral abducens nucleus were destroyed. Animal 13 gave an average of 93% CRs before surgery and 100% during the retention test. Measures of response topography failed to uncover a significant effect of the lesions. Amplitude of the CR averaged 8.7 mm (SD=3.0, R=l-13) before surgery and 10.9 mm (SD=3.5, R=2-15) on the retention test. Latency of the CR averaged 156 msec (SD=48) before surgery and 162.0 msec (SD=48) during retention. Average UR amplitude before surgery was 10.9 mm (SD=3.2, R=I-13) and 13.6 mm (SD=I.8, R=9-18) on the retention test. A n i m a l 16
Animal 16 sustained a total ablation of the ipsilateral abducens nucleus (see Figs. 1 and 7), yet the conditioned and unconditioned responses were not eliminated. Figure 2 suggests a reduction of the amplitude of the response, and this tended to be confirmed by comparing the average amplitude from the session immediately before surgery with those on the retention test. The CR amplitude averaged 6.4 mm (SD=3.7, R=l-15) before surgery and 1.4 mm (SD=0.6, R = 1-4) after surgery. Latency of CRs, however, was unaffected by the lesion, averaging 207 msec (SD=38) before surgery and 170 msec (SD=69) during the retention test. Before surgery UR amplitude averaged 12.9 mm (SD=4.7, R=8-21) and after surgery it averaged 4.4 mm (SD=I.4, R = 1-8). Although the unconditioned NMR was apparent on 100% of trials during the retention test, CRs occurred on 75% of trials compared to 87% before surgery. This decrease was due largely to the backshock trials which, unlike tone or light trials, yielded only 60% of CRs after surgery compared to 93% before surgery. Although the average UR amplitude in Animal 16 after surgery (4.4 mm) was only 34% of the average UR amplitude for this animal before surgery (12.9 mm), an average UR amplitude of this extent suggests that some eyeball retraction had occurred. Given that the UR amplitude of the response ranged as high as 8 mm, it seems unlikely that NM extension was due solely to the effects of eyelid closure. (Cegavske et al. [4] found that the mean amplitude of the unconditioned response following section of the sixth nerve was 1.2 mm, compared with 19.0 mm before transection. The authors attributed this small response to the animals' attempt to close their eyes against the eyelid retractors employed in their study.) GENERAL DISCUSSION
Motoneurons of the abducens nucleus evidently do not provide the only efferent control of the rabbit NMR. Ninety-one percent destruction of the abducens nucleus in one case and complete destruction in another case failed to eliminate the conditioned or unconditioned responses (Animals 13 and 16). How can these findings be reconciled with
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the evidence cited in the introduction implicating abducens motoneurons in the NMR? One possibility is that retractor bulbi muscles are innervated by the contralateral (spared) abducens nucleus. Another possibility is that other extraocular muscles in addition to the retractor bulbi muscles participate in the NMR. Finally, the motoneurons innervating the retractor bulbi muscles may not originate in the abducens nucleus but in some other nuclear group. Crossed efferent control might be ruled out as an explanation because anatomists have not reported contralateral projections of the abducens nerve. This is consistent with our observations that stimulation of the abducens nucleus (e.g., in Animals 13 and 16 prior to lesioning) evokes retraction of the ipsilateral eyeball but no reaction in the contralateral eyeball. Far more likely is the possibility that other extraocular muscles in addition to the retractor bulbi muscles participate in eyeball retraction. Thus, while the retractor bulbi muscles may be sufficient to retract the eyeball and produce NM extension [12], these muscles may not be necessary, at least when paraorbital shock is employed as the eliciting stimulus. Lorente de N6 [10] stated that the rabbit NMR is produced by "simultaneous contraction of the retractor bulbi and the six extrinsic ocular muscles ([10], p. 704)." This contention is supported by evidence that peripheral trigeminal stimulation elicited contraction of all six extrinsic muscles. Contraction of the four rectus muscles was more pronounced than contraction of the oblique muscles ([10], p. 706, Fig. 1). The negligible role of oculomotor and trochlear motoneurons in eyeball retractions reported by Cegavske et al. [4] might reasonably be attributed to the use of a corneal airpuff as the eliciting stimulus in their study in contrast to the use of electrical shock by Lorente de N6 [10] and paraorbital shock in the present investigation. The possibility that the retractor bulbi muscles are not innervated by motoneurons of the abducens nucleus seems plausible in light of the recent HRP-studies with cats that report the retractor bulbi muscles to be innervated by the accessory abducens nucleus [2] which lies dorsal to the superior olive [7, 8, 16]. However, investigators differ as to whether other nuclei besides the accessory abducens innervate the retractor bulbi muscles. Spencer [16] concluded that the retractor bulbi is innervated by the abducens, accessory abducens, and oculomotor nuclei. Grant et al. [7] and Guegan, Geritaud and Horcholle-Bossavit [8] contend that the retractor bulbi is innervated solely by the accessory abducens nucleus. In all cases reported in the present experiment the accessory ahducens nucleus was spared. Further research in our laboratory is proceeding on the assumption that as many as five distinct nuclear groups may be involved in eyeball retraction and the NMR: (a) the ipsilateral abducens nucleus innervating the lateral rectus muscle, (b) the ipsilateral oculomotor nucleus innervating the medial and inferior rectus muscles and the inferior oblique, (c) the contralateral oculomotor nucleus innervating the superior rectus muscle, (d) the trochlear nucleus innervating the superior oblique, and (e) the accessory abducens nucleus, should its function in rabbit be the same as in cat, innervating the retractor bulbi muscles. This model of efferent control of the rabbit NMR can be reconciled with the available evidence. Cegavske et al. [4] reported that stimulation of the oculomotor nucleus produced retraction of the NM rather than extension. This finding does not rule out the possibility that extrinsic muscles
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innervated by the third nerve participate in eyeball retraction when elicited by noxious peripheral stimulation [10]. Stimulation of the area of sixth nucleus [4, 13, 17, 18] could conceivably produce eyeball retraction through either (a) spread of current to the sensory trigeminal nucleus or (b) direct stimulation of the sixth nerve containing axons from an accessory abducens nucleus projecting to the retractor bulbi muscles. Finally, the widely reported observation of elec-
trophysiological unit activity (multiple unit activity) from abducens motoneurons correlated with the conditioned and unconditioned NMR [4, 9, 17] would be consistent with the notion that the lateral rectus muscle, as well as the retractor bulbi muscles, participates in eyeball retraction. Alternatively, these studies might have been recording action potentials from axons of an accessory abducens nucleus which, in the cat, course through the abducens nucleus [7].
REFERENCES 1. Akagi, Y. The localization of the motor neurons innervating the extraocular muscles in the oculomotor nuclei of the cat and rabbit, using horseradish peroxidase. J. comp. Neurol. 181: 745-762, 1978. 2. Ariens Kappers, C. U., G. C. Huber and E. C. Crosby. The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. Volume One. New York: Harrier, 1936. 3. Berger, T. W. and R. F. Thompson. Neuronal plasticity in the limbic system during classical conditioningof the rabbit nictitating membrane response. I. The hippocampus. Brain Res. 145: 323--346, 1978. 4. Cegavske, C. F., R. F. Thompson, M. M. Patterson and I. Gormezano. Mechanisms of efferent neuronal control of the reflex nictitating membrane response in rabbit (Oryctolagus cuniculus). J. comp. physiol. Psychol. 90: 411-423, 1976. 5. Delgado-Garcia, J. M., R. Baker, K. Alley and R. McCrea. Axotomy and physiology of axotomized cat abducens motoneurons. Society for Neuroscience Abstracts 4: 168, 1978. 6. Gormezano, I. Classical conditioning. In: Experimental Methods and Instrumentation in Psychology, edited by J. B. Sidowski. New York:. McGraw-Hill, 1966. 7. Grant, K., P. Gutritaud, G. Horcholle-Bossavit and S. Ty~Dumont. Anatomical and electrophysiological identification of motoneurons supplying the cat retractor bulbi muscle. Expl Brain Res. 34: 541-550,_1979. 8. Gutgan, M., J-P. Gutritaud and G. Horcholte-Bossavit. Localisation des motoneurons du muscle retractor bulbi par transport rttrograde de peroxydase exogtne chez le chat. C. R. Acad. Sci. Paris (D). 286: 1355-1357, 1978. 9. Harrison, T. A. and C. F. Cegavske. Neural activity recorded in the abducens and oculomotor nuclei during nictitating membrane conditioning in the rabbit. Society for Neuroscience Abstracts 4: 259, 1978.
10. Lorente de Nt, R. The interaction of the corneal reflex and vestibular nystagmus. Am. J. Physiol. 103: 704-711, 1933. 11. Messen, H. and J. A. Olszewsky. A Cytoarchitectonic Atlas of the Rhombencephalon of the Rabbit. Basal: Karger, 1949. 12. Mis, F. W. A midbrain-brainstem circuit for conditioned inhibition of the nictitating membrane response in the rabbit (Oryctolagus cuniculus). J. comp. physiol. Psychol. 91: 975-988, 1977. 13. Mis, F. W., I. Gormezano, D. Rosewall and J. A. Harvery. Electrical stimulation of the abducens nucleus in classical conditioning of the rabbit's nictitating membrane response. Society for Neuroscience Abstracts 4: 261, 1978. 14. Moore, J. W. Brain processes and conditioning. In: Mechanisms of Learning and Motivation. A Memorial Volume to Jerzy Konorski, edited by A. Dickinson and R. A Boakes. Hillsdale, NJ: Erlbaum, 1979. 15. Moore, J. W., H. G. Marchant III, J. B. Norman and S. E. Kwaterski. Electrical brain stimulation as a Pavlovian conditioned inhibitor, Physiol, Behav. 10: 581-587, 1973. 16. Spencer, R. F. Identification and localization of motoneurons innervatingthe cat retractor bulbi muscle. Society for Neuroscience Abstracts 4: 168, 1978. 17. Thompson, R. F. The search for the engram. Am. Psychologist 31: 209-227, 1976. 18. Young, R. A., C. F. Cegavske and R. F. Thompson. Toneinduced changes in excitability of abdueens motoneurons and the reflex path of nictitating membrane response in rabbit (Oryctolagus cuniculus). J. comp. physiol. Psychol. 90: 424-434, 1976.
