Amygdala Stimulation Enhances the Rat Eyeblink Reflex Through a

Behavioral Neuroscience 1996, Vol. 110, No. 1, 51-59

Copyright 1996 by the American Psychological Association, Inc. 0735-7044/96/$3.00

Amygdala Stimulation Enhances the Rat Eyeblink Reflex Through a Short-Latency Mechanism Turhan Canli and Thomas H. Brown Yale University Amygdala stimulation was shown to enhance the trisynaptic (fast, R1) component of the electromyogram recorded in the rat orbicularis oculi (oo) muscle, which is responsible for the active force generating eyelid closure. The eyeblink was elicited via direct electrical stimulation of the supraorbital branch of the trigeminal nerve. Possible mechanisms responsible for the effect of amygdala stimulation on the eyeblink reflex were evaluated by measuring the amount of R1 enhancement as a function of the interstimulus interval (ISI) between the onset of amygdala and trigeminal nerve stimulation. Amygdala stimulation produced significant R1 enhancement at ISis that imply short-latency excitation of the eyeblink circuit by way of a fast-acting neurotransmitter.

The eyeblink reflex exhibits several experience- or usedependent modifications, including habituation or sensitization (Basso, Strecker, & Evinger, 1993; Sanes & Ison, 1983; Shahani, 1970); adaptive gain (Evinger & Manning, 1988); and modulation by context, stimulus anticipation, or discrete stimuli such as tones or lights (Brandon, Bombace, Falls, & Wagner, 1991; Canli, Detmer, & Donegan, 1992; Donegan, 1981; Ison & Leonard, 1971; Ison, Sanes, Foss, & Pinckney, 1990; Sanes & Ison, 1979, 1982; Steinmetz, Lavond, Ivkovich, Logan, & Thompson, 1992; Weisz, Harden, & Xiang, 1992; Weisz & Mclnerney, 1990; Young, Cegavske, & Thompson, 1976). The rabbit eyeblink can be influenced by at least two, apparently different, forms of Pavlovian conditioning (Konorski, 1967; Rescorla & Solomon, 1967; Thompson et al., 1986; Wagner & Brandon, 1989; Weinberger, 1982). The first form is thought to depend on cerebellar circuitry (Steinmetz et al., 1992; Thompson & Krupa, 1994; Yeo, 1991) and results in an anticipatory conditioned eyeblink response to the conditioned stimulus (CS). The second form of conditioning does not depend on the cerebellum (Weisz & LoTurco, 1988) but may instead rely on circuitry of the amygdala (Weisz et al., 1992) or closely related structures, and it results in a conditioned enhancement of the eyeblink reflex (Brandon et al., 1991; Canli et al., 1992; Wagner & Brandon, 1989). Lesioning the amygdala blocks the maintenance of CS-produced eyeblink enhancement (Weisz et al., 1992), while electrical stimulation

of the central nucleus of the amygdala causes enhancement of the eyeblink reflex (Whalen & Kapp, 1991). We have been pursuing analogous studies of amygdaladependent eyeblink enhancement in the rat, which offers certain practical advantages, especially for combining in vitro (Chapman, Kairiss, Keenan, & Brown, 1990; Chattarji & Brown, 1994; Chatterji, Faulkner, & Brown, 1995; Faulkner & Brown, 1995; Faulkner, Chattarji, & Brown, 1994) and in vivo analyses (Canli & Brown, 1994; Evinger, Manning, & Sibony, 1991; Lam, Wong, Canli, & Brown, 1995; Skelton, 1988; Stanton, Freeman, & Skelton, 1992). The R1 component of the rat eyeblink reflex is biomechanically and neurophysiologicaUy relatively simple. Stimulation of the supraorbital (SO) branch of the trigeminal nerve produces contraction of the orbicularis oculi (oo) muscle, which is exclusively responsible for generating the active force that closes the eyelid (reviewed in Pellegrini, 1993). The electromyogram (EMG) of oo activity shows a fast (R1) response with an onset latency of about 4-7 ms and a slower (R2) response with an onset latency of roughly 14-20 ms (Evinger et al., 1993). The shortest pathway mediating the R1 response involves only three synapses: from the trigeminal (5th) nerve to the 5th nucleus; from the 5th nucleus to the facial (7th) nucleus; and from the 7th nucleus to the oo muscle (Hiraoka & Shimamura, 1977; Pellegrini, 1993). Here we examine the effects on the R1 response of electrical stimulation of the central nucleus of the amygdala, which has been shown to modulate the rabbit eyeblink reflex (Whalen & Kapp, 1991) and the rat acoustic startle response (Rosen & Davis, 1988). We demonstrate that electrical stimulation of the central nucleus of the rat amygdala (CeA) enhances the R1 component of the eyeblink reflex through a short-latency mechanism that is consistent with a relatively direct projection to the reflex circuit mediated by a fast-acting neurotransmitter. Some of this work was previously presented in abstract form (Canli & Brown, 1994).

Turhan Canli, Department of Psychology; Thomas H. Brown, Department of Psychology and Department of Cellular and Molecular Physiology, Yale University. This article was supported by grants from the National Institutes of Health and the Office of Naval Research. We thank James Monckton and Billie Faulkner for contributions to the early stages of this work, Craig Evinger for teaching us his surgical procedures, Ronald Skelton and Richard Thompson for describing or demonstrating pertinent in vivo electrophysiological methods, and Joseph LeDoux for useful comments on the manuscript. Correspondence concerning this article should be addressed to Thomas H. Brown, Department of Psychology, Yale University, P.O. Box 208205, New Haven, Connecticut 06520.

Method

Subjects Nineteen male Sprague-Dawley rats were housed individually and kept on a 12-hr dark-light cycle with ad libitum access to food and 51

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water. All procedures were performed during the light hours of the cycle. At the time of surgery, the animals weighed between 250 and 380 g. The American Psychological Association's Guidelines for Ethical Conduct in the Care and Use of Animals was strictly followed.

pulse generator (A.M.P.I. Master-8). Electrical stimulation of the 5th nerve and amygdala were delivered by two battery-powered, constantcurrent, stimulus isolation units (SIUs; BAK, model BSI-2) that were controlled by the programmable pulse generator.

Surgical Procedures

Electrical Stimulation and Recording

Subjects were anaesthetized with an intraperitoneal (ip) injection of sodium pentobarbitol (65 mg/kg). Anesthesia was maintained with hourly intraperitoneal injections of sodium pentobarbitol at one-half of the initial dose. Boric acid ophthalmic ointment was applied to both eyes to prevent drying of the cornea. Body temperature was maintained during surgery by placing the subject on a heating pad. Surgical techniques for implanting the nerve cuff and EMG electrodes were adapted from Evinger and colleagues (Basso et al., 1993; Evinger et al., 1993; Horn, Porter, & Evinger, 1993). The supraorbital branch of the trigeminal (5th) nerve was isolated and cuffed with a piece of longitudinally split PE50 tubing containing a pair of stainless steel stimulating electrodes, which were uninsulated on the inner surface of the tubing. The only departure from Evinger's procedure was that instead of suturing the split cuff in place around the nerve, we sealed it closed using Corning RTV-11 (room temperature vulcanizing silicon). The latter modification, developed by James Monckton, increased our success rate and seemed to reduce unwanted current spread. Unipolar EMG electrodes (single-strand stainless steel wire coated with Teflon except for 1 mm at the tip) were inserted into the nasal and medial parts of the right oo muscle. Amygdala stimulation was done using a unipolar electrode (00 stainless steel insect pin, insulated with epoxylite except for 0.5 mm at the tip) that was stereotaxically targeted at the CeA (2.8 mm posterior to bregma, 4.0 mm lateral of midline, and 8.0 mm ventral to bregma with the skull surface level) of the right amygdala. Three small screws fixed to the skull helped anchor the head set (below). Two of the three skull screws, the amygdala and nerve cuff stimulation electrodes, and the EMG recording electrodes were attached to male amphenol connector pins and cemented onto the skull with dental acrylic to form a head set that could be electrically connected to the neurophysiological equipment (see below). Subjects were returned to their home cage after surgery and given a minimum of 10 days to recuperate.

Nerve stimulation and muscle recording methods were similar to those developed by Evinger and colleagues (Basso et al., 1993; Evinger et al., 1993; Horn et al., 1993). The supraorbital branch of the trigeminal nerve was directly stimulated by the implanted cuff. Nerve cuff stimulation (NCS) of the trigeminal nerve consisted of a single biphasic square-wave pulse (the duration of each phase was 0.1 ms) set at a current (range = 0.3-2.0 mA) sufficient to produce consistent R1 eyeblink responses. The second phase of the biphasic stimulation was used to reduce the duration of the stimulation artifact. Electrical brain stimulation (EBS), targeted at the central nucleus of the amygdala, consisted of a single biphasic square-wave pulse (the duration of each phase varied from 0.1 to 0.4 ms, depending on the subject, and the first phase was equal to or longer than the second). The amplitude of the first phase was fixed at 400 ~A, comparable to that used by Rosen and Davis (1988) for CeA stimulation. The monopolar stimulating electrode was connected to the negative terminal of the SIU, and one skull screw was connected to the positive terminal. Another skull screw was used as a system ground. The two leads connected to the nerve cuff were attached to a second SIU for independent electrical stimulation. EMG responses were recorded differentially from the two EMG electrodes placed in the oo muscle. The output from those two EMG electrodes was connected to the differential amplifier. EMG eyeblink responses were amplified (1K) and filtered (0.3 kHz and 5 kHz) before being digitized (10 kHz). Software running on the 486 Gateway 2000 was used to control presentation and timing of EBS and NCS, sample and store the digitized EMG response, and rectify and integrate the waveforms (Custom Lab Software, Centerbrook, CT). Data were sampled for 300 ms during each trial of the long interstimulus interval (ISI) test session and 200 ms during each trial of the short ISI test session (see below). These sampling periods are long in relation to the R1 response latency and duration (see Figure 2). Hardcopy reproductions of unrectified and rectified EMG waveforms were generated by converting the original digitized raw data files to ASCII files that were imported to a Macintosh-based drawing program. The R1 amplitude was measured as the integral of the full-wave rectified EMG response (see Figure 2) because (a) this is a conventional and reliable measure of eyeblink responding; (b) a substantial literature exists on this measure; and (c) there is a strong and well-documented correlation between the integral of the rectified EMG in the oo muscle and eyeblink amplitude, as measured by the downward distance of eyelid movement (Evinger et al., 1991; Manning & Evinger, 1986; for review, see Pellegrini, 1993). More generally, integration of a monophasic response is often preferred in electrophysiological recordings because it can improve the signal-to-noise ratio and reduce trial-to-trial variability. The analysis was entirely restricted to the R1 component of the response because the underlying circuitry is relatively simple.

Apparatus The training and testing apparatus, similar to that used by Skelton and colleagues (Skelton, 1988; Stanton et al., 1992), had a standard grid floor consisting of parallel stainless steel rods (5 mm diameter and 15 mm spacing) with interior dimensions of 22 x 28 cm. To provide additional head room for the freely moving rat, we increased the height of the chamber from the value furnished by the manufacturer (Med Associates, model ENV-001) to 42 cm (measured from the grid floor to the commutator at the top). The chamber was placed within a sound-attenuating cubicle (Med Associates, model ENV-018XX). The headset of the subject was connected by amphenol pins to a lead that attached to the commutator (Airflyte CAY-960-6) at the top of the chamber. The lead connecting the headset to the commutator, which relayed output EMG signals from the oo muscle and input current for nerve cuff and brain stimulation, was sufficiently long to permit unrestricted movement of the animal. The EMG signals were fed to a differential ac amplifier (AM Systems, model 1700), the output of which was displayed on a digital oscilloscope (Gould, model 450) and also digitized (Keithley, model DAS-40) for storage on a computer (486 Gateway 2000). The computer controlled the data collection via the A/D converter and controlled the sequence of trial types by triggering the programmable

Behavioral Procedures Habituation and pretest. After 10 days of recuperation following surgery, subjects were habituated for 60 min to the test chamber. A pretest was conducted on the next day to determine the appropriate parameters for NCS and EBS. The significance of the pretest was to identify a set of stimulation parameters that could produce eyeblink enhancement in at least one ISI condition without evoking an EMG response. The subsequent ISI testing was designed to assess how those

53

AMYGDALA-PRODUCED EYEBLINK ENHANCEMENT stimulation parameters affect the eyeblink amplitude across a range of ISis. To find the appropriate value at which NCS could produce a reliable but submaximal R1 eyeblink response, we applied a single biphasic square-wave pulse (0.1 ms duration for each phase) to the SO branch of the trigeminal nerve at an initial current of 0.25 mA. Current was gradually increased in subsequent trials until R1 eyeblink responses were reliably elicited by NCS. At this stimulation intensity, the long-latency R2 response was often not present. Once the current intensity for NCS was determined, a pretest was conducted to identify EBS parameters that would enhance the R1 response when EBS and NCS were presented simultaneously. The parameter space for EBS was limited by fixing the current intensity at 400 ixA and varying only the duration of the single biphasic pulse. The purpose of the second phase of the pulse was to minimize the stimulus artifact. The total duration of each biphasic EBS ranged from 0.2 to 0.8 ms. On the basis of extensive pilot data, combined with the findings of Rosen and Davis (1988) in an analogous experiment, we expected that these parameters would usually be effective in enhancing the eyeblink without directly eliciting an EMG response. There were two criteria for proceeding to the testing stage of the experiment. First, during the pretest period, EBS had to produce at least a 20% enhancement of the R 1 eyeblink response when presented simultaneously with NCS. This requirement ensured that there was a minimum facilitory effect of EBS in at least one 1SI condition. The second criterion was that eyeblink enhancement had to be evident in the absence of any EMG activity induced by EBS alone. The concern was that EBS-induced EMG activity could confound the modulation of motor responses with the direct generation of motor responses. Indeed, one of the reasons for constraining the EBS parameter space to 400 p.A and single pulses of 0.8 ms or less was that pilot data showed that amygdala stimulation alone could evoke EMG activity in the oo muscle when stimulating outside these limits. No further electrophysiological data were collected if these two conditions could not be satisfied, but the location of all the electrode sites was verified histologically. Testing. Subjects were tested for the effects of amygdala stimulation on eyeblink enhancement in two test sessions, which were conducted on 2 consecutive days. Short ISis between EBS onset and NCS onset (from - 2 to 2 ms) were tested in one session, while long ISis (from 5 to 200 ms) were assessed in another session (a negative ISI means that NCS preceded EBS, whereas a positive ISI means the reverse). The order of these two test sessions was counterbalanced across subjects. The basic design of a test session consisted of seven trial types, which were presented for a total of 38 trials. Six of these trial types were presented six times each in a balanced Latin-square design: One of these six trial types entailed presentation of NCS alone (nerve stimulation in the absence of EBS); the other five trial types involved presentation of EBS and NCS at varying ISis. The seventh trial type consisted of presentation of EBS alone (brain stimulation in the absence of NCS). This trial type was presented once at the beginning and again at the end of the test session to document what effects, if any, brain stimulation alone had on the overt expression of behavior or on electrical activity in the oo muscle. For the short ISI test session, EBS and NCS were presented such that EBS (a) followed NCS by either I ms or 2 ms (termed the -1-ms and -2-ms ISI conditions, respectively), (b) was presented simultaneously with NCS (0-ms ISI), or (c) preceded NCS by 1 ms or 2 ms (1-ms and 2-ms 1Sis, respectively). ISis more negative than - 2 ms were not explored because pilot data revealed occasional contamination of the R1 response by the EBS artifact. In the long ISI session, EBS preceded NCS by 5, 10, 20, 100, or 200 ms. EBS current was kept constant at 400 ~A. The mean intertrial interval (ITI) was 15 s (range of 10-20 s, uniformly distributed). This ITI was chosen to avoid enhancement of the R1 eyeblink response due to sensitization. Sanes

and Ison (1983) have shown that sensitization of the R1 response only occurs when the eyeblink is elicited at 1- or 2-s ITIs, but not at longer intervals.

Histology After behavioral testing was concluded, subjects were anaesthetized with Halothane, and electrode placements were marked with small lesions made by applying 100 I~A cathodal and anodal current for 5 s each. Subjects were then overdosed with sodium pentobarbitol or Halothane and intracardially perfused with physiological saline, followed by 8% formalin. The brains were removed from the skull and kept in 8% formalin solution for 3 days. The tissue was then frozen, sectioned in 45-txm thick slices, and stained with cresyl violet. Electrode placements were determined, using a compound light microscope at low power (3.2x objective), from the atlas of Paxinos and Watson (1986).

Statistical Data Analysis and Data Presentation Recall that the ITI was chosen to avoid sensitization-produced enhancement of the R1 eyeblink response, which we did not expect to occur at the chosen ITIs (Sanes & Ison, 1983). A repeated measures analysis of variance (ANOVA) was used to verify that sensitizationproduced enhancement did not in fact occur within or between sessions. This analysis was done on the NCS-alone trial type, on the basis of the integral of the rectified EMG. A repeated measures ANOVA was also performed on the integral of the rectified EMG to evaluate the effects of EBS on eyeblink amplitude at different ISis. Planned, single-degree-of-freedom contrasts were performed between the different ISI conditions and the NCS-alone condition. A subsequent repeated measures ANOVA tested for the effect of EBS on the eyeblink onset latency. All ANOVA p values were calculated using the Geisser-Greenhouse correction for the sphericity assumption (Keppel, 1991). The less conservative Huynh-Feldt (Keppel, 1991) correction was also applied, but the results are not presented here because this correction did not cause any changes in the pattern of statistical significance that was seen with the Geisser-Greenhouse correction (only the level of significance was altered in some cases). To illustrate graphically the effects of EBS on the eyeblink amplitude at different ISis, we normalized the raw data before plotting (see Figure 3). The mean response amplitude in each IS1 condition (Misi) was obtained for each subject and then adjusted with respect to the mean amplitude in the NCS-alone condition (MNcA) for that subject. The normalized eyeblink amplitude at a given ISI was calculated as [(Mlsl/MNcA) -- 1]. Thus a score of 0.0 implies that the mean R1 response to NCS plus EBS was not different from that produced by NCS alone and a score of 0.2 indicates a 20% enhancement. Results

Histology The p l a c e m e n t s of electrodes targeted at the C e A o f the amygdala are shown in Figure 1. Solid circles r e p r e s e n t electrode locations (n = 12) that satisfied our two selection criteria for EBS (see the M e t h o d section) and were t h e r e f o r e tested at the various ISis. H a t c h e d circles are electrode locations ( n - - 7 ) from which no ISI data were collected because EBS failed to satisfy our criteria. In 5 of these 7 animals, EBS failed to p r o d u c e the r e q u i r e d 20% R I e n h a n c e ment. In the remaining 2 animals, C e A stimulation g e n e r a t e d E M G activity in the absence o f NCS. The latter two sites are

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CANLI AND BROWN

\ • Effective P 0 Ineffective

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1993). Figure 2 shows a representative record of the R1 component of the eyeblink E M G when NCS was applied alone. The top trace (Figure 2A) shows the unrectified recording, and the bottom trace (Figure 2B) shows the full-wave rectified waveform. The initial deflection is the stimulus artifact. Recall that the NCS-alone trial type was presented six times in each of the two testing sessions on successive days. To assess whether the eyeblink amplitudes changed during the course of a test session or across days of testing, we performed a repeated measures A N O V A to test the effect of time (first trial vs. last trial in a test session) and session (Day 1 vs. Day 2) on the R1 response amplitude in the NCS-alone condition. The analysis revealed no significant effect of time, F(1, 11) = 1.84, p > .05, or session, F(1, 11) = 1.46,p > .05, and there was no significant Time × Session interaction, F(1, 11) = 3.28, p > .05. Before proceeding further, it is worth mentioning another trial type, EBS delivered in the absence of NCS, which was conducted at the beginning and end of both sessions. This trial type was included to verify that EBS alone did not elicit any E M G . Although ISI testing was done only on animals that did

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/ Figure 1. A schematic representation of coronal sections -1.8 to -3.3 mm from (posterior to) bregma, indicating electrode placements. Solid circles show 12 electrode placements that were used in the interstimulus interval (ISI) test. Hatched circles represent 7 electrode placements at which electrical brain stimulation failed to produce at least 20% enhancement during pretest or produced overt electromyogram responses in the orbicularis oculi muscle. No ISI data were collected from these sites. BL = basolateral nucleus; BM = basomedial nucleus; BLV = basolateral nucleus, ventral; Ce = central nucleus; and La = lateral nucleus.

1 mVI B

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the medial locations indicated on the section labeled -3.3 (3.3 mm posterior to bregma in Paxinos & Watson, 1986).

Rat Eyeblink Responses

Figure2. Typical electromyogram (EMG) recording in the rat orbicu-

For the 12 subjects that satisfied our testing criteria, the mean latency for the onset of the R1 response to NCS alone was 4.8 ms (range = 3.5-6.3 ms; SEM = 0.2), similar to the results reported by Evinger and colleagues (Evinger et al.,

laris oculi (oo) muscle in response to nerve cuff stimulation. SA indicates the stimulation artifact, and R1 denotes the fast (R1) component of the eyeblink response. A: An example of the unrectified (raw) EMG response in the oo muscle. B: Illustration of the same EMG signal from the oo muscle following full-wave rectification.

AMYGDALA-PRODUCED EYEBLINK ENHANCEMENT

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Figure3. Effectof amygdalastimulation on eyeblink enhancement. Eyeblink responses at different interstimulus intervals (ISis) were normalized in relation to the nerve cuff stimulation alone (NCSa) condition (see the Method section). A score of 0.0 means that the average R1 response to NCS plus electrical brain stimulation (EBS) was not different from that produced by NCS alone, and a score of 0.2 indicates a 20% enhancement. Vertical error bars indicate the standard error of the mean (SEM). A: Amount of reflex enhancement at relatively short ISis ( - 2 ms to 5 ms), Asterisks denote statistically significant (p < .05) effects of EBS. B: Similar plot for relativelylong ISis (from 2 ms to 200 ms). As is indicated, EBS produced no significant effects at these longer ISls. To provide visual continuity between the upper and lower graphs, we plotted the last two data points illustrated in the upper graph (ISis of 2 and 5 ms) as the first two data points in the lower graph. not show any EMG response to EBS alone in the pretest phase of the experiment, we wanted to verify that no changes occurred between the pretest and the test phase. Indeed, in none of the 12 animals did EBS alone cause an EMG response during the testing phase of the experiment.

Eyeblink Enhancement Two repeated-measures one-way ANOVAs tested the effects of EBS on enhancement of the R1 response amplitude, as measured by the integral of the rectified EMG: one for data collected in the short ISI test session ( - 2 , - 1 , 0, 1, and 2 ms and NCS alone), the other for data collected in the long ISI test session (5, 10, 20, 100, and 200 ms and NCS alone). For short ISI data, the analysis showed a significant main effect of ISI, F(5, 55) = 5.03, p < .05. Planned single-degree-offreedom contrasts showed significant enhancement at three ISis: - 2 ms (p < .005), - 1 ms (p < .05), and 0 ms (p < .005). For long ISI data, there was no overall significant main effect

for ISI, F(5, 55) = 2.75,p > .05, nor any significant difference between any of the ISI conditions and the NCS-alone condition (F values ranged from 0.52 to 4.11,p values from .08 to .33). The ISI-enhancement function is plotted in Figure 3. Note that the plotted data are normalized with respect to eyeblink amplitude in the NCS-alone condition (see the Method section). Normalization reduced the among-subjects variability and presented the data in terms of the decimal equivalent of a percentage, which is more informative than the absolute value of the integral of the rectified EMG. Because of the wide range of tested intervals, data obtained in the shorter and longer ISI sessions are plotted separately. To provide visual continuity between these graphs, we included (repeated) data from two ISI conditions (2 and 5 ms) in both plots. The 2-ms ISI data point was actually collected in the "short ISI session" and the 5-ms ISI data point was collected in the "long ISI session" (see the Method section). As is indicated in Figure 3A, only the first three data points achieved statistical significance (denoted by the asterisk). None of the longer intervals produced a statisti-

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CANLI AND BROWN

cally significant effect on the eyeblink response (Figures 3A and 3B, no asterisk). Across all ISis, the maximum mean R1 enhancement was 44%. Most important, for interpreting the underlying mechanism, significant enhancement was documented (Figure 3A and above) even when the amygdala was stimulated 1 or 2 ms after 5th-nerve stimulation ( - 1 and - 2 ms ISis, respectively). Next we attempted a more fine-grained temporal analysis of the effect of EBS on the R1 response. In particular, we wanted to know whether EBS affected the R1 response latency, because such information could add further constraints on the possible circuitry between the CeA and the eyeblink circuit. For each subject, we determined the mean R1 response latency for NCS alone and for NCS plus EBS at ISis of - 2 ms, - 1 ms, and 0 ms. These are the three ISis that produced significant overall R1 enhancement (see Figure 3). Mean (++_SEM)response latencies for the four conditions were as follows: NCS alone (4.8 --- 0.2 ms); - 2 ms ISI (4.9 --- 0.3 ms); - 1 ms ISI (4.6---0.3 ms); and 0 ms ISI ( 4 . 4 - 0.3 ms). A repeated measures one-way A N O V A revealed a significant main effect of ISI on R1 latency, F(3, 33) = 4.60, p < .05. Planned single-degree-of-freedom contrasts showed a significant latency effect of EBS only at an ISI of 0 ms (p < .05). Discussion The primary purpose of this experiment was to determine whether electrical stimulation of the central nucleus of the amygdala can enhance the R1 component of the rat eyeblink reflex. The results demonstrate unequivocally that it can do so and under conditions in which amygdala stimulation by itself produces no EMG response. That is, amygdala stimulation can have a modulatory effect on the R1 component of the rat eyeblink reflex. The secondary purpose of this experiment was an attempt to place constraints on the possible mechanisms whereby this reflex modulation might occur. The results suggest that the observed R1 enhancement reflects a relatively direct projection from the amygdala to the reflex circuit and that this effect is mediated by a fast-acting neurotransmitter.

Effective and Ineffective Electrode Placements Of the 19 electrodes that were assessed in pretest, 12 produced at least 20% enhancement of the eyeblink in the 0-ms ISI condition without generating EMG activity in the oo muscle. In all 12 cases the electrode tip was clearly within or touching the periphery of the central nucleus (Figure 1, solid circles). In these effective sites, subsequent testing at different ISis demonstrated significant enhancement of the eyeblink response over a range of short ISis (from - 2 ms to 0 ms) but not long ISls. Thus the effect persisted from the pretest to the test session and generalized to more negative ISis. EBS also produced a small but statistically significant reduction in the response latency at an ISI of 0 ms. In two of the seven cases that did not meet our pretest criteria, and were therefore not used in ISI testing, EBS induced EMG activity in the absence of NCS. Had we proceeded to ISI testing in these cases, there might have been an enhancement of the E M G response at some ISI, but the

results would have been difficult to interpret in terms of reflex modulation. In the remaining five cases, electrical stimulation failed to produce any effect during pretest. It is important to recognize that these electrodes were not tested at EBS currents greater than 400 txA and that only a single biphasic stimulus was delivered. Higher current intensity, longer current pulse duration, or multiple pulses might well have produced reflex enhancement at these electrode sites. However, our pilot experiments suggested that such variations would increase the chances that EBS would directly produce EMG responses, which we wanted to avoid. Moreover, the current intensity used in this experiment is comparable to that used in the only other analogous study of the effects of rat CeA stimulation on reflex enhancement (Rosen & Davis, 1988). That study found that stimulation of the central nucleus usually required 300 to 400 ~A to enhance the acoustic startle response. One wonders why 5 of the 19 electrodes failed to produce any apparent enhancement effect within the range of stimulation parameters we explored. In some cases, the electrode tips were clearly not within the central nucleus of the amygdala. Possibly the current spread was insufficient to activate neurons in CeA. Alternatively, for those electrodes that border the CeA, one could imagine that stimulation may have activated not only excitatory but also inhibitory reflex modulation systems. Such inhibitory effects on the eyeblink amplitude have been observed in the rabbit, in which some electrodes placed in the central amygdala nucleus did in fact inhibit the nictitating membrane response (Whalen & Kapp, 1991). Stimulation of the lateral part of CeA and other parts of the amygdala has also been reported to inhibit the masseteric reflex (Gary Bobo & Bonvallet, 1975).

Significance of R1 Response Enhancement We recently demonstrated conditioned enhancement of the rat R1 response by a tone CS that had been previously paired with an aversive unconditioned stimulus (US), an effect that required explicit CS-US pairings (Lam et al., 1995). As an expression system for monitoring such conditioning, the R1 component of the rat eyeblink response offers the following advantages: It has been carefully studied, and it is biomechanically and neurophysiologically relatively simple (reviewed in Evinger et al., 1991, and Pellegrini, 1993). The active force generating the eyelid closure in the rat is produced by a single muscle, the oo, which is well understood in terms of its kinetics and biophysics (Evinger & Manning, 1988; Evinger, Shaw, Peck, Manning, & Baker, 1984; Evinger et al., 1991; Horn et al., 1993; Manning & Evinger, 1986). The shortest pathway from the trigeminal nerve back to the oo muscle is thought to involve only two central synapses (Hiraoka & Shimamura, 1977; reviewed in Pellegrini, 1993). Importantly, we have been able to determine the location and characterize the three-dimensional structure of the oo motoneurons using confocal laser scanning microscopy applied to thick brain slices (Lofthus & Brown, 1993). The oo motoneurons, which were fluorescently labeled in vivo before obtaining the brain slices, are located along the rostral-caudal extent of the dorsal crest of the facial nucleus. With the use of dual

AMYGDALA-PRODUCED EYEBLINK ENHANCEMENT labeling, it may be possible using these methods to trace the particular connections to the oo motoneurons that are hypothesized to be involved in modulating the eyeblink reflex.

Implications of R1 Enhancement at Short ISis A wide range of ISis was examined (Figure 3), ranging from - 2 ms (EBS followed NCS by 2 ms) to 200 ms (EBS preceded NCS by 200 ms). Significant enhancement was evident only at short ISis ( - 2 ms to 0 ms). This eliminates one obvious possible mechanism, namely, a serotonergic action that increases motoneuron excitability. The facial nucleus is known to receive heavy serotonergic innervation, and serotonin is known to increase the excitability of these motoneurons (Aghajanian, 1990; Aghajanian & Rasmussen, 1989; Aghajanian, Sprouse, Sheldon, & Rasmussen, 1990; Larkman & Kelly, 1992; Rasmussen & Aghajanian, 1990; VanderMaelen & Aghajanian, 1980). However, all such G-protein-mediated synaptic effects develop too slowly to be manifest this quickly. The results also place loose constraints on the possible circuitry. The significant enhancement observed when stimulation of the amygdala followed trigeminal nerve stimulation by 1 or 2 ms ( - 1 ms and - 2 ms ISI, respectively; see Figure 3) raises the possibility of a relatively direct connection. The directness of the connection between CeA and the eyeblink circuit is important in terms of ultimately understanding the system to the point of being able to furnish a neurophysiologically realistic quantitative model. For this purpose one wants the simplest possible effector (expression) system as a monitor of learning-related changes in the amygdala and related structures.

Neurophysiological Evidence Regarding R1 Enhancement The mean (+-SEM) onset latency for the R1 eyeblink response to NCS was 4.8 _ 0.2 ms, placing an upper limit on the available time a signal has to be transmitted from the CeA to influence the R1 onset. A significant decrease in the latency was seen only at an ISI of 0 ms. To interpret this effect, we must take into account the time it takes for a signal to propagate from the facial nucleus to the oo muscle. In the rat, the latency from stimulating the facial nerve to an EMG response in the oo muscle is slightly greater than 2 ms (C. Evinger, 1994, personal communication) and in the cat the latency is 2-3 ms (Tamai, Iwamoto, & Tsujimoto, 1986). This leaves less than 2.8 ms for local excitation and propagation to the eyeblink circuit, consistent with the possibility of a relatively direct (monosynaptic or disynaptic) connection from CeA to the oo motoneurons. Fanardjian and Manvelyan (1987) measured the latency of intracellularly recorded excitatory postsynaptic potentials (EPSPs) in facial nucleus motoneurons in response to electrical stimulation of CeA in the cat. The EPSPs that they judged to be monosynaptic responses had latencies in the range of 0.9-2.8 ms.

Neuroanatomical Evidence Regarding R1 Enhancement There is anatomical evidence that the amygdala does project directly or indirectly onto motor nuclei. Light and electron

57

microscopic evidence reveals connections to the dorsal motor nucleus of the vagus nerve, seemingly onto preganglionic neurons (Takeuchi, Matsushima, Matsushima, & Hopkins, 1983). This direct connection is presumably responsible for some of the autonomic activity that is modulated by the amygdala (Amaral, Price, Pitkanen, & Carmichael, 1992; Kapp, Frysinger, & Gallagher, 1979; Kapp, Whalen, Supple, & Pascoe, 1992; Schwaber, Kapp, Higgins, & Rapp, 1982). There is some neuroanatomical evidence regarding the possible pathway from the amygdala to the oo motoneurons. Whalen and Kapp (1991) reported anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (HRP) from CeA to the entire rostrocaudai extent of the lateral tegmental field (which includes the region of the facial nucleus). Hopkins (1975) found retrogradely labeled neurons in the amygdala (mainly CeA) after injecting HRP into different tegmental areas in the rat, cat, and rhesus monkey. In particular, injections of HRP in the medulla oblongata at the level of the facial nucleus produced retrogradely labeled CeA neurons in the rat amygdala. One tenable hypothesis is that EBS-induced enhancement of the rat R1 response involves activation of interneurons in the lateral tegmental field (LTF) that in turn project to oo motoneurons. There is a projection from CeA to the LTF (Hopkins & Holstege, 1978; Hopkins, McLean, & Takeuchi, 1981; Krettek & Price, 1978; Post & Mai, 1980; Price & Amaral, 1981; Whalen & Kapp, 1991) and the medial part of LTF sends projections to the region of the facial nucleus where the oo motoneurons are located (Holstege, Kuypers, & Dekker, 1977; Lofthus & Brown, 1993). A similar projection pattern from the amygdala onto motor systems is believed to underlie amygdala-dependent modulation of other facial movements. Stimulation of the amygdala can facilitate or inhibit masticatory movements and the trigeminai motoneurons controlling these movements (Gary Bobo & Bonvallet, 1975; Nakamura & Kubo, 1978; Ohta, 1984). Anatomical evidence based on retrogradely labeled neurons and anterogradely degenerating fibers shows that there are neurons in the LTF that receive input from the central nucleus of the amygdala and project to the trigeminal motor nucleus (Takeuchi, Satoda, Tashiro, Matsushima, & Uemura-Sumi, 1988). Similarly, the enhancement of the rabbit nictitating membrane (NM) response produced by CeA stimulation is thought to involve interneurons in the LTF. The LTF receives input from the CeA (Whalen & Kapp, 1991) and projects to the accessory abducens nucleus (Harvey, Land, & McMaster, 1984), which controls the NM response (Berthier & Moore, 1983; Harvey, Land, & McMaster, 1984).

Generality of the Reflex Enhancement Effect Our results, along with others cited earlier, suggest that the effect of amygdala stimulation on reflex modulation is somewhat general, being neither species specific nor peculiar to a particular reflex or motor unit. This conclusion is consistent with the generally recognized importance of the amygdala in responding to fear-eliciting stimuli (Davis, 1992; LeDoux, 1992, 1993) and in producing a state of vigilance (Gallagher & Holland, 1992; Kapp et al., 1992).

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R e c e i v e d J a n u a r y 31, 1995 Revision received April 13, 1995 A c c e p t e d May 18, 1995 •