Behavioral Neuroscience
1991, Vol. 105, No. 1, 141-153
Copyright 1991 by the American Psychological Association, Inc.
0735-7044/9I/S3.00
Contributions of the Amygdaloid Central Nucleus to the
Modulation of the Nictitating Membrane Reflex in the Rabbit
Paul J. Whalen and Bruce S. Kapp
University of Vermont
The contributions of the amygdaloid central nucleus (ACe) to the modulation of the amplitude
of the nictitating membrane reflex (NMR) were determined. Experiment 1 demonstrated that
low-level electrical stimulation of the ACe enhances the amplitude of the NMR when administered immediately preceding the elicitation of the reflex by an eyelid stimulus. In Experiment
2 the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase
determined that the ACe projects to the entire rostrocaudal extent of the lateral tegmental field
(LTF), the brainstem area in which the multisynaptic component of the unconditioned NMR
pathway is believed to be located. These results are consistent with the hypothesis that the ACe,
via its projections to the LTF, modulates reflex sensitivity during conditioned arousal and may
contribute to the associative enhancement of the unconditioned NMR that occurs early during
Pavlovian nictitating membrane conditioning.
ably mediate these modulatory effects (Hopkins & Holstege,
1978; Price & Amaral, 1981; Schwaber, Kapp, Higgins, &
Rapp, 1982).
Recently, Weisz and LoTurco (1988) have demonstrated
that during Pavlovian conditioning of the nictitating membrane reflex (NMR) in the rabbit, the unconditioned NMR
in response to an airpuff unconditioned stimulus (US) is
enhanced in amplitude when the tone CS immediately precedes the US than when the reflex is elicited in the absence
of the CS. This enhancement (a) has been demonstrated to
be a function of an associative learning process (Weisz &
Mclnemey, 1990) and (b) develops within few CS-US pairings. The rapid development of this associative enhancement
is similar to the rapid development of associative cardiodeceleration in the rabbit during Pavlovian conditioning, a response markedly attenuated by lesions of the ACe as just
described. Because lesions of the cerebellum, which block
the development of the conditioned NMR (McCormick &
Thompson, 1984), do not affect the acquisition of the enhancement of the unconditioned NMR (Weisz & LoTurco,
1988), the brain structures mediating this associative enhancement have yet to be identified. Given the research indicating that (a) the ACe contributes to reflex modulation,
(b) associative enhancement of the unconditioned NMR and
associative cardiodeceleration demonstrate similar acquisition rates in the rabbit, and (c) lesions of the ACe markedly
attenuate the latter conditioned response, the ACe would
appear to be a prime candidate. Experiment 1, therefore, was
designed to determine whether low-level electrical stimulation of the ACe enhances the amplitude of the NMR when
administered immediately preceding the elicitation of the
reflex by an eyelid stimulus (ES). In Experiment 2, the anterograde transport of wheat germ agglutinin conjugated to
horseradish peroxidase (WGA-HRP) was used to determine
whether the ACe projects to the brainstem area in which the
multisynaptic component of the unconditioned NMR pathway is believed to be located.
The amygdaloid complex, and particularly the amygdaloid
central nucleus (ACe), has long been implicated in the expression of species-typical behaviors indicative of emotional
arousal (Kaada, 1972). For example, lesions in the region of
the ACe in the rat attenuate conditioned blood pressure and
freezing responses to the presentation of a fear-arousing conditioned stimulus (CS; LeDoux, Iwata, Cichetti, & Reis, 1988),
whereas in the rabbit lesions attenuate the magnitude of conditioned cardiodeceleration to such a stimulus (Gentile, Jarrell, Teich, McCabe, & Schneiderman, 1986; Kapp, Frysinger, Gallagher & Haselton, 1979). Consistent with these
observations are the findings that stimulation of the ACe
induces responses similar to those observed in response to
the presentation of emotionally arousing stimuli (Applegate,
Kapp, Underwood, & McNall, 1983; Iwata, Chida, & LeDoux,
1987; Senior, Stumpf, & Stock, 1984). Further, the ACe appears to contribute to the modulation of reflexes during emotional arousal because lesions of the ACe in the rat block
both (a) the potentiation of the acoustic startle reflex to the
presentation of a fear-arousing CS (Hitchcock & Davis, 1986)
and (b) the arousal-induced inhibition of the baroreceptor
reflex in rats presented with a mouse (Printz et al., 1983).
The contribution of the ACe to reflex modulation is supported by observations that stimulation of the ACe modulates the gain of somatomotor and autonomic reflexes (Gary
Bobo & Bonvallet, 1975; Pascoe, Bradley, & Spyer, 1989;
Rosen & Davis, 1988; Schlor et al., 1984). Widespread projections from the ACe throughout the brainstem most prob-
This research was supported by Public Health Services Institutional Grant 7125-89/6.
A preliminary report of these data was presented at the 18th annual
meeting of the Society for Neuroscience, Toronto, Canada, November 1988.
Correspondence concerning this article should be addressed to
Paul J. Whalen, Department of Psychology, University of Vermont,
Burlington, Vermont 05405-0134.
141
142
PAUL J. WHALEN AND BRUCE S. KAPP
Experiment 1
Method
Subjects. Nine experimentally naive New Zealand albino rabbits
(Oryctolagus cuniculus) that weighed 5.0-5.5 Ibs. (2.3-2.5 kg) at the
beginning of the experiment were used. They were maintained on a
12:12-hr light-dark cycle (lights on at 7:00 a.m.), given food and
water ad libitum and handled daily for 1 week before surgery. The
American Psychological Association's Guidelines for Ethical Conduct in the Care and Use of Animals were strictly followed as were
the principles for the care and use of laboratory animals as outlined
by the U. S. Public Health Service (National Institutes of Health
Publication No. 85-23, 1985).
Surgical procedure. After application of topical lidocaine to the
skin overlying the marginal ear vein, all animals were sedated with
chlorpromazine hydrochloride (20 mg/kg iv) and anesthetized with
Nembutal (sodium pentobarbital; 30-60 mg/kg iv). Two monopolar
stimulating electrodes, constructed from stainless steel insect pins
and insulated with Epoxylite except for 200 ion at the tip, were
positioned in the ACe bilaterally. The animals were given 10 days
to recover before beginning the experiment.
Apparatus. During the experiment, animals were placed in a
Plexiglas rabbit restrainer with an adjustable headstock, padded earclamps, and backplate. The restrainer was located inside a soundattenuating chamber equipped with a ventilating fan and speaker.
The chamber was located within a shielded, soundproof room (Industrial Acoustics Co.). Brain stimulation was administered using a
Nuclear-Chicago 7150 constant-current stimulator programmed to
deliver monophasic square-wave cathodal pulses. The electrocardiogram (EKG) was recorded via stainless steel wire loops positioned
subcutaneously, one dorsomedial to the left scapula and the other
dorsomedial to the right haunch. A stainless steel wire loop subcutaneously positioned anterior to the right scapula served as a ground
electrode for EKG recording. The EKG was amplified using a Grass
Model 7P511 amplifier, fed into a Grass 7P4C Tachograph preamplifier, and recorded by a Grass Model 78 polygraph.
The NMR was elicited by an ES that consisted of a 0.4-mA stimulus train (60 Hz) of 100-ms duration applied to two stainless steel
dresshooks, the curved portion of which held the eyelids open. The
NMR was recorded using the methodology described by VanDecar,
Swadlow, Elster, and Schneiderman (1969), which consisted of a
photocell and a 12-V incandescent light bulb connected to a muzzle
which was placed over the head of the animal. This NMR measurement apparatus was positioned at a distance of approximately
1.0 cm from the animal's eye. With this technique, the light measured
by the photocell is decreased as the nictitating membrane sweeps
across the eyeball in response to the ES. This change is transduced
into a voltage change, amplified using a Grass Model 7P511 amplifier, and recorded on the polygraph as a pen deflection. It is important to note that the distance of the NMR measurement apparatus
from each animal's eye varied across animals due to differences in
muzzle fit across animals. Thus, in terms of pen deflection the absolute amplitude of the NMR varied across animals. This variation
dictated that changes in the absolute amplitude of the NMR be
converted to a percent change score for statistical analysis. This
conversion is discussed in detail in the Results section.
Habiluation procedure and determination of brain stimulation parameters. After surgical recovery, the animals were habituated to
a restrainer for 30 min/day for 5 days. During the final 4 days of
restrainer habituation, they were placed into the sound-attenuating
chamber and habituated to the NMR measurement apparatus.
Bradycardia is characteristic of stimulation of the ACe in the
anesthetized and awake rabbit (Applegate et al., 1983; Cox et al.,
1986; Kapp, Gallagher, Underwood, McNall, & Whitehorn, 1982)
and is a measure of the activation of the ACe projections to the
lower brainstem. Therefore, before examining the eflects of stimulation of the ACe on the NMR, a determination was made of the
extent to which stimulation elicited bradycardia. This was performed
to ascertain the optimal current level necessary to activate ACe
projections to the lower brainstem and was done on the 3rd day of
habituation to restraint. During this procedure, current was delivered
through each electrode in turn at the following parameters: 50 jjA,
1-s train duration, 100 Hz, 0.5-ms pulse duration. If no heart rate
decrease was elicited, then the current intensity was increased in 50j*A increments until a 10% or more peak decrease in heart rate from
prestimulation baseline was observed or a maximum of 200 ^A was
reached. The interstimulation interval was 2 min. The current level
used to determine whether ACe stimulation affects the amplitude of
the NMR for each animal was the intensity that elicited a shortlatency bradycardiac response equal to or greater than 10% of prestimulation baseline with the exception that the duration of the
stimulus train was decreased to 400 ms. Electrode placements that
did not produce at least a 10% decrease in heart rate were examined
for stimulus-induced eflects on the amplitude of the NMR using
various stimulation amplitudes (50-200 i*A) and a stimulus train
duration of 400 ms. These various amplitudes matched those used
at placements that elicited a 10% decrease in heart rate upon stimulation.
Procedure for examining the effects of stimulation on the
NMR. Each stimulation session consisted of 27 trials. Two trials
were administered initially in which stimulation of the ACe was
presented alone to determine whether stimulation per se produced
movement of the nictitating membrane at the predetermined current
parameters. These 2 trials were followed by 9 trials in which the ES
was applied alone using a variable intertrial interval (ITI) of 90 (75,
90, and 105 s), and the NMR was recorded. The rationale for these
ES-alone trials derived from pilot data that suggested that the amplitude of the NMR in some animals was highly variable during
initial ES-alone presentations and that this variability declined over
repeated ES presentations. Finally, 16 test trials were given, 8 trials
during which the ES was presented alone (no-stim trials) and 8 trials
during which stimulation of the ACe began 400 ms before the ES
and ended with its onset (stim trials). The 16 test trials were presented
in a pseudorandom sequence using a variable ITI of 90 s. After the
session, the animal was returned to its home cage for a period of 13 days, after which it was returned to the apparatus for stimulation
at the second electrode placement. If a statistically significant ((test
for related measures, p < .05) change in the amplitude of the NMR
was demonstrated at the 400-ms stimulation duration, subsequent
sessions were administered in which the stimulation duration was
decreased to determine the minimal stimulus train duration necessary to affect the reflex.
Histological analysis. After completion of the experiment, small
direct-current lesions were made through the stimulating electrodes
(300 (iA, 3 s). All animals were then euthanized using an overdose
of Nembutal (sodium barbital; 150 mg/kg iv) and perfused with
physiological saline, which was followed by 10% formal-saline. Frozen sections (60 *im) were taken through the extent of the electrode
tract area and stained with thionin. The location of each stimulating
electrode tip was determined by microscopic examination of stained
sections and with reference to a stereotaxic atlas of the rabbit brain
(Urban & Richard, 1972). A determination was then made of whether a placement was located within the ACe (group ACe) or outside
of the ACe (group non-ACe).
Results
Histological analysis of electrode placements. The electrical stimulation procedure and subsequent histological
AMYGDALOID MODULATION OF THE NMR
analysis were completed for 18 electrode placements, the
locations of which are depicted in Figure 1. Eight of these
placements were located within the ACe as defined by Schwaber et al. (1982) in the rabbit, and the remaining 10 placements were located outside the ACe in adjacent structures,
including the internal capsule, globus pallidus, optic tract,
stria terminalis, and the amygdaloid lateral nucleus.
Stimulation-induced heart rate changes. Stimulation of
six of eight placements located within the ACe elicited a 10%
or more peak decrease in heart rate from prestimulation
baseline at current levels of 200 iiA or less. At the two remaining sites, stimulation produced a peak tachycardiac response of less than 10% of prestimulation baseline at current
levels of 200 /*A or less. These latter two sites were excluded
from the analysis based on the following: (a) Stimulation at
these two sites during heart rate recording did not elicit the
characteristic bradycardiac response in the rabbit as reported
by Applegate et al. (1983), Cox et al. (1986), and Kapp et al.
(1982), and (b) both of these sites were located on the periphery of the ACe, one at the anterior border (see Figure
I A) and one at the posteromedial border (see Figure 1C).
This raises the possibility that the tachycardia evoked from
these sites was a function of stimulation of adjacent areas.
Thus, based on previous research in the rabbit, these two
sites were not regarded as "true" ACe placements.
Of the remaining 10 sites located outside the ACe, stimulation did not produce heart rate changes meeting the justdescribed criterion in 9 of the sites at current levels of 200
MA or less. At the 10th site, stimulation elicited a peak tachycardiac response of less than 10% of prestimulation baseline.
This site was located within the stria terminalis adjacent to
the posteromedial border of the ACe. Interestingly, this site
was located proximal to one of the sites that was located on
the periphery of the ACe and that, when stimulated, elicited
a tachycardiac response as just described. In summary, there
were a total of 16 placements included in this analysis, 10
lying outside of the ACe and 6 lying within the ACe.
Stimulation-induced effects on the amplitude of the
NMR. There was no movement of the nictitating membrane for any animal when stimulation was administered
alone at the start of each session, nor was any movement
observed during the 400-ms period of ACe stimulation before the onset of the ES. Thus, stimulation of the ACe per
se did not directly produce movements of the nictitating
membrane at the chosen parameters.
To determine whether stimulation of placements located
within the ACe (group ACe) versus stimulation of those lying
outside of the ACe (group non-ACe) affected the amplitude
of the NMR, the absolute amplitudes of the NMRs in terms
of pen deflection were first converted to percent change scores
for all animals. For this conversion, the amplitude of the
NMR on each trial during which stimulation preceded the
elicitation of the reflex (stim trial) was compared with the
amplitude of the NMR on the temporally adjacent trial during which stimulation did not precede elicitation of the NMR
(no-stim trial), which served as a baseline. As just described,
this conversion was necessary due to the between-subjects
variability inherent in the use of the NMR measurement
apparatus. A Group x Trials interaction analysis of variance
143
on these percent change scores was then used to determine
whether the amplitude of the NMR was aifected by stimulation of the ACe versus non-ACe placements. The data are
presented in Figure 2 as group mean percent changes in NMR
amplitude from no-stimulation baseline trials across each of
the eight stimulation trials. A significant difference was present between the ACe and non-ACe groups, demonstrating
that stimulation of placements within the ACe before elicitation of the NMR produced an increase in the amplitude
of the NMR, F(\, 14) = 21.17, p < .001. Note from Figure
2 that stimulation of placements located outside the ACe
(group non-ACe) did not significantly affect the amplitude
of the NMR. There was no significant effect of trials (F <
1).
In summary, when compared with stimulation of placements located outside of the ACe, stimulation at the six ACe
placements that produced a bradycardiac response also produced an increase in the amplitude of the NMR when stimulation preceded the ES. The mean increase for the six placements was 52% and ranged from 13% to 92%. Interestingly,
the three sites, which when stimulated resulted in a tachycardiac response (see Figure 1), resulted in a statistically significant decrease in the NMR when stimulation preceded the
ES (t test for related measures, all ps < .05).
The data from an individual animal in which stimulation
of the ACe increased the amplitude of the NMR is shown in
Figure 3 A. This figure depicts a percent change score for each
of the eight stimulation trials when compared with the temporally adjacent baseline trials in which elicitation of the
NMR was not preceded by stimulation. Note that (a) stimulation of the ACe produced a 92% mean increase in the
amplitude of the NMR across the eight trials and (b) this
increase was produced by a stimulus train duration of 100
ms. An increase in amplitude of the NMR was also obtained
with a 50-ms stimulus train duration. The electrode placement in this animal is shown (Figure 3B), as is the bradycardiac response (Figure 3C) resulting from the administration of a 100-(iA stimulus train (100 Hz) for a 3-s duration.
Experiment 2
Anatomical evidence demonstrates that the ACe possesses
widespread projections to a variety of brainstem regions. For
example, in the cat Hopkins and Holstege (1978) demonstrated an extensive projection from the ACe to the lateral
tegmental field (LTF), a continuous area that extends from
the caudal medulla rostrally to the parabrachial region. The
LTF is considered to be the brainstem homologue of the
dorsal aspects of the intermediate zone of the spinal cord
(Holstege & Kuypers, 1977). Neurons of the LTF in the cat
have been shown to project to a variety of cranial nerve
nuclei, including the trigeminal, facial, abducens, accessory
abducens, and hypoglossal nuclei (Holstege, Kuypers, & Dekker, 1977; Holstege, Tan, van Ham, & Graveland, 1986).
The LTF is believed to subserve a variety of cranial nerve
somatomotor reflexes (Holstege & Kuypers, 1977).
The existence of a projection from the ACe to the LTF
presents a substrate by which the ACe might contribute to
the modulation of the NMR in the rabbit. This is particularly
144
PAUL J. WHALEN AND BRUCE S. KAPP
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Figure I. A and B: Most-anterior placements of stimulation electrode tips within and adjacent to
the amygdaloid central nucleus (ACe) as depicted on coronal sections through the amygdala of the
rabbit. (Placements within the ACe are represented by solid symbols. Placements outside the ACe are
represented by open symbols. Of 18 placements shown, 8 are within the region of the ACe, and 10
are outside the ACe. Heart rate response to stimulation at each site is indicated by circles [bradycardia],
triangles [tachycardia], and squares [<10% change in heart rate]. En = endopiriform nucleus; La =
amygdaloid lateral nucleus; Me = amygdaloid medial nucleus; ot = optic tract; P = putamen; PC =
prepiriform cortex; PMC = amygdaloid posteromedial cortical nucleus; SI = substantia innominata;
st = stria terminalis; SO = supraoptic nucleus.)
145
AMYGDALOID MODULATION OF THE NMR
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Figure 1 (continued). C and D: Most-posterior placements of stimulation electrode tips within and
adjacent to the amygdaloid central nucleus (ACe) as depicted on coronal sections through the amygdala
of the rabbit. (BL = amygdaloid basolateral nucleus; BM = amygdaloid basomedial nucleus; En =
endopiriform nucleus; GP = globus pallidus; La = amygdaloid lateral nucleus; Me = amygdaloid
medial nucleus; ot = optic tract; P = putamen; PC = prepiriform cortex; PMC = amygdaloidposteromedial cortical nucleus; st = stria terminalis.)
146
PAUL J. WHALEN AND BRUCE S. KAPP
TO
Percent
Change
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120
so
Mean
Percent
Change
40
30
20
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1
2
3
4
5
6 7
8
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Trials
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Figure 2. Graphic representation of the effects of stimulation of
sites within the amygdaloid central nucleus (ACe) and. outside (NonACe) of the ACe on the amplitude of the nictitating membrane reflex
(NMR) when a mean percent change score is used. (This score expresses each trial in which stimulation is administered immediately
before eh'citation of the NMR, as a percent change from its temporally adjacent baseline trial in which the NMR is elicited in the
absence of stimulation. A zero percent change indicates no effect on
the amplitude of the NMR.)
significant in light of recent research in the rabbit that has
demonstrated that neurons located within the pontine and
medullary lateral tegmentum, areas that comprise components of the LTF, project to the accessory abducens nucleus
(ACC; Harvey, Land, & McMaster, 1984). The ACC is the
primary motor neuron pool involved in the expression of the
unconditioned NMR (Berthier & Moore, 1983; Harvey et
al., 1984; Marek, McMaster, Gormezano, & Harvey, 1984).
Because areas of the pontine and medullary lateral tegmentum that project to the ACC are recipient of projections from
the trigeminal pars oralis, which in turn is recipient of primary afferent projections from the periorbital region, it is
believed that neurons in the pontine and medullary lateral
tegmentum represent a multisynaptic component of the unconditioned NMR (Harvey et al., 1984).
Although the projections from the ACe to the LTF have
been described in the cat, the existence of this projection in
the rabbit has yet to be described, particularly with reference
to the region of the LTF demonstrated to contain neurons
that project to final path motor neurons of the ACC. Experiment 2 was designed to determine whether the ACe in the
rabbit projects to the region of the LTF in which neurons
believed to represent the1 multisynaptic component of the
unconditioned NMR pathway are located {Harvey et al.,
1984). The anterograde transport of WGA-HRP was used to
determine the existence of this projection. In addition, the
location of transported label from the ACe with respect to
the ACC was determined by the use of an acetylcholinesterase
(AChE) stain previously demonstrated to demarcate the location of the ACC in the rabbit (Disterhoft, Quinn, Weiss,
La
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-200
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Figure 3. A: Data from an individual animal in which facilitation
of the amplitude of the nictitating membrane reflex (NMR) was
obtained from stimulation at a site located within the amygdaloid
central nucleus (ACe). (The Kaxis represents the percent change in
reflex amplitude when stimulation of the ACe preceded elicitation
of the NMR compared with the temporally adjacent baseline trial
in which stimulation did not precede the elicitation of the NMR.)
B: A thionin-stained coronal section illustrating the electrode placement that when stimulated produced facilitation of the NMR. (The
arrow denotes the placement of the ventral tip of the electrode where
stimulation was applied, located in the dorsal region of the posterior
ACe. BL = amygdaloid basolateral nucleus; BM = amygdaloid basomedial nucleus; En = endopiriform nucleus; La = amygdaloid
lateral nucleus; ot - optic tract.) C: The bradycardtac response upon
stimulation at this site. (The bar represents a 3-s stimulus duration.)
AMYGDALOID MODULATION OF THE NMR
147
& Shipley, 1985). Evidence for such a projection in the rabbit
would present additional support for the hypothesis that the
ACe may directly influence the NMR reflex pathway and
thereby contribute to the modulation of the sensitivity of this
reflex.
Method
Animals. Experimentally naive New Zealand albino rabbits
(Oryctolagus cuniculus) that weighed 5.0-5.5 Ibs (2.3-2.5 kg) at the
beginning of the experiment were used. They were maintained on a
12:12-hr light-dark cycle (lights on at 7:00 a.m.) and given food and
water ad libitum.
Surgical procedure. All animals were sedated with chlorpromazine hydrochloride (20 mg/kg iv) and anesthetized with Nembutal
(sodium pentobarbital; 30-60 mg/kg iv). Injections of WGA-HRP
into the ACe were made through a glass pipette (40-60-/xm tip diameter) cemented to the end of a 26-gauge needle of a Hamilton
1-jil syringe. Bilateral injections were made in each rabbit because
our previous unpublished observations in the rabbit demonstrated
that the contralateral projections of the ACe to the brainstem are
particularly sparse (Kapp, 1986). Similar observations in the cat have
also been reported (Hopkins & Holstege, 1978). Hence, transported
label in the brainstem ipsilateral to the injection site represents label
transported predominantly from that injection site. From 10 to 50
nl of WGA-HRP (1%) were injected over a 20-40-min period.
Histological analysis. Seventy-two hours after the injections, all
animals were euthanized using an overdose of Nembutal (sodium
pentobarbital; 150 mg/kg iv) and perfused with physiological saline,
which was followed by a 3% paraformaldehyde fixative (Gibson,
Hansma, Houk, & Robinson, 1984). Frozen sections (40 om) were
taken through the injection site and through the brainstem from the
obex rostrally to the parabrachial region. Alternate coronal sections
through the injection site were processed for WGA-HRP or stained
with thionin. Alternate coronal sections through the brainstem were
(a) processed for WGA-HRP, (b) stained for AChE to determine the
location of the ACC (Mesulam, 1982), or (c) stained with thionin.
A diaminobenzidine HRP-processing procedure (La Vail & La Vail,
1976) was used to visualize HRP at the injection sites, whereas a
tetramethylbenzidine HRP-processing procedure (Mesulam, 1982)
was used on brainstem sections.
The location of WGA-HRP label and AChE-stained neurons within the pontine and medullary LTF was examined under bright- and
darkfield microscopy with an Olympus BHA photomicroscope
equipped with a camera lucida. Photomicrographs were taken at
various levels along the rostrocaudal extent of the LTF to depict
HRP label and AChE staining.
Results
Components of the L TF. The LTF, as denned by Holstege
and Kuypers (1977), is comprised of neurons that project to
a variety of cranial nerve motor nuclei. It is comprised rostrally of Area H, as defined by Meessen and Olszewski (1949),
which is located in the caudal pons just posterior to the
parabrachial complex and which surrounds the motor nucleus of the trigeminal nerve. The LTF extends from Area
H in a caudal direction as a continuous region into the rostral
medulla where it is comprised of the parvicellular reticular
area. The parvicellular reticular area extends throughout the
rostrocaudal extent of the medulla, and at anterior and midmedullary levels it is located adjacent and dorsal to the facial
motor nuclear complex. At the level of the caudal medulla,
Figure 4. A line drawing of an injection site of 10 nl wheat germ
agglutinin-horseradish peroxidase within the amygdaloid central nucleus (stippled area). (GP = globus pallidus; Me = amygdaloid medial
nucleus; ot = optic tract; PMC = amygdaloid posteromedial cortical
nucleus; SO = supraoptic nucleus.)
the LTF is comprised of the dorsal and ventral reticular areas,
which merge with the dorsal and lateral aspects of the intermediate zone of the spinal cord. All of the areas just mentioned, as described by Holstege and Kuypers (1977) in the
cat, were readily discernible in the rabbit in the present study.
Anterograde transport of WGA-HRP to the LTF. Figure
4 depicts an injection site from a representative animal (Case
3) in which an injection of WGA-HRP was located within
the ACe, with minimal spread to the overlying globus pallidus. This injection resulted in fiber and terminal labeling
within the entire rostrocaudal extent of the LTF.
Figure 5A depicts an AChE-stained coronal section from
this case at the level of the motor nucleus of the trigeminal
nerve. Figure SB is a darkfield photomicrograph of a section
neighboring that shown in Figure 5A and illustrates the distribution of transported WGA-HRP within Area H seen here
surrounding the motor nucleus of the trigeminal nerve. Retrogradely labeled neurons resulting from an injection of HRP
into the ACC are located within the area surrounding this
nucleus (see Figure 1, Level P15 of Harvey et al., 1984).
Figure 6A depicts a more caudal AChE-stained coronal
section from this case than that shown in Figure 5, at the
level of the ACC. Figure 6B is a darkfield photomicrograph
of a section neighboring that shown in Figure 6A and illustrates the distribution of transported WGA-HRP within the
LTF. Note the fascicles of labeled axons lateral to the ACC
and what appears to be terminal labeling characterized by
its granular appearance immediately dorsal, lateral, and ventral to the ACC. Harvey et al. (1984) reported retrogradely
labeled neurons within the LTF surrounding the ACC after
HRP injections into the ACC in the rabbit (see Figure 1,
Level PI 7 of Harvey et al., 1984).
Figure 7A depicts a more caudal AChE-stained coronal
section from this case than those shown in Figures 5 and 6.
Note the large darkly stained neurons of the facial nuclear
complex lying immediately ventrolateral to the parvicellular
reticular area of the LTF. Figure 7B is a darkfield photo-
148
PAUL J. WHALEN AND BRUCE S. KAPP
&
.
«
\«
,
Figure 5. Photomicrographs of coronal sections of the lateral tegmental field (LTF) through Area H
(h) surrounding the nucleus of the trigerninal nerve (Mo V). (A: Acetylcholinesterase-stained LTF
brainstem section. B: Neighboring wheat germ agglutinin-horseradish peroxidase [WGA-HRP]-processed section depicting the anterograde transport of WGA-HRP 72 hr after injection into the amygdaloid central nucleus.)
micrograph of a section neighboring that shown in Figure
7 A and illustrates the distribution of transported WGA-HRP
within the parvicellular reticular area of the LTF. The parvicellular reticular area of the LTF was demonstrated by Harvey et al. (1984) to contain retrogradely labeled neurons after
injections of HRP into the ACC in the rabbit (see Figure 1,
Level PI 8 of Harvey et al., 1984).
Injection sites lying outside of, but adjacent to, the ACe
(e.g., globus pallidus, optic tract, amygdaloid basolateral nucleus) did not result in transported label to the LTF.
Stimulation-Induced Modulation of the Amplitude of
the NMR
Electrical stimulation of 6 of 8 sites within the ACe produced a bradycardiac response characteristic of electrical
stimulation of the ACe in the rabbit (Applegate et al., 1983;
Cox et al., 1986; Kapp et al., 1982). As a group, stimulation
at these 6 sites produced a statistically significant increase in
the amplitude (facilitation) of the NMR. Interestingly, stimulation of the 2 sites within the ACe that produceda tachycardiac response resulted in a decrease in the amplitude (inhi-
General Discussion
Two major findings emerged from this research. First, electrical stimulation of the ACe prior to the elicitation of the
NMR by the ES affected the amplitude of the reflex. Second,
the ACe in the rabbit was shown to innervate the entire
rostrocaudal extent of the LTF as denned by Holstege and
Kuypers (1977). The significance of each of these findings
will be discussed in detail.
Figure 6. Photomicrographs of coronal sections of the lateral tegmental field (LTF) at the level of the accessory abducens nucleus
(ACC), (A: Acetylcholinesterase-stained LTF brainstem section. B:
Neighboring wheat germ agglutinin-horseradish peroxidase [WGAHRP]-processed section depicting the anterograde transport of WGAHRP 72 hr after injection into the amygdaloid central nucleus. VII
= seventh cranial [facial] nerve.)
AMYGDALOID MODULATION OF THE NMR
ACC 1 *'
v.
*•
'% -*
149
150
PAUL J. WHALEN AND BRUCE S. KAPP
»
rfrv,
c
Figure 7. Photomicrographs of coronal sections of the lateral tegmental field (LTF) at the level of
the facial nuclear complex (FN). (A: Acetylcholinesterase-stained LTF brainstem section. B: Neighboring wheat germ agglutinin-horseradish peroxidase [WGA-HRP]-processed section depicting the
anterograde transport of WGA-HRP 72 hr after injection into the amygdaloid central nucleus.)
bition) of the NMR. However, because (a) these sites were
located on the periphery of the ACe, (b) they yielded tachycardia upon stimulation, (c) previous research has demonstrated that stimulation of the ACe consistently produces
bradycardia in the rabbit (Applegate et al., 1983; Cox et al.,
1986; Kappetal., 1982), and (d) stimulation of a site medially
adjacent to the ACe produced this same tachycardia-inhibition profile, the possibility exists that the decreased amplitude of the NMR after stimulation at these two sites is a
function of stimulation of adjacent areas. Of the 10 sites
located outside of the ACe, stimulation at only I (described
in d] yielded an effect on the NMR to stimulation. This site
was located medially adjacent to the posterior ACe in the
stria tenninalis lying just under the ventral edge of the optic
tract. One of the 2 tachycardia-inhibition sites located just
within the ACe was proximal to this placement.
The present findings confirm previous research demonstrating that stimulation of the amygdala, particularly in the
vicinity of the ACe, modulates reflex sensitivity. For ex-
ample, Gary Bobo and Bonvallet (1975) demonstrated both
facilitation and inhibition of the masseteric reflex to stimulation of this region in the cat. Comparable to the present
findings, they demonstrated facilitation in sites located in the
central and lateral regions of the ACe and report a bradycardiac response to stimulation of sites located within these
regions (Bonvallet & Gary Bobo, 1972). Gary Bobo and Bonvallet (1975) reported inhibition from sites lying medially
within the ACe, which is consistent with the present study.
They also reported inhibition from sites lying just medial to
the ACe under the ventral edge of the optic tract, comparable
to the site in the present study located within the stria terminalis.
The fact that facilitation of the NMR was observed in the
rabbit only from stimulation of sites that produced bradycardia was interesting not only in light of Bonvallet and Gary
Bobo's (1972) findings but also with respect to more recent
findings by Pascoe et al. (1989). They demonstrated that
stimulation of sites located within the ACe in the rabbit
AMYGDALOID MODULATION OF THE NMR
facilitated the magnitude of the baroreceptor reflex and also
produced bradycardia. In contrast, however, Schlor et al.
(1984) observed inhibition of the baroreceptor reflex in the
cat to stimulation of the ACe, but this stimulation produced
a tachycardiac response. In light of the findings of Bonvallet
and Gary Bobo (1972) and Gary Bobo and Bonvallet (1975),
the differences between the effects on the baroreceptor and
heart rate responses in the cat and rabbit may be a function
of differences in stimulation sites within the region of the
ACe. Species differences, however, may also contribute to
the disparate findings.
Finally, the facilitation of the NMR during stimulation of
the ACe is consistent with the findings of Rosen and Davis
(1988) who demonstrated facilitation of the acoustic startle
response to stimulation of the ACe in the rat. The present
study compliments this research with a demonstration of
modulation of the sensitivity of the NMR, a reflex for which
the circuitry has been well established and that is extensively
used in neurobiological investigations of the neural substrates
that contribute to the conditioning of the reflex (Gormezano,
Prokasy, & Thompson, 1987).
Projections of the ACe to the LTF
After an injection of WGA-HRP into the ACe, dense areas
of transported label were observed throughout the LTF of
the pons and medulla, a result similar to that reported by
Hopkins and Holstege (1978) in the cat and Price and Amaral
(1981) in the primate. Much of the label appeared to represent terminal labeling from anterograde transport because
of its widespread distribution within the LTF and its fine
granular appearance that made it distinguishable from labeled fiber bundles. In addition, the fact that the observed
label in the LTF was due to transport from neurons located
in the ACe is supported by several observations. First, no
other amygdaloid nuclei or structures lying immediately dorsal to the amygdala (e.g., globus pallidus, putamen) have been
shown to project extensively to the LTF (Hopkins & Holstege, 1978). Second, in the present study injections lying
outside of the ACe and located within the globus pallidus
and amygdaloid basolateral nucleus did not result in label
within the LTF. Third, no label was observed within cell
bodies of the LTF, thus ruling out the possibility that the
label within the LTF was retrogradely transported.
Harvey et al. (1984) have demonstrated in the rabbit that
neurons located in an extensive region of the pons and medulla, a region that corresponds to the LTF as defined by
Holstege and Kuypers (1977), project to the ACC. These
same regions receive direct projections from the pars oralis,
a subcomponent of the sensory trigeminal nucleus, which in
turn is a recipient of sensory information from the periorbital
region of the eye. These observations, together with electrophysiological investigations suggesting that there exists a short
latency disynaptic component and a longer latency multisynaptic component of the NMR in the rabbit (Berthier &
Moore, 1983), have led to the suggestion that two NMR
circuits exist (Harvey et al., 1984). The disynaptic circuit is
comprised of sensory projections to the pars oralis that in
turn projects directly to the ACC, whereas the multisynaptic
151
circuit is comprised of a projection from the pars oralis to
the LTF and from the LTF to the ACC as already described.
The present results demonstrate that the ACe projects to the
entire rostrocaudal extent of the LTF and overlaps the regions
of the LTF that send projections to the ACC as described by
Harvey et al. (1984). This projection offers a neuroanatomical substrate by which stimulation of the ACe may exert its
facilitatory effect on the NMR. However, the exact synaptic
relationships between ACe terminals and neurons within the
LTF that project to the ACC must await further analysis
using combined electron microscopy and conventional tracttracing techniques. Likewise, the exact physiological mechanism by which stimulation of the ACe modulates the NMR
must await further research using single cell electrophysiological techniques.
Finally, although the ACe-to-LTF projection may form a
substrate for the facilitation of the NMR, it is important to
realize that other projections from the ACe to the brainstem
also may be involved. For example, it has been demonstrated
that the ACe projects to the ventrocaudal region of the periaqueductal gray in the cat (Hopkins & Holstege, 1978) and
in the rabbit (Kapp, Wilson, Schwaber, & Bilyk-Spaffbrd,
1986). Furthermore, Coburn (1986) suggests that in the rabbit the nucleus centralis caudalis (retractor palpebrae tertiae
motor neuron pool), which lies in the ventrocaudal region
of the periaqueductal gray at the most dorsal edge of the
oculomotor nucleus, is responsible for contraction of the retractor palpebrae tertiae muscle via the oculomotor nerve.
This muscle originates at the superior edge of the optic foramen and inserts onto the upper margin of the nictitating
membrane, functioning to both anchor the membrane and
actively retract it into the nasal canthus (Prince & Eglitis,
1964). This suggestion raises two interesting possibilities.
First, in addition to facilitating the NMR via projections to
the LTF, the ACe might also facilitate this reflex via projections that actively inhibit the nucleus centralis caudalis, leading to an enhanced extension of the membrane. Second, it
is also possible that stimulation of placements that produced
inhibition of the NMR exerted their effect through this circuit. Thus, it is possible that a synergistic network exists that
could functionally modulate the NMR in either of two directions depending on the context of a particular situation
encountered by the animal.
Functional Implications of Facilitation of the NMR by
the ACe
In addressing the functional implications of the contributions of the ACe to reflex facilitation, the question arises
concerning the conditions under which such facilitation occurs in the behaving organism. Several lines of investigation
are pertinent to the answer. For example, Bohlin and Graham
(1977) have demonstrated that the eyeblink reflex in humans
is facilitated during an anticipatory warning stimulus in a
signaled reaction time task. More recent research has demonstrated that reflex facilitation occurs during a similar warning stimulus when assessed in muscles of limbs both involved
and not involved in the reflex (Brunia, Scheirs, & Haagh,
1982). Brunia et al. (1982) suggest that this generalized fa-
152
PAUL J. WHALEN AND BRUCE S. KAPP
cilitation is indicative of a heightened state of arousal during
the warning stimulus. Because heightened arousal is generally
associated with increased sensory intake, reflex facilitation
during the warning stimulus may be secondary to increased
sensory flow within the reflex circuit as has been suggested
by Bohlin and Graham (1977) to account for the facilitation
of the eyeblink reflex. Alternatively, reflex facilitation may
also reflect arousal-induced potentiation of the motor system
to facilitate motor output.
Reflex facilitation also occurs during heightened arousal
in mammals other than the human. For example, the acoustic
startle reflex is facilitated when elicited during a CS previously paired with shock (Davis, 1986). Presumably, the CS
elicits heightened arousal in the form of fear. Furthermore,
recall that early in Pavlovian conditioning of the NMR in
the rabbit, the amplitude of the unconditioned NMR elicited
by the US is enhanced when the tone CS immediately precedes the US as compared with when the reflex is elicited in
the absence of the CS (Weisz & LoTurco, 1988). This facilitation of the unconditioned NMR is believed to be a function
of an associative learning process (Weisz & Mclnerney, 1990).
In the context of reflex facilitation during arousal, it may be
argued that the associative facilitation of the unconditioned
NMR is reflective of a conditioned increase in arousal. If this
argument proves valid, then the possibility arises that the
ACe contributes to responses indicative of conditioned
arousal, including associative facilitation of reflexes such as
the unconditioned NMR. These responses would serve to
enhance the detection and processing of sensory information
or, in the case of unconditioned NMR facilitation, would be
indicative of increased sensory intake similar to that proposed by Bohlin and Graham (1977) for the enhancement
of the human eyeblink reflex.
Indeed, as recently reviewed by Kapp, Wilson, Pascoe,
Supple, and Whalen (in press), many of the responses elicited
by electrical stimulation of the ACe, including electroencephalographic desynchronization, pupillodilation, pinna
orientation, and bradycardia can be considered to reflect
heightened arousal and serve to enhance the detection and
processing of sensory information. Further, lesions of the
ACe have been found to block conditioned bradycardia during a fear-arousing CS in the rabbit (Gentile et al., 1986;
Kapp et al., 1979) as well as the facilitation of the acoustic
startle reflex during a fear-arousing CS in the rat (Hitchcock
& Davis, 1986). Given that these responses are indicative of
a conditioned increase in arousal, then the results are consistent with the notion that the ACe contributes to this conditioned increase (Kapp et al., in press), the expression of
which may be mediated by the extensive projections of the
ACe to the brainstem (see Kapp et al., in press). The validity
of this contribution awaits further research.
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Received June 25, 1990
Revision received August 13, 1990
Accepted August 13, 1990