Brain Research, 556 (1991) 317-320 Elsevier Science Publishers B.V. ADONIS 000689939124773X
317
BRES 24773
Selective involvement of the spinal trigeminal nucleus in the conditioned nictitating membrane reflex of the rabbit Vlastislav Bracha, Jin-Zi Wu, Shelley Cartwright and James R. Bloedel Division of Neurobiology, Barrow Neurological Institute, Phoenix, A Z (U.S.A.) (Accepted 30 April 1991) Key words: Nictitating membrane; Trigeminal nuclei; Conditioning; Learning; Brainstem; Rabbit; Lidocaine
These experiments were performed to test the hypothesis that a region associated with the trigeminal nuclear complex is selectively involved in mediating the classically conditioned nictitating membrane reflex in the rabbit. Mieroinjections of Lidocaine were used to produce a temporary, localized block of neural activity following the conditioning of the reflex using a standard tone/air puff-paired stimulus paradigm. Data indicate that the injection of Lidocaine at the medial pars oralis/retieular formation junction results in a selective suppression of the conditioned reflex The substrate for the plastic changes underlying various types of motor learning has been a topic of great interest during the past decade. Considerable focus has been placed on the possible role of the cerebellum in this process for at least two reasons. First, theoretical and experimental studies have implicated the climbing fiberPurkinje cell synapse as a potential site for such changes n'14. Second, lesions in specific cerebellar regions have been reported to block the acquisition as well as the performance of classically conditioned reflexes, most notably the conditioned nictitating membrane reflex (NMR) of the rabbit 24'2s. In addition, unitary responses of cerebellar neurons correlated with the conditioned response have also been reported 15. However, recent studies have challenged this view by showing that the conditioning of this reflex can occur in the absence of the cerebellum in decerebrate rabbits 12 and that at least some of the effects of cerebellar lesions in intact rabbits are due to a performance deficit rather than a learning deficit 22' 2a. Based on these studies and previous experiments implicating the supratrigeminal region 7 and the red nucleus s'18--2° in this process, we have focused our attention on the role of brainstem sites in the performance of these reflexes. However, our initial studies on the effects of temporary lesions in the vicinity of the red nucleus indicated that this structure is likely involved in the performance of both the conditioned and unconditioned reflexes 3. In contrast, recent data from Gormezano's laboratory ~6 suggest that a region associated with the pars oralis of the spinal trigeminal nucleus may be involved in the conditioned NMR.
The experiments reported here were performed to examine this possibility by assessing the effects of microinjections of Lidocaine in the pars oralis of the spinal trigeminal nucleus and the adjacent reticular formation on the performance of the conditioned and unconditioned NMR. This portion of the trigeminal nucleus receives the majority of the afferents activated by corneal air puff stimuli 2"5"9, the unconditioned stimuli employed in these experiments. The data will show that injection of a caudal-medial region of this nucleus can produce a selective reduction in the amplitude as well as the occurrence of the conditioned NMR. For the purposes of this manuscript, the term selective will refer to non-parallel effects of an agent or procedure on the conditioned and unconditioned responses. During a sterile surgical procedure in which adult albino New Zealand rabbits were anesthetized with a mixture of ketamine (50 mg/kg), xylazine (6 mg/kg) and acepromazine (1.5 mg/kg), guide tubes were implanted to within 3.5 mm above the injection sites in the region of the left trigeminal nucleus (AP = -18 mm, ML = 3 mm, DV = 19-20 mm). Stereotaxic coordinates were based on the position of bregma, which was placed 1.5 mm below lambda. The guide tubes were placed above the trigeminal nucleus ipsilateral to the eye receiving the unconditioned stimulus. In addition hardware was secured to the animal's skull to permit the attachment of a device that held both the lever arm system used to monitor the movement of the nictitating membrane bilaterally and the tube through which the air puff was delivered. Once the animals were adapted to a restraint box, the
Correspondence: J.R. Bloedel, Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ 85013, U.S.A.
318 animals were conditioned in a standard delay paradigm using a 1-kHz, 85-dB tone as the conditioned stimulus (CS). The tone was applied in a 5-pulse, 450-ms duration train (65 ms on and 35 ms off) which was superimposed on a continuous 70 dB white noise background. A n air puff 100 ms in duration and coterminal with the CS was used as the unconditioned stimulus (US). The air, 1.7 psi (11.876 x 103 N/m 2) at the outlet, was delivered to the left cornea through a tube placed 8 m m from the cornea. Each training session consisted of 100 paired trials presented in 19 to 21-s intervals. Animals were trained until they reached at least a 90% incidence of conditioned responses (CRs) across a daily session. Once criterion was reached, the effect of Lidocaine injection in the trained animals was determined using a succession of 3-trial sequences: one paired trial, one air puff-only trial, and one light-only trial. The light US (200 ms in duration) was emitted from a small flashlight bulb mounted to the air puff delivery tube. The light-only trials were used to evaluate the effect of Lidocaine on an unconditioned response (UR) that did not require the trigeminal nuclei for its performance. This control made it possible to determine if the local anesthetic spread to the motor nuclei, a circumstance that would result in the suppression of both the light-evoked as well as the air
puff-evoked U R . The needle used to administer the Lidocaine was inserted at the beginning of each microinfusion experiment. Prior to the injection of Lidocaine 20 three-trial sequences were performed. Then a 4% Lidocaine solution was administered through the injection tube in quantities ranging from 0.2 to 1.0/~1 over a period of 5 min. If no effect was observed, a minimum of 30 three-trial sequences was administered. If a change in the response did o c c u r a succession of three-trial sequences were performed until the animal's U R and C R were qualitatively similar to those observed prior to Lidocaine injection. Control experiments were performed the next day using the injection of pH-adjusted saline administered in the same quantity and to the same site used for lidocaine one day previously. Prior to sacrificing the animal, the position of the site at which the maximum effect was observed was marked by reinserting the injecting tube at the site and injecting 0.5-1.0 #1 of a colloidal iron solution. The location of the injection site was then determined histologically after the animal was perfused with a 1% ferrocyanide and 10% buffered formalin solution and the tissue stained with Perls iron reaction and Neutral red. These sites were used to localize the end of the injection tube. The spread of the
NMR Changes After Lidocaine Injection into the Trigeminal Nucleus
A
C URs in Air Puff Only Trials
L i d o c a i n e ~njection ~"
10o
,c~
3
~ g
2 ,
2-
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0
. . 1
.
. 3
.
. . 5
.
. 7
.
0 0
9
600
12()0
Number of Trials ( b l o c k s o f tS)
B
D URs in Light Only Trials
CRs in Tone+Air Puff Trials
"~.~
Q "
2
I I
,,
2
8
cO 0
0 0
600
1200
t
6O0
1200 ms
Before I n j e c t i o n .....
After Injection
...... Recovery
Fig. 1. The effect of injecting 1.0/~1 of Lidocaine at the medial border of the pars oralis of the spinal trigeminal nucleus. A: incidence of the CR following the injection of Lidocaine. Each data block represents the average of 10 consecutive trials. B: 3 superimposed responses illustrating the CRs and URs observed in paired trials before, immediately after, and following recovery from the injection. C: 3 superimposed trials illustrating the UR in air puff-only trials at the same times during the experiment indicated in B. D: superimposed URs to the fight-only stimuli, also at the same times indicated in B.
319 Lidocaine was estimated on the basis of the physiological data of Sandkiihler et al. 21. The findings fell into two categories. In the first the Lidocaine injection produced a selective effect on the CR. In all of the rabbits in this group (n = 4), the injection resulted in an immediate decrease of CR incidence and amplitude which lasted approximately 10-30 min, depending on the volume injected and the site of infusion. The injection sites in this group of animals were all located in the dorsal half of the medial spinal trigeminal nucleus at its junction with the reticular formation just caudal to the facial nucleus. An example of the observations in this group of animals is shown in Fig. 1. The effect of the injection of 1/~1 of Lidocaine on the incidence of the CR is shown in A. No change in UR incidence was found over the same period. The superimposed records in B illustrate the CR and UR in paired trials before, immediately after, and following recovery from the Lidocaine microinjection. Notice the substantial reduction of the CR immediately following the injection. The effect of the injection on the UR is best seen in the US alone trials (C). In the example shown, there was a slight increase in the amplitude of the UR. Although increases in the amplitude of this response were observed, no reductions were found following Lidocaine injection at this location. Lidocaine injection did not affect the UR evoked by the light (D). The site at which these effects were observed was well localized within the brainstem. It was not possible to duplicate these findings with injections 1.5 mm above or below the site at which these observations were produced. On the group level the CR amplitude dropped after the injection to 20.5 + 5.2% (mean + S.E.M.) of its preinjection level. The CR latency slightly increased to 127.8 + 12.3%. The UR amplitude and latency in the US alone trials did not change significantly (115.3 + 16.0% and 108.3 + 5.6% respectively) when compared to the performance before Lidocaine infusion. Furthermore, saline control injections at the primary site failed to produce significant changes in either the CR or UR. In the second group of animals (n = 6) the injections produced a reduction in both the UR and CR. The CR and UR amplitudes were reduced to 25.3 + 7.4% and 45.0 + 11.1% as compared to the level before injection. In all of these animals the injection site was located lateral to the region described above at which selective effects were produced. These findings demonstrate that Lidocaine microinjec-
tions into a specific region of the pars oralis of the spinal trigeminal nucleus can produce a selective depression of the CR, namely a depression that was not paralleled by a decrease in the amplitude of the UR. Furthermore this effect was produced by blocking sensory components of the NMR circuitry, an observation consistent with recent studies examining the effects of using electrical stimulation of various brainstem sites as a substitute for the US 16. Unequivocally, the findings presented here do not establish the pars oralis as the site at which the plastic changes required for the conditioning of the NMR occur. In fact microinjections of local anesthetic into the pontine nuclei also have been reported to produce selective effects on the CR 13. However, the observations do provide a basis for hypothesizing that this trigeminal nuclear region and/or the adjacent reticular formation is a putative site for these modifications. This view is supported by the fact that this region of the trigeminal complex not only receives afferents activated by corneal air puff stimuli 5'9'1° but also receives convergent inputs activated by auditory stimuli 17. Second, the red nucleus, a structure likely involved in modulating the execution of the conditioned reflexs'18-2°, also may project to the pars oralis ~'6. It is not likely that the selective effect produced in these experiments is due to interrupting rubral projections to motor neurons, a possibility suggested by reported effects of red nucleus lesions 18-2° since the Lidocaine injections were caudal to the motor nuclei. The fact that injections in more lateral regions of the pars oralis resulted in a reduction of both the CR and UR probably results from either a block of the afferent input required for both the CR and U R or to the action of Lidocaine on the final common path for the NMR. The latter alternative seems to be less probable because the decrease of the CR and the air puff-elicited U R was not accompanied by changes in the unconditioned response to the light (see Fig. 1D). On a more general issue, at present it is not clear whether the conditioned NMR engram resides in one particular site or whether it is distributed among several structures, as recently discussed by Byrne 4. Our data do not formally address this point. However, combined with our previous study showing that NMR conditioning can occur in the absence of the cerebellum 12, the findings do provide an alternative to the cerebellar hypothesis.
1 Bander, R., Evidence for a bilateral 'glomerular' projection from the red nucleus to the spinal nucleus of the trigeminalnerve in the cat, Neurosci. Lett., 8 (1978) 211-217. 2 Berthier, N.E. and Moore, J.W., The nictitating membrane response: an electrophysiologicalstudy of the abducens nerve
and nucleus and the accessoryabducens nucleus in rabbit, Brain Research, 258 (1983) 201-210. 3 Bracha, V., Stewart, S.L. and Bloedel, J.R., Temporary blockade of red nucleus in the rabbit affects performance of conditioned and unconditioned nictitating membrane responses,
This research was supported by NIH Grant NS 21958.
320 Soc. Neurosci. Abstr., 15 (1989) 507. 4 Byrne, J.H., Cellular analysis of associative learning, Physiol. Rev., 67 (1987) 329-439. 5 Cegavske, C.E, Harrison, T.A. and Torigoe, Y., Identification of the substrates of the unconditioned response in the classically conditioned rabbit, nictitating-membrane preparation. In I. Gormezano, W.E Prokasy and R.E Thompson (Eds.), Classical Conditioning, Lawrence Edbaum, Hillsdale, NJ, 1987, pp. 65-92. 6 Davis, K.D. and Dostrovsky, J.O., Modulatory influences of red nucleus stimulation on the somatosensory responses of cat trigeminal subnucleus oralis neurons, Exp. Neurol., 91 (1986) 80-101. 7 Desmond, J.E. and Moore, J.W., A supratrigeminal region implicated in the classically conditioned nictitating membrane response, Brain Res. Bull., 10 (1983) 765-773. 8 Haley, D.A., Thompson, R.E and Madden IV, J., Pharmacological analysis of the magnocellniar red nucleus during classical conditioning of the rabbit nictitating membrane response, Brain Research, 454 (1988) 131-139. 9 Harvey, J.A., Land, T. and McMaster, S.E., Anatomical study of the rabbit's corneal-Vlth nerve reflex: connections between cornea, trigeminal sensory complex, and the abducens and accessory abducens nuclei, Brain Research, 301 (1984) 307-321. 10 Hiraoka, M. and Shimamura, M., Neural mechanisms of the corneal blinking reflex in cats, Brain Research, 125 (1977) 265-275. 11 Ito, M., The role of the cerebellum during motor learning in the vestibular reflex: different mechanisms and different species, Trends Neurosci., 5 (1982) 416. 12 Kelly, T.M., Zuo, C.-C. and Bloedel, J.R., Classical conditioning of the eyeblink reflex in the decerebrate-decerebellate rabbit, Behav. Brain Res., 38 (1990) 7-18. 13 Knowlton, B.J. and Thompson, R.E, Microinjections of local anesthetic into the pontine nuclei reduce the amplitude of the classically conditioned eyelid response, Physiol. Behav., 43 (1988) 855-857. 14 Marr, D., A theory of cerebellar cortex, J. Physiol., 202 (1969) 437-470. 15 McCormick, D.A. and Thompson, R.E, Neuronal responses of the rabbit cerebellum during acquisition and performance of a
classically conditioned nictitating membrane-eyelid response, J. Neurosci., 4 (1984) 2811-2822. 16 Nowak, A.J. and Gormezano, I., Electrical stimulation of brainstem nuclei: elicitation, modification and conditioning of the rabbit nictitating membrane response, Behav. Neurosci., 104 (1990) 4-10. 17 Ricciardi, T.N., Richards, W.G. and Moore, J.W., Single unit activity in spinal trigeminal oralis and adjacent reticular formation during classical conditioning of the rabbit nictitating membrane response, Soc. Neurosci. Abstr., 15 (1989) 507. 18 Rosenfield, M.E. and Moore, J.W., Red nucleus lesions impair acquisition of the classically conditioned nictitating membrane response but not eye-to-eye savings or unconditioned response amplitude, Behav. Brain Res., 17 (1985) 77-81. 19 Rosenfield, M.E. and Moore, J.W., Red nucleus lesions disrupt the classically conditioned nictitating membrane response in rabbits, Behav. Brain Res., 10 (1983) 393-398. 20 Rosenfield, M., Dovydaitis, A. and Moore, J.W., Brachium conjunctivum and rubrobulbar tract: brainstem projections of red nucleus essential for the conditioned nictitating membrane response, Physiol. Behav., 34 (1985) 751-759. 21 Sandkiihler, J., Maisch, B. and Zimmerman, M., The use of local anesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block, Exp. Brain Res., 68 (1987) 168-178. 22 Welsh, J.P. and Harvey, J.A., Cerebellar lesions and the nictitating membrane reflex: performance deficits of the conditioned and unconditioned response, J. Neurosci., 9 (1989) 299-311. 23 Welsh, J.P. and Harvey, J.A., Intra-cerebellar lidocaine: dissociation of learning from performance, Soc. Neurosci. Abstr., 15 (1989) 639. 24 Yeo, C.H., Hardiman, M.J. and Glickstein, M., Classical conditioning of the nictitating membrane response of the rabbit. I. Lesions of the cerebellar nuclei, Exp. Brain Res., 60 (1985) 87-98. 25 Yeo, C.H., Hardiman, M.J. and Glickstein, M., Classical conditioning of the nictitating membrane response of the rabbit. II. Lesions of the cerebellar cortex, Exp. Brain Res., 60 (1985) 99--113.
317
BRES 24773
Selective involvement of the spinal trigeminal nucleus in the conditioned nictitating membrane reflex of the rabbit Vlastislav Bracha, Jin-Zi Wu, Shelley Cartwright and James R. Bloedel Division of Neurobiology, Barrow Neurological Institute, Phoenix, A Z (U.S.A.) (Accepted 30 April 1991) Key words: Nictitating membrane; Trigeminal nuclei; Conditioning; Learning; Brainstem; Rabbit; Lidocaine
These experiments were performed to test the hypothesis that a region associated with the trigeminal nuclear complex is selectively involved in mediating the classically conditioned nictitating membrane reflex in the rabbit. Mieroinjections of Lidocaine were used to produce a temporary, localized block of neural activity following the conditioning of the reflex using a standard tone/air puff-paired stimulus paradigm. Data indicate that the injection of Lidocaine at the medial pars oralis/retieular formation junction results in a selective suppression of the conditioned reflex The substrate for the plastic changes underlying various types of motor learning has been a topic of great interest during the past decade. Considerable focus has been placed on the possible role of the cerebellum in this process for at least two reasons. First, theoretical and experimental studies have implicated the climbing fiberPurkinje cell synapse as a potential site for such changes n'14. Second, lesions in specific cerebellar regions have been reported to block the acquisition as well as the performance of classically conditioned reflexes, most notably the conditioned nictitating membrane reflex (NMR) of the rabbit 24'2s. In addition, unitary responses of cerebellar neurons correlated with the conditioned response have also been reported 15. However, recent studies have challenged this view by showing that the conditioning of this reflex can occur in the absence of the cerebellum in decerebrate rabbits 12 and that at least some of the effects of cerebellar lesions in intact rabbits are due to a performance deficit rather than a learning deficit 22' 2a. Based on these studies and previous experiments implicating the supratrigeminal region 7 and the red nucleus s'18--2° in this process, we have focused our attention on the role of brainstem sites in the performance of these reflexes. However, our initial studies on the effects of temporary lesions in the vicinity of the red nucleus indicated that this structure is likely involved in the performance of both the conditioned and unconditioned reflexes 3. In contrast, recent data from Gormezano's laboratory ~6 suggest that a region associated with the pars oralis of the spinal trigeminal nucleus may be involved in the conditioned NMR.
The experiments reported here were performed to examine this possibility by assessing the effects of microinjections of Lidocaine in the pars oralis of the spinal trigeminal nucleus and the adjacent reticular formation on the performance of the conditioned and unconditioned NMR. This portion of the trigeminal nucleus receives the majority of the afferents activated by corneal air puff stimuli 2"5"9, the unconditioned stimuli employed in these experiments. The data will show that injection of a caudal-medial region of this nucleus can produce a selective reduction in the amplitude as well as the occurrence of the conditioned NMR. For the purposes of this manuscript, the term selective will refer to non-parallel effects of an agent or procedure on the conditioned and unconditioned responses. During a sterile surgical procedure in which adult albino New Zealand rabbits were anesthetized with a mixture of ketamine (50 mg/kg), xylazine (6 mg/kg) and acepromazine (1.5 mg/kg), guide tubes were implanted to within 3.5 mm above the injection sites in the region of the left trigeminal nucleus (AP = -18 mm, ML = 3 mm, DV = 19-20 mm). Stereotaxic coordinates were based on the position of bregma, which was placed 1.5 mm below lambda. The guide tubes were placed above the trigeminal nucleus ipsilateral to the eye receiving the unconditioned stimulus. In addition hardware was secured to the animal's skull to permit the attachment of a device that held both the lever arm system used to monitor the movement of the nictitating membrane bilaterally and the tube through which the air puff was delivered. Once the animals were adapted to a restraint box, the
Correspondence: J.R. Bloedel, Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ 85013, U.S.A.
318 animals were conditioned in a standard delay paradigm using a 1-kHz, 85-dB tone as the conditioned stimulus (CS). The tone was applied in a 5-pulse, 450-ms duration train (65 ms on and 35 ms off) which was superimposed on a continuous 70 dB white noise background. A n air puff 100 ms in duration and coterminal with the CS was used as the unconditioned stimulus (US). The air, 1.7 psi (11.876 x 103 N/m 2) at the outlet, was delivered to the left cornea through a tube placed 8 m m from the cornea. Each training session consisted of 100 paired trials presented in 19 to 21-s intervals. Animals were trained until they reached at least a 90% incidence of conditioned responses (CRs) across a daily session. Once criterion was reached, the effect of Lidocaine injection in the trained animals was determined using a succession of 3-trial sequences: one paired trial, one air puff-only trial, and one light-only trial. The light US (200 ms in duration) was emitted from a small flashlight bulb mounted to the air puff delivery tube. The light-only trials were used to evaluate the effect of Lidocaine on an unconditioned response (UR) that did not require the trigeminal nuclei for its performance. This control made it possible to determine if the local anesthetic spread to the motor nuclei, a circumstance that would result in the suppression of both the light-evoked as well as the air
puff-evoked U R . The needle used to administer the Lidocaine was inserted at the beginning of each microinfusion experiment. Prior to the injection of Lidocaine 20 three-trial sequences were performed. Then a 4% Lidocaine solution was administered through the injection tube in quantities ranging from 0.2 to 1.0/~1 over a period of 5 min. If no effect was observed, a minimum of 30 three-trial sequences was administered. If a change in the response did o c c u r a succession of three-trial sequences were performed until the animal's U R and C R were qualitatively similar to those observed prior to Lidocaine injection. Control experiments were performed the next day using the injection of pH-adjusted saline administered in the same quantity and to the same site used for lidocaine one day previously. Prior to sacrificing the animal, the position of the site at which the maximum effect was observed was marked by reinserting the injecting tube at the site and injecting 0.5-1.0 #1 of a colloidal iron solution. The location of the injection site was then determined histologically after the animal was perfused with a 1% ferrocyanide and 10% buffered formalin solution and the tissue stained with Perls iron reaction and Neutral red. These sites were used to localize the end of the injection tube. The spread of the
NMR Changes After Lidocaine Injection into the Trigeminal Nucleus
A
C URs in Air Puff Only Trials
L i d o c a i n e ~njection ~"
10o
,c~
3
~ g
2 ,
2-
rO
0
. . 1
.
. 3
.
. . 5
.
. 7
.
0 0
9
600
12()0
Number of Trials ( b l o c k s o f tS)
B
D URs in Light Only Trials
CRs in Tone+Air Puff Trials
"~.~
Q "
2
I I
,,
2
8
cO 0
0 0
600
1200
t
6O0
1200 ms
Before I n j e c t i o n .....
After Injection
...... Recovery
Fig. 1. The effect of injecting 1.0/~1 of Lidocaine at the medial border of the pars oralis of the spinal trigeminal nucleus. A: incidence of the CR following the injection of Lidocaine. Each data block represents the average of 10 consecutive trials. B: 3 superimposed responses illustrating the CRs and URs observed in paired trials before, immediately after, and following recovery from the injection. C: 3 superimposed trials illustrating the UR in air puff-only trials at the same times during the experiment indicated in B. D: superimposed URs to the fight-only stimuli, also at the same times indicated in B.
319 Lidocaine was estimated on the basis of the physiological data of Sandkiihler et al. 21. The findings fell into two categories. In the first the Lidocaine injection produced a selective effect on the CR. In all of the rabbits in this group (n = 4), the injection resulted in an immediate decrease of CR incidence and amplitude which lasted approximately 10-30 min, depending on the volume injected and the site of infusion. The injection sites in this group of animals were all located in the dorsal half of the medial spinal trigeminal nucleus at its junction with the reticular formation just caudal to the facial nucleus. An example of the observations in this group of animals is shown in Fig. 1. The effect of the injection of 1/~1 of Lidocaine on the incidence of the CR is shown in A. No change in UR incidence was found over the same period. The superimposed records in B illustrate the CR and UR in paired trials before, immediately after, and following recovery from the Lidocaine microinjection. Notice the substantial reduction of the CR immediately following the injection. The effect of the injection on the UR is best seen in the US alone trials (C). In the example shown, there was a slight increase in the amplitude of the UR. Although increases in the amplitude of this response were observed, no reductions were found following Lidocaine injection at this location. Lidocaine injection did not affect the UR evoked by the light (D). The site at which these effects were observed was well localized within the brainstem. It was not possible to duplicate these findings with injections 1.5 mm above or below the site at which these observations were produced. On the group level the CR amplitude dropped after the injection to 20.5 + 5.2% (mean + S.E.M.) of its preinjection level. The CR latency slightly increased to 127.8 + 12.3%. The UR amplitude and latency in the US alone trials did not change significantly (115.3 + 16.0% and 108.3 + 5.6% respectively) when compared to the performance before Lidocaine infusion. Furthermore, saline control injections at the primary site failed to produce significant changes in either the CR or UR. In the second group of animals (n = 6) the injections produced a reduction in both the UR and CR. The CR and UR amplitudes were reduced to 25.3 + 7.4% and 45.0 + 11.1% as compared to the level before injection. In all of these animals the injection site was located lateral to the region described above at which selective effects were produced. These findings demonstrate that Lidocaine microinjec-
tions into a specific region of the pars oralis of the spinal trigeminal nucleus can produce a selective depression of the CR, namely a depression that was not paralleled by a decrease in the amplitude of the UR. Furthermore this effect was produced by blocking sensory components of the NMR circuitry, an observation consistent with recent studies examining the effects of using electrical stimulation of various brainstem sites as a substitute for the US 16. Unequivocally, the findings presented here do not establish the pars oralis as the site at which the plastic changes required for the conditioning of the NMR occur. In fact microinjections of local anesthetic into the pontine nuclei also have been reported to produce selective effects on the CR 13. However, the observations do provide a basis for hypothesizing that this trigeminal nuclear region and/or the adjacent reticular formation is a putative site for these modifications. This view is supported by the fact that this region of the trigeminal complex not only receives afferents activated by corneal air puff stimuli 5'9'1° but also receives convergent inputs activated by auditory stimuli 17. Second, the red nucleus, a structure likely involved in modulating the execution of the conditioned reflexs'18-2°, also may project to the pars oralis ~'6. It is not likely that the selective effect produced in these experiments is due to interrupting rubral projections to motor neurons, a possibility suggested by reported effects of red nucleus lesions 18-2° since the Lidocaine injections were caudal to the motor nuclei. The fact that injections in more lateral regions of the pars oralis resulted in a reduction of both the CR and UR probably results from either a block of the afferent input required for both the CR and U R or to the action of Lidocaine on the final common path for the NMR. The latter alternative seems to be less probable because the decrease of the CR and the air puff-elicited U R was not accompanied by changes in the unconditioned response to the light (see Fig. 1D). On a more general issue, at present it is not clear whether the conditioned NMR engram resides in one particular site or whether it is distributed among several structures, as recently discussed by Byrne 4. Our data do not formally address this point. However, combined with our previous study showing that NMR conditioning can occur in the absence of the cerebellum 12, the findings do provide an alternative to the cerebellar hypothesis.
1 Bander, R., Evidence for a bilateral 'glomerular' projection from the red nucleus to the spinal nucleus of the trigeminalnerve in the cat, Neurosci. Lett., 8 (1978) 211-217. 2 Berthier, N.E. and Moore, J.W., The nictitating membrane response: an electrophysiologicalstudy of the abducens nerve
and nucleus and the accessoryabducens nucleus in rabbit, Brain Research, 258 (1983) 201-210. 3 Bracha, V., Stewart, S.L. and Bloedel, J.R., Temporary blockade of red nucleus in the rabbit affects performance of conditioned and unconditioned nictitating membrane responses,
This research was supported by NIH Grant NS 21958.
320 Soc. Neurosci. Abstr., 15 (1989) 507. 4 Byrne, J.H., Cellular analysis of associative learning, Physiol. Rev., 67 (1987) 329-439. 5 Cegavske, C.E, Harrison, T.A. and Torigoe, Y., Identification of the substrates of the unconditioned response in the classically conditioned rabbit, nictitating-membrane preparation. In I. Gormezano, W.E Prokasy and R.E Thompson (Eds.), Classical Conditioning, Lawrence Edbaum, Hillsdale, NJ, 1987, pp. 65-92. 6 Davis, K.D. and Dostrovsky, J.O., Modulatory influences of red nucleus stimulation on the somatosensory responses of cat trigeminal subnucleus oralis neurons, Exp. Neurol., 91 (1986) 80-101. 7 Desmond, J.E. and Moore, J.W., A supratrigeminal region implicated in the classically conditioned nictitating membrane response, Brain Res. Bull., 10 (1983) 765-773. 8 Haley, D.A., Thompson, R.E and Madden IV, J., Pharmacological analysis of the magnocellniar red nucleus during classical conditioning of the rabbit nictitating membrane response, Brain Research, 454 (1988) 131-139. 9 Harvey, J.A., Land, T. and McMaster, S.E., Anatomical study of the rabbit's corneal-Vlth nerve reflex: connections between cornea, trigeminal sensory complex, and the abducens and accessory abducens nuclei, Brain Research, 301 (1984) 307-321. 10 Hiraoka, M. and Shimamura, M., Neural mechanisms of the corneal blinking reflex in cats, Brain Research, 125 (1977) 265-275. 11 Ito, M., The role of the cerebellum during motor learning in the vestibular reflex: different mechanisms and different species, Trends Neurosci., 5 (1982) 416. 12 Kelly, T.M., Zuo, C.-C. and Bloedel, J.R., Classical conditioning of the eyeblink reflex in the decerebrate-decerebellate rabbit, Behav. Brain Res., 38 (1990) 7-18. 13 Knowlton, B.J. and Thompson, R.E, Microinjections of local anesthetic into the pontine nuclei reduce the amplitude of the classically conditioned eyelid response, Physiol. Behav., 43 (1988) 855-857. 14 Marr, D., A theory of cerebellar cortex, J. Physiol., 202 (1969) 437-470. 15 McCormick, D.A. and Thompson, R.E, Neuronal responses of the rabbit cerebellum during acquisition and performance of a
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