The effect of medial frontal cortex lesions on cardiovascular conditioned emotional responses in the rat

BRAIN RESEARCH ELSEVIER

Brain Research 643 (1994) 181-193

Research Report

The effect of medial frontal cortex lesions on cardiovascular conditioned emotional responses in the rat Robert J. Frysztak *, Edward J. Neafsey Department of Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, 2160 S. First Ave., Maywood, IL 60153, USA (Accepted 28 December 1993)

Abstract The effect of ventral medial frontal cortex (MFC) lesions on heart rate and blood pressure during conditioned emotional responses (CER) was investigated. Male Sprague-Dawley rats were divided into two groups: MFC-lesioned rats (n = 11) sustained bilateral lesions of the infralimbic and ventral prelimbic regions of the MFC via microinjection of the neurotoxin N-methyl-D-aspartate; Controls (n = 13) received sterile saline. Following a 2-week recovery period, all animals were trained; one of two tones served as the conditioned stimulus (CS) and a 2 mA footshock served as the unconditioned stimulus (US). The CS + tone was consistently paired with the US, while the CS - tone was randomly paired with the US. Heart rate and blood pressure were recorded during CS + and CS - presentations before and after administration of the following pharmacological agents: atropine, atenolol, and atropine + atenolol. All animals responded to the CS + with increased BP compared to baseline; the increase was not significantly different between groups. Controls responded to the CS + with increased HR, while MFC-lesioned animals displayed a bimodal H R response which was not significantly different from baseline, but was significantly different from Controls. Pharmacological blockade of the H R response revealed coactivation of the sympathetic and parasympathetic nervous systems during the CS + , with a significant decrease (52%) in the sympathetic tachycardia component of the CS + H R response in MFC-lesioned rats as compared to Controls; the parasympathetic bradycardia component was not altered by MFC lesions. In all cases, CS - responses were smaller than the CS + responses. Pharmacological analysis revealed that the C S - H R response was mediated by the sympathetic component only, which was also significantly reduced in MFC-lesioned animals as compared to Controls. This significant reduction in the sympathetically mediated HR component of both the reinforced CER (CS + ) and the unreinforced CER (CS - ) following ventral MFC lesions implies that the MFC is necessary for complete sympathetic activation of cardiovascular responses to both severely and mildly stressful stimuli. The role of the MFC in emotion is also discussed.

Key words: Stress; Blood pressure; Heart rate; Motivation; N M D A lesion

1. Introduction I n 1927 C a n n o n [10] p o s t u l a t e d t h a t w h e n an o r g a n ism faces a t h r e a t e n i n g situation, b o d i l y r e s o u r c e s a r e m o b i l i z e d to p r e p a r e t h e a n i m a l for ' f i g h t or flight'. It is now well e s t a b l i s h e d t h a t aversive e m o t i o n a l a r o u s a l a n d stress a r e a c c o m p a n i e d by w i d e s p r e a d s y m p a t h e t i c activation in b o t h e x p e r i m e n t a l a n i m a l s a n d man. F o r

* Corresponding author. Present address: Department of Neurobiology and Anatomy, University of Texas Health Science Center, PO

Box 20708, Houston, TX 77225, USA. Fax: (1) (713) 792-5795. E-Mail address: [email protected]. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 0 5 0 - M

example, beta-adrenergic sympathetic blockade comp l e t e l y e l i m i n a t e s t h e aversively c o n d i t i o n e d t a c h y c a r d i a s e e n in b a b o o n s [57] a n d dogs [4]. I n m a n , O b r i s t e t al. [45] f o u n d t h e e x p e c t a t i o n o f stress was also associa t e d with a s y m p a t h e t i c a l l y m e d i a t e d t a c h y c a r d i a . This is f u r t h e r s u p p o r t e d by e v i d e n c e which directly r e c o r d e d i n c r e a s e s in n e u r a l activity f r o m single sympathetic cardiac postganglionic neurons during condit i o n i n g [12]. This s y m p a t h e t i c a l l y m e d i a t e d t a c h y c a r d i a is p r e s u m a b l y driven by d e s c e n d i n g activity f r o m s u p r a s p i n a l levels. L e s i o n s of t h e h y p o t h a l a m u s a n d a m y g d a l a , which p r o j e c t directly to c a r d i o v a s c u l a r ' c e n t e r s ' such as t h e solitary n u c l e u s a n d n u c l e u s a m b i g u u s l o c a t e d in t h e b r a i n s t e m a n d t h o r a c i c i n t e r m e d i o l a t e r a l cell c o l u m n

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R.J. Frysztak et al. / Brain Research 643 (1994) 181-193

of the spinal cord, result in attenuation of the blood pressure (BP) and heart rate (HR) conditioned emotional responses (CER) [6,11,20,23,32,46,51,56]. The infralimbic (IL) and ventral prelimbic (PL) regions of the medial frontal cortex (MFC) also project to autonomic regions such as the solitary nucleus [44,58,60,62] and the intermediolateral cell column [3,26], but no studies of the effects of lesions of the MFC following conditioning have been performed. Electrical or chemical stimulation of the ventral MFC elicits prominent cardiovascular and other autonomic changes [8,9,24,27, 28,31,38,59]. In addition, stimulation of the ventral MFC prior to defense responses induced via electrical stimulation of the amygdala results in attenuation or elimination of the defense response [40]. Previous work in our laboratory studying the behavioral aspects of the conditioned emotional response (CER) found a dramatic decrease in freezing behavior and ultrasonic vocalizations in response to the CS + following ventral MFC lesions [21]. For these reasons, the present study investigated the effect of lesions of the infralimbic and ventral prelimbic portions of the MFC on the conditioned cardiovascular responses. Aspiration lesions were performed initially to determine if lesions .of the MFC would significantly alter cardiovascular components in the rat. This work was then followed by more specific and localized lesions using the neurotoxin NMDA. In order to distinguish the learned (associative) components of the CER from the unlearned (nonassociative) components of the orienting response, the responses to a second tone (CS - ) that was randomly paired with the footshock were also studied.

2. Materials and methods

(n = 13) defined the normal response patterns during the CER, while rats with bilateral chemical lesions of the ventral medial frontal cortex (n = 11) served to define the effects of M F C lesions on the CER. Animals were anesthetized with ketamine HC1 (100 m g / k g ) and placed in a stereotaxic frame fitted with blunted earbars to prevent damage to the rat's eardrums. A small b u r r hole was made in the skull bilaterally (anterior 3.5 m m from bregma, lateral 0.75 m m from midline). A micropipette attached to a Hamilton syringe was lowered 4.0 m m from the dura into the ventral medial frontal cortex. Lesions were created by bilateral microinfusions (0.4 /xl over 10 min) of the neurotoxin N-methyl-o-aspartate (100 nM in sterile saline) [65]. N M D A destroys neuronal cell bodies while sparing axonal fibers of passage [67]. All controls received sterile saline. T h e pipette was then removed, the holes in the skull filled with gel-foam, and the wound sutured. T h e animal was returned to his own cage to recover for 2 weeks.

2.3. Conditioning parameters Each animal was conditioned to a tone paired with a footshock in a clear, lighted plexiglass box with a metal grid floor and an overhead speaker, using procedures previously outlined by LeDoux et al. [36]. A differential conditioning procedure included both a C S + tone (950 Hz) that was consistently paired with a footshock, and a C S tone (300 Hz) that was randomly associated with footshock [49]. C S - responses were recorded and compared to C S + responses within each group to determine the contribution of non-associative (unreinforced) responses. Forty tones (averaging 20 tones each for C S + and C S - ) were presented, with the order of presentation randomly generated by a computer. Each tone (950 or 300 Hz) was randomly presented through the overhead speaker for 10 s at 80 db at variable intervals (10 s to 5 min). The C S + tone was always followed immediately by a 0.5-s, 2-mA footshock (US) delivered through the grid floor. The C S - tone was randomly associated with the footshock, where the unconditioned footshock was equally likely to occur either before, during or after the C S - tone. However, neither the unconditioned footshock nor the C S - tone overlapped with the presentation of the CS + and its associated footshock.

2.4. Cannula implantation

2.1. MFC aspiration lesion study A pilot study involving aspiration lesions of the M F C was performed to determine whether any change in the cardiovascular response pattern would be found. The medial frontal cortex was removed bilaterally from 2 to 5 m m anterior to bregma, 1 to 2 m m laterally from the midline, and 3 to 5 m m ventral from the surface of the dura in 11 ketamine HC1 (100 m g / k g ) anesthetized rats. Controls (n = 11) consisted of sham-operated animals. The animals were allowed to recover for 2 weeks. Following reccvery, the animals were then conditioned to a single C S + tone (800 Hz, 10-s duration) followed immediately by a 0.5-s, 2 m A scrambled footshock. The animals received 10 orientation trials (no footshock), followed by 30 acquisition trials (tone paired with footshock). The intertrial interval was approximately 2 min. Electrodes and cannulas for recording H R and BP were implanted immediately after conditioning (see below). On the following day, BP and H R were recorded during three C E R trials in which only the CS was presented (no footshock).

2.2. MFC chemical lesion study Twenty-four male Sprague-Dawley rats weighing 350-500 g were used in this experiment and were divided into two groups. Controls

Immediately following conditioning, the animal was anesthetized with ketamine HC1 (100 m g / k g ) and restrained in a supine position on a rat board. Electrocardiogram (ECG) leads, composed of teflon coated, multi-stranded stainless steel wires, were implanted in the chest through small stab wounds. The free ends were tunnelled subcutaneously to the base of the neck and exteriorized through a puncture wound. The ventral hindlimb thigh was incised, and the femoral artery and vein carefully dissected free from the neurovascular sheath and surrounding fat. The artery was clamped and incised, and polyethylene tubing (PE10) was threaded inside the vessel approximately 11 cm. The free end of the PE10 was mated to PE50 in order to facilitate connection to the transducer during recording. After determining patency of the catheter and collateral circulation around it, the catheter was flushed with heparinized saline (100 U / m l ) and heat sealed to prevent leakage. T h e vein was also cannulated with a polyethylene tube (PE50) approximately 5 cm. After determining the patency of the venous catheter, it was flushed with heparinized saline (50 U / m l ) and heat sealed to prevent leakage. The free end of each catheter was tunnelled subcutaneously to the base of the neck, exteriorized through the puncture wound previously m a d e for the E C G leads, and marked for future reference. The wound in the leg was sutured and swabbed with bacteriocidal soap.

R.J. Frysztak et al. / Brain Research 643 (1994) 181-193 2.5. Data acquisition O n the day following conditioning, each animal was brought to the recording room in its own cage where it remained [33,36]. Previous recording sessions in the conditioning chamber revealed significant levels of stress (as measured by increases in blood pressure, heart rate and ultrasonic vocalizations) prior to delivery of any tones, which could be associated to the conditioning chamber itself (context conditioning). In addition, Phillips and LeDoux [48] have shown that conditioning to a 2-mA footshock results in the contextual response being undistinguishable from the CS response during the early phase of extinction testing. To avoid this interference and obtain a true m e a s u r e of the learned response, all recordings were performed in the animal's h o m e cage. The catheters and E C G leads were connected and 30 min were allowed to pass before any CS were presented to allow the animal to return to a 'resting' state. Blood pressure and heart rate were continuously recorded on a polygraph; in addition, during the actual C E R trials, BP and H R were input to the computer's digital or analog ports for storage and analysis. Computer sampling of BP and H R took place at the occurrence of each R wave in the E C G signal (approximately 5 Hz). Heart rate (bpm) was derived by detecting R waves in the E C G signal with a window discriminator. Blood pressure was recorded via the arterial catheter attached to a Statham blood pressure transducer. Sampling of blood pressure at the occurrence of the R wave of the H R signal results in an approximation of m e a n arterial pressure, since the peak of the R wave occurs at approximately the midpoint of the systolicdiastolic pressor wave. During C E R trials, only the tone (CS + or C S - ) was presented to the rat; no footshocks were delivered. A speaker placed over the rat's home cage produced the tones (10 s, 80 db, 950 or 300 Hz). After determining that the animal was at a 'resting' or baseline status (via inspection of H R and BP), the trial was initiated. Data were recorded for 750 data points (approximately 2 min), with the tone CS beginning at second 21; seconds 9 to 19 served as the baseline for that trial. T h u s each trial included a 20-second pre-CS

183

epoch, a 10-s CS presentation epoch, and a 90-s post-CS epoch. The normal responses to the C S + and C S - tones were characterized first, followed by presentation of these same tones after administration of various pharmacological agents. During the C E R ' s in the second condition (pharmacological manipulations), Control and MFC-lesioned rats were randomly divided into three sub-categories (n = 4 for each). C E R ' s were tested 5 min after the intravenous administration of one of the following drug combinations: (a) the muscarinic cholinergic blocker methyl atropine (1 m g / k g ) ; (b) the cardio-selective beta 1 antagonist atenolol (1 m g / k g ) ; (c) methyl atropine + atenolol (1 m g / k g each). Following all CS + and C S - trials, the animal was placed in the acquisition chamber and cardiovascular responses were recorded during the delivery of the footshock alone (US). At the completion of all testing, the rat was anesthetized with an overdose of sodium pentobarbital (300 m g / k g ) administered through the venous line. T h e rat was perfused transcardially with a 10% formalin solution, and the brain removed and allowed to sink in 30% sucrose in formalin solution. Frozen 50-p.m sections were cut and stained with Cresyl violet for histological reconstruction of the lesion sites.

2.6. Data analysis The average response patterns for BP and H R measured during the behavioral and pharmacological trials were calculated for each group and compared as described below. All responses are reported as the mean_+ 1 S.E.M.. Responses were considered significantly different if the calculated P value was less than 0.05. Blood pressure and heart rate were averaged within each trial on a second by second basis over the entire 120 s of data collection for each animal, To permit across animal averaging, all responses were determined as the net change from second 20 (immediately prior to CS onset). These individual responses were then averaged second by second for each group to yield the m e a n change per second (as represented in Fig. 6-A1). In addition, the m e a n BP and H R values were calculated for the baseline (pre-CS), CS, and post-CS periods for each animal. an

Fig. 1. M F C aspiration lesions. Coronal sections taken every 0.5 m m between 2 and 4 m m anterior to bregma. Shaded areas represent a typical Dorsal + Ventral M F C lesion on the left (A), and a Dorsal-only M F C lesion on the right (B). Note sparing of infralimbic cortex (*) in B. AC, anterior cingulate cortex; AgL, agranular lateral cortex; AgM, agranular medial cortex; CPu, caudate-putamen; IL, infralimbic cortex; Ins, insular cortex; PL, prelimbic cortex; SI, primary somatosensory cortex; SII, secondary somatosensory cortex. Bar represents 1 mm. N u m b e r s at right denote m m anterior to bregma of each section.

R.J. Frysztak et al. /Brain Research 643 (1994) 181-193

184

Baseline values were determined as the m e a n value of the parameter studied (BP or H R ) in seconds 9 to 19; CS responses were determined as the m e a n during seconds 21 to 31; post-CS responses were determined as the m e a n during seconds 40 to 120. Mean group BP and H R responses during each of the three periods were determined from the previously calculated individual animal means, thus yielding the m e a n change per period for each group (as represented in Fig. 6-A2). Comparisons within each group were made by A N O V A to determine if baseline, CS, and post-CS responses differed significantly over time; if so, comparisons between the m e a n baseline responses and the CS and the post-CS responses for both BP and H R were performed using Bonferroni t-tests. Comparisons between groups were made by A N O V A , followed by pre-selected individual (Bonferroni) t-tests comparing the same epochs (baseline, CS, or Post-CS) between groups for both BP and H R [68]. For the second condition studied (CER with pharmacologic blockade), data from each sub-category were first pooled, forming three sub-groups (atropine, atenolol, or atropine + atenolol). H R and BP responses were then averaged as previously described. The m e a s u r e m e n t of variability for both BP and H R were calculated for each of the three periods (baseline, CS, and post-CS responses) by determining the standard deviation (SD) from the original, raw data from each individual animal during each period. The overall m e a n variability for each group (Control vs. MFC) was then calculated from these individual animal m e a s u r e m e n t s of SD, yielding a m e a s u r e of m e a n variability+S.E.M, for each period for both BP and HR.

the infralimbic portion of the MFC (IL) was intact in the dorsal group, but was totally eliminated in the dorsal + ventral group. All animals (control, dorsal MFC, dorsal + ventral MFC) responded during the CS with an average blood pressure increase of approximately 10 mm Hg. However, heart rate responses for the three groups differed significantly, as shown in Fig. 2. Controls (n = 11) responded during the CS with a mean heart rate increase of 13.1 + 3.4 bpm. The dorsal MFC-lesioned group responded during the CS with an enhanced tachycardia, with a mean increase of 21.2 + 12.1 bpm. The dorsal + v e n t r a l MFC-lesioned group responded during the CS with a clear bradycardia, with a mean decrease of - 2 1 . 1 + 8.5 bpm. This bradycardia was significantly different from baseline, Control, and Dorsal MFC-lesioned responses.

3.Z MFC chemical lesion study Lesion analysis Fig. 3 is a photomicrograph of a coronal section through the lesion site of animal QM10 at the level of the infralimbic cortex, illustrating that the neuronal cell bodies of the infralimbic cortex were destroyed, but the more dorsal regions of the MFC were not disturbed. Fig. 4 illustrates the areas common to all lesions (hatched area) surrounded by the largest lesion achieved (dotted line). These lesions did not affect the resting BP (108.2 + 3.1) or H R (359.4 + 4.9) values when compared with resting Control values (107.2 + 2.5 and 364.4 + 5.0).

3. Results

3.1. MFC aspiration lesion study Histological reconstruction of the brains of animals with aspiration lesions found two distinct groups: animals with lesions restricted to the dorsally located agranular medial (AgM) and PL areas of the medial frontal cortex (Dorsal lesions, n = 5), and animals with lesions of the entire MFC including the AgM, PL and IL regions (Dorsal + Ventral lesions, n = 6). Fig. 1 illustrates coronal sections through dorsal + ventral (A) and dorsal-only (B) MFC-lesioned animals. Note that

CER responses Representative polygraph tracings of the cardiovascular responses recorded during single CS + or CS trials are illustrated in Fig. 5. The Control CS + response was a pressor response paired with a tachycar-

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Fig. 2. M e a n change per second and m e a n change per period in heart rate responses to the CS + following aspiration lesions of the MFC. Mean c h a n g e / s e c responses are plotted as change from second 9; m e a n c h a n g e / p e r i o d responses are the average H R response for baseline (seconds 4 - 8 ) and CS + (seconds 10-20). Dotted l i n e / o p e n bars represent Control response; dashed l i n e / s h a d e d bars represent response following Dorsal-only M F C lesion; solid line/solid bars represent response following Dorsal + Ventral M F C lesion. All CS + responses were significantly different from baseline ( P < 0.05, a). The Dorsal-only lesion group CS + response (tachycardia) was significantly greater that the Control CS + response ( P < 0.05, c); the Dorsal + Ventral lesion group CS + bradycardia was significantly different from both the Control and Dorsal-only CS + responses ( P < 0.05, b).

R.J. Frysztak et al. / Brain Research 643 (1994) 181-193

185

followed by a bradycardia beginning at second 25. Due to this bimodality, the m e a n M F C CS + H R response (1.7 + 4.4 bpm) was not significantly different from baseline, but was significantly less than that of Controis. During the post-CS + phase (seconds 40 to 120), the mean BP and H R responses were not significantly different from baseline or between Controls and MFC-lesioned animals. H e a r t rate variability in Controis during the post-CS + phase increased significantly ( 9 . 9 + 1.3) compared to baseline (4.1 + 1.1), however heart rate variability for MFC-lesioned rats was not significantly different from baseline during the post-CS + phase. The average blood pressure responses to the CS for both groups consisted in mild pressor responses which were not significantly different between Control (3.9 + 1.7 m m H g ) and MFC-lesioned (3.5 + 1.9 m m H g ) animals or from baseline, but were significantly less than their corresponding CS + responses. The average C S - heart rate response (Fig. 6B) did not differ significantly from baseline at any time throughout the trial for either Controls or MFC-lesioned animals. H e a r t rate variability was not significantly different from baseline during the p o s t - C S - phase for either Controls or MFC-lesioned animals.

Fig. 3. Photomicrograph of the rat medial frontal cortex taken 2.5 mm anterior to bregma following N M D A lesion in subject QM10. Note both the lack of cells within the infralimbic and ventral prelimbic cortices (Arrow 2), and the integrity of the overlying anterior cingulate cortex (Arrow 1). Small arrows delineate cytoarchitectonic boundaries. IL, infralimbic cortex; PL, prelimbic cortex; AC, anterior cingulate cortex; tt, taenia tecta. Bar = 1 mm.

dia (A). The MFC-lesioned CS + response (C) was a pressor response and, in this case, a bradycardia. The increased beat-to-beat H R variability seen in Controls following the CS + is also conspicuously lacking in MFC-lesioned rats. The cardiovascular responses to the CS - presentation (Fig. 5B and D) were typically smaller than those observed during the CS + . Following the CS - offset, BP and H R displayed considerably less beat-to-beat variability than CS + responses.

Auerage responses The average blood pressure responses to the CS + for both groups consisted of pressor responses which were not significantly different between Control (7.6 + 1.9 m m H g ) and MFC-lesioned (6.7 + 1.7 m m H g ) animals, but were significantly greater than baseline. The average Control CS + H R response (Fig. 6A) was a tachycardia with a mean response of 13.1 + 3.4 bpm. The average MFC-lesioned CS + H R response was bimodal, consisting of a small initial increase in H R

CS + pharmacological blockade Table 1 shows the m e a n resting blood pressure and heart rate measurements for both Control and MFClesioned animals prior to C E R testing. Methyl atropine significantly raised the m e a n resting H R compared to untreated animals for both Control and MFC-lesioned animals ( P < 0.01) as well as notably increasing variability (as seen in the S.E.M.). Mean resting blood pressure was not significantly affected by atropine administration, but variability was also increased. Atenolol did not significantly affect the mean resting BP or H R in any of the animals tested; variability was not affected. The combination of atropine and atenolol did not significantly alter BP or H R from untreated levels. The average CS + blood pressure response under methyl atropine was not significantly different between Control and MFC-lesioned rats. The magnitude of the average heart rate response in MFC-lesioned rats, however, was significantly reduced as compared to Controls (Fig. 7A). The mean atropine-treated Control heart rate response during the CS + was 17.2 + 3.8 bpm, while MFC-lesioned animals responded with only an 8.1 + 1.8 b p m increase, representing a 52% decrease as compared to the Control response. Controls remained significantly above baseline throughout the remainder of the trial, while the MFC-lesioned response returned quickly to baseline. The average CS + blood pressure responses under atenolol were not significantly different between Control and MFC-lesioned rats. The heart rate response

186

R.Z Frysztak et al. /Brain Research 643 (1994) 181-193 II f l

Fig. 4. Coronal view of left and right rat medial frontal cortex representing the extent of the area common to all lesions (dark grey shading), and the largest composite lesion (light grey shading). Note that the infralimbic portion of the MFC was removed in all cases. AC, anterior cingulate cortex; Acb, nucleus accumbens; AgL, agranular lateral cortex; AgM, agranular medial cortex; CPu, caudate-putamen; IL, infralimbic cortex; Ins, insular cortex; PL, prelimbic cortex; SI, primary somatosensory cortex; SII, secondary somatosensory cortex. Bar represents 1 ram. Numbers at right denote mm anterior to bregma of each section.

(Fig. 7B) for b o t h g r o u p s w a s a s m a l l initial d e c r e a s e ( a r r o w ) , f o l l o w e d by a l a r g e r , s l o w e r - o n s e t b r a d y c a r d i a , with the peak decrease occurring near the offset of the C S + . T h e initial d e c r e a s e m a y b e d u e to b a r o r e c e p t o r a c t i v a t i o n , w h i l e t h e m o r e slowly d e v e l o p i n g b r a d y c a r d i a w a s m o s t likely d u e to d i r e c t p a r a s y m p a t h e t i c activ a t i o n . T h e C o n t r o l C S + H R r e s p o n s e was n o t signifi-

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..'30O 2°t Fig. 5. Representative polygraph tracings of individual Control and MFC-lesioned CS + and C S - cardiovascular responses. Bars denote presentation of 10 s tone. BP, blood pressure in mm Hg; HR, heart rate in beats per minute. A: control responses during the CS + presentation. Note the increase in BP and HR at the CS + onset and the high degree of variability in the HR persisting following termination of the CS + tone. B: control responses during the C S - presentation. Both BP and HR responses are reduced compared to the CS + response. C: MFC-lesioned responses during the CS + presentation. Note the decrease in HR at the CS + onset, which persists throughout the remainder of the trial. D: MFC-lesioned responses during the CS - presentation. Note the slight increase in HR during the CS - tone.

R.Z Frysztak et al. / Brain Research 643 (1994) 181-193 Table 1 Resting blood pressure and heart rate values

CS -

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Blood pressure

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Untreated Control(13) MFC(12)

107.2+ 2.5 108.2_+ 3.1

364.4_+ 5.0 359.4_+ 4.9

Methyl atropine Control (5) MFC (4)

109.4_+ 11.7 110.1 _+ 12.9

423.1 _+ 17.4 * 437.6 _+22.7 *

Atenoiol Control(4) MFC(4)

105.1_+ 3.2 104.7_+ 3.8

361.2_+ 5.8 357.3_+ 5.1

Atropine + atenolol Control (4) MFC(4)

105.6_+ 1.7 106.1_+ 1.6

362.3_+ 2.8 361.7_+ 2.0

Resting blood pressure and heart rate values-+l S.E.M. before (untreated) and after selective pharmacologic blockade. * denotes responses significantly different ( P < 0.05) from corresponding Untreated group.

cantly different between Control and MFC-lesioned rats at any of the time points studied or from baseline (Fig. 7C).

187

pharmacological blockade

Pharmacological analysis of the CS - H R responses is shown in Fig. 8. The average C S - blood pressure responses in Control and MFC-lesioned rats following administration of methyl atropine were not significantly different from baseline or each other. Following the administration of atropine, the MFC-lesioned CS H R response was significantly reduced compared to C S - Controls, corresponding to a 73% reduction in sympathetic tone during the C S - for MFC-lesioned animals (Fig. 8A). Both Control and MFC-lesioned heart rate responses returned to baseline during the late CS - phase (sec 40-120) and did not differ significantly from each other. Following administration of atenolol, BP responses were not significantly different from one another or from baseline during the C S - . The heart rate response (Fig. 8B) displayed only a small decrease during the C S - for both Control and MFC-lesioned groups which was not significantly different from baseline. Note, however, that C S - H R responses were significantly smaller from CS + H R responses under atenolol (Fig. 7B), indicating a lack of parasympathetic activa-

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188

R.J. F r y s z t a k et al. / Brain Research 643 (1994) 1 8 1 - 1 9 3

back into the conditioning chamber resulted in noticeable increases in BP, H R and respiration prior to the administration of either the CS or the US. Each animal received one US (2 mA footshock, 0.5 sec duration). The US response in Control and MFC-lesioned animals consisted of a large increase in blood pressure paired with a strong tachycardia, and was most often accompanied by ultrasonic vocalizations. The response pattern in MFC-lesioned animals was not significantly different from those seen in Controls during the US or from MFC-lesioned rats. However, the tachycardia observed in MFC-lesioned animals during the US trial was significantly larger than the biphasic H R response

tion during the CS - . Both Control and MFC-lesioned HR responses displayed increased variability during the late CS - phase, resulting in an elevated heart rate compared to baseline. Following administration of atropine + atenolol, both blood pressure and heart rate showed little or no changes in response to the CS - as compared to baseline (Fig. 8C).

Unconditioned stimulus (US) responses Following all CER trials, the rats were returned to the plexiglass shock chamber where acquisition ocCurred. As previously described, placing the animals

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R.J. Frysztak et aL / Brain Research 643 (1994) 181-193

seen in MFC-lesioned animals during the CS + presentation in the home cage.

mally act to decrease HR during stress. This is consistent with reports that electrical and chemical stimulation sites in the dorsal MFC attenuate or inhibit the defense response and the associated cardiovascular responses evoked by amygdala stimulation [40,55,69]. This area of the prefrontal cortex has been classified as 'sympatho-inhibitory' by Maskati and Zbrozyna [40], and probably plays an essential role in the suppression of cardiovascular and behavioral changes activated by either fear or aggression [69]. In contrast, aspiration lesions of the dorsal + ventral MFC (anterior cingulate, prelimbic and infralimbic areas) resulted in a bradycardia to the CS + , indicating that the ventral MFC may normally act to increase HR during stress. Chemical

4. Discussion 4.1. Associative responses

Lesions of the MFC in rats altered the sympathetic component of the HR responses, dependent on the extent and placement of the lesion. Aspiration lesions of the dorsal MFC (anterior cingulate and prelimbic areas) resulted in an increased tachycardia response to the CS + , indicating that the dorsal MFC may nor-

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lesions of primarily the infralimbic portion of the MFC (with variable destruction of the prelimbic region) resuited in a bimodal HR response. In these chemically lesioned animals, sympathetic activation during the CS + was reduced by 52% compared to Controls, further supporting the idea that the ventral MFC is normally involved in activation of a significant number of sympathetic neurons during stress [28]. The rat MFC has recently been shown to project directly to the thoracic intermediolateral cell column [3,26], the site of origin for preganglionic cardiac sympathetic neurons. Through its projections to the solitary nucleus and lateral hypothalamus [58,60,62,63], the MFC also has indirect access to the sympathetic nervous system. Thus the MFC has significant access to sympathetic outflow. The Control responses presented here agree with all previous reports of cardiovascular responses during the CS + in freely moving rats [5,15,17,29,36,37,42], namely a pressor response paired with a tachycardia. The major cause of the aversively conditioned tachycardia in Controls appears to be increased sympathetic outflow, apparently 'buffered' by a slow-onset activation of the parasympathetic component during the CS + . The 'sympatho-inhibitory' effects of the dorsal MFC appear to be overcome by the increases in sympathetic outflow achieved by the activation of the ventral MFC (and presumably the hypothalamus and amygdala). In addition, parasympathetic activity may also be inhibited early in the CER ('parasympathetic withdrawal' [13]). Sympathetic outflow is dominant initially, and the resulting response is a pronounced tachycardia. Parasympathetic activation occurs more slowly, and the system returns to a buffered, 'steady state' equilibrium. The CS + response is therefore characterized by sequential activation of the sympathetic and parasympathetic systems. In MFC-lesioned animals, activation of the sympathetic component by the ventral prelimbic and infralimbic cortices is lost, resulting in significant attenuation of the sympathetic component responsible for HR increases during stress. Since activation (both direct and indirect) of the parasympathetic system is left intact, the parasympathetic system becomes dominant, and a slow onset bradycardia is observed late in the CS + that persists after termination of the CS + tone. The result is the bimodal HR response observed. The blood pressure responses in Control and MFClesioned animals, both to the CS + and the C S - , were not significantly different. Presumably, the pressot response to the CS is also produced by sympathetic activation. LeDoux and colleagues have shown that the conditioned pressor response is a reliable indicator of stress in freely moving rats. Lesions of the lateral hypothalamus a n d / o r the amygdala result in a decrease in the conditioned pressor response [30,35], indicating that these areas may be important sites for

the maintenance and expression of blood pressure responses to the CS. If the infralimbic area were responsible for producing changes in blood pressure, one would expect that there would be a decrease in the magnitude of the BP response following MFC lesions. Lesions of the ventral MFC, however, failed to produce a similar reduction in the conditioned pressor response. While the MFC has been shown to modulate both heart rate and blood pressure in electrical [8,40,59] and chemical [1] stimulation experiments, the expression of a learned pressor response appears to be controlled from elsewhere in the CNS, perhaps from the lateral hypothalamus [30,35] a n d / o r the amygdala [22,23,41,51]. 4.2. N o n - a s s o c i a t i v e responses

Our CS + and CS - BP responses agree with data presented by Iwata and LeDoux [29], who described significant attenuation of the BP response with all forms of nonassociative conditioning (random (CS - ), backwards, US-alone, naive) compared to associative (paired; CS + ) conditioning. Our CS + and CS - HR responses, however, differ from those of Iwata and LeDoux, who found that the HR response to associative conditioning (CS + ) was not significantly larger than that seen with non-associative conditioning ( C S - ) . They concluded that the BP response clearly and reliably reflects the associative conditioning process in rats, whereas the HR response reflects a nonassociative process. In contrast, our results show a significant reduction in the HR response to random (non-associative) conditioning compared to that of paired (associative) conditioning. However, our findings are in agreement with several other studies that have reported HR responses which differ significantly between associative and non-associative trials [6,7,18,39]. This disparity over whether the heart rate response is a reliable indicator of conditioning may be explained, at least in part, by noting that Iwata and LeDoux [29] averaged the effects of three trials in determining both associative and non-associative responses, a procedure that increases the significance of the second and third trials during which extinction is occurring. Our study utilized only the initial trial, and therefore the effects of extinction were not averaged into the results. Blood pressure and heart rate responses in MFC-lesioned animals during the C S - were significantly smaller than the CS + responses. The CS - response, mediated only by the sympathetic component, resulted in a tachycardia in MFC-lesioned rats. These results are consistent with those of Iwata and LeDoux [29], who determined that the CS + response was due to coactivation of the sympathetic and parasympathetic systems, whereas the C S - response was due only to sympathetic activation.

R.J. Frysztak et al. / Brain Research 643 (1994) 181-193

The responses associated with the US indicate that the 'learned' responses to the CS + and C S - are mediated by brain areas other than or in addition to the brain areas mediating the US response. During the presentation of the footshock (US) alone, it is likely that pain pathways and other sensory input contributed to the profound autonomic (pressor response and tachycardia) land behavioral (ultrasonic vocalizations) responses observed in both Control and MFC-lesioned animals. 4.3. Ultrasonic vocalizations and heart rate variability

When a rat is placed in a naturally stressful situation, such as defeat [19], the rat initially becomes immobile ('freezes'), pants at a very rapid rate (approximately 5 Hz), and heart rate and blood pressure are generally increased. This initial phase is followed abruptly by a period of slow, deep breathing ( < 1 Hz) that lasts for several min and is often accompanied by ultrasonic vocalizations (USV) and continued freezing. This latter phase is also characterized by elevated blood pressure and pronounced fluctuations in blood pressure and heart rate corresponding to the 'forced breathing' associated with USV [19], which results in increased heart rate variability. This increase in H R variability is evident in the raw polygraph tracings of Controls (Fig. 5A). The increased H R variability expressed late is directly related to the emotional and behavioral reactions (USV and freezing) occurring during this phase [19,21]. Following MFC lesions, both the autonomic (sympathetic outflow and H R variability) and behavioral (USV and freezing) [21] responses are significantly reduced in response to the CS + , resulting in an animal which is significantly less affected by previous aversive experiences or associations (footshock). Consequently, H R variability was not significantly different from baseline. Similarly, the CS - was not previously associated with the aversive stimulus, and subsequent presentations of the C S - do not result in USV [21]. Since neither Controls (Fig. 5C) nor MFC-lesioned (Fig. 5D) animals exhibited USV following the CS - , H R variability during the late phase was not significantly different from baseline. 4.4. L i m b i c system and visceral control

Lesions of a variety of limbic structures within the CNS significantly alter the autonomic a n d / o r behavioral conditioned responses to stress. Both electrolytic and chemical lesions of the lateral hypothalamus significantly decreased the conditioned arterial pressure response mediated by the sympathetic nervous system [30,35]. Behavioral CER responses, such as immobility, were not affected by hypothalamic lesions, [30] but were reduced by lesions of the central gray [29]. Le-

191

sions of the amygdala, which projects to both the lateral hypothalamus and the central gray, attenuated both the autonomic (cardiovascular) and behavioral (freezing) CER responses to stress [22,23,35,51,64]. It is interesting to note that the MFC projects to all of these areas of the CNS [3,25,26,50]. For example, the infralimbic portion of the MFC projects directly to the medial subdivision of the central nucleus of the amygdala [3,26,50], which in turn has extensive connections with the parabrachial nucleus [52], the dorsal motor vagal complex [54], and the nucleus of the solitary tract [63]. These structures have been shown to play an influential role in cardiovascular regulation [47], and may allow the infralimbic MFC to modulate cardiovascular function. In addition, other cardiovascular sites within the CNS to which the infralimbic MFC has projections include the bed nucleus of the stria terminalis [26,50,66], parabrachial nucleus [52], nucleus ambiguus [26], the rostral ventrolateral medulla [26,61], the nucleus of the solitary tract [58,60,62], and lamina I of the spinal cord [26]. The infralimbic MFC, therefore, has extensive projections to many central autonomic nuclei, and may serve a primary role in modulating cardiovascular responses to conditioned stimuli and stress. 4.5. Frontal cortex and emotion

Nauta [43] proposed that lesions of the frontal cortex in human subjects result in an 'interoceptive agnosia', characterized by an absence of normal visceral responses. According to Nauta, the frontal cortex plays a large role in producing visceral responses; without the frontal cortex, these responses and the associated 'gut feelings' would be absent or significantly reduced, resulting in an individual who has become 'ignorant' of the cues or signals his body would normally be providing. The profound significance of this absence of normal visceral responses in humans following bilateral medial frontal and orbitofrontal cortex lesions is dramatically illustrated in the case of EVR [16]. EVR and similar cases manifest marked personality changes, disturbances of social conduct, poor decision-making skills, poor planning, and loss of normal motivation and affect [2,14,16], despite normal or above average memory, intelligence, etc. [53] The only observable neurophysiological deficit in these subjects is an absence of sympathetically mediated skin conductance responses (SCR) during passive viewing of visual stimuli charged with high emotional value. It has been proposed by Damasio et al. [14] that these behavioral and personality deficits stem from defects in somatic states in response to stimuli which normally produce punishment or reward and whose experience may trigger states of pleasure or pain. In other words, EVR's poor decisions and judgement can be explained not because he has

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R.J. Frysztak et al. /Brain Research 643 (1994) 181-193

'lost his mind', but rather because he has 'lost his body'. Our findings in the rat reinforce and support this view, and suggest a critical role for the medial frontal cortex in emotional expression and experience.

5. Acknowledgements The authors would like to thank Dr. D. Euler for his comments. This work was supported by a Loyola University Potts Estate Fund Grant.

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