role of catecholamines in the frontal cortex in the modulation of basal and stress-induced autonomic...

BRAIN RESEARCH

E L S E V I E R Brain Research 741 (1996) 220 229

R e s e a r c h r e p o r t

Role of catecholamines in the frontal cortex in the modulation of basal and stress-induced autonomic output in rats

Douglas Funk, Jane Stewart "

Center for Studies in Behacioral Neurobioloy, y, Department ~/'Psychology. Coneordia Unil,ersio,, 1455 DeMaisonneut,e Bh~d. W., Montreal. Quebec, H3G 1MS, Canada

Accepted 30 July 1996

Abstract

Exposure of animals to noxious or stressful stimuli increases heart rate (HR) and blood pressure through activation of the autonomic nervous system (ANS) and elicits the release of the catecholamines noradrenaline (NA) and dopamine (DA) in the frontal cortex. Subregions of the frontal cortex, such as the medial frontal cortex (MFC) and agranular insular cortex (AIC) project directly to brainstem nuclei involved in autonomic control. It may be hypothesized that catecholamines in the frontal cortex could influence autonomic output through actions on these descending pathways. To evaluate this hypothesis, the effects of intracortical microinjections of drugs acting at NAergic and DAergic receptors were assessed on an autonomically mediated response, the increase in HR induced by tail pinch, in rats anesthetized with urethane, lntra-MFC or AIC injections of an antagonist of [3-adrenoceptors reduced the magnitude of the HR response to pinch. Injections of an agonist of [3-adrenoceptors into these regions increased basal HR but did not affect the pinch response. Injections of drugs acting at ~x-adrenoceptors were without effect. When injected alone, drugs acting at DAergic receptors did not effect basal HR or the response to pinch, but intra-AIC injections of a combination of a D 2 antagonist and an agonist of [3-adrenoceptors increased the magnitude of the pinch response. These results suggest that catecholamines, especially NA, released in the frontal cortex are important modulators of the basal and stress-induced output of the ANS.

Keywords: Heart rate; Autonomic nervous system; Stress: Medial frontal cortex: Agranu[ar insular cortex; Noradrenaline: Dopamine: [3-Adrenoceptor

I. Introduct ion

Increases in heart rate (HR) and blood pressure elicited by noxious or stressful stimuli are mediated by the autonomic nervous system (ANS). The central nervous control of these responses in rats and other animals has been the subject of intensive study [7,9,22]. Stressors also activate central pathways containing the neuromodulatory catecholamines noradrenaline (NA) and dopamine (DA), resulting in the release of these transmitters throughout the tore- brain, including the frontal cortex [ 1,2,5,8,10,18,19,24,28].

Two regions of the frontal cortex, the medial frontal cortex (MFC) and agranular insular cortex (AIC), are known to influence the output of the ANS through direct and indirect projections to brainstem nuclei involved in autonomic control, including the nucleus of the solitary tract (NTS), the parabrachial nucleus and the rostral ventrolateral medulla (RVLM) [ 16,32,41 ]. Despite this knowl-

* Corresponding author. Fax: + 1 (514) 848-2817.

edge, the possibility that the catecholaminergic innervation of the frontal cortex modulates the basal output or the stress-induced activation of the ANS through actions on these projections has not been addressed.

Electrical stimulation of the MFC or AIC in the rat decreases basal HR and blood pressure [3,4,13]. These hypotensive responses to stimulation are blocked when the output of the sympathetic, but not parasympathetic division of the ANS is disrupted, suggesting that the projections from these two regions of the frontal cortex modulate, in an inhibitory manner, the basal output of the sympathetic division of the ANS [13].

In agreement with these findings, electrical or chemical stimulation of the MFC blocks the increases in HR and blood pressure induced by stimulation of the amygdala or reticular formation [3,44], while destruction of the MFC blocks the decreases in HR and blood pressure induced by stimulation of the hippocampus [17,20,31]. These results suggest that projections from the MFC also exert an inhibitory influence during conditions when the output of the ANS is increased.

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 9 3 1 - 6

D. Funk, J. Stewart/Brain Research 741 (1996) 220-229 221

Aspiration of the MFC, on the other hand, does not affect the HR and blood pressure responses elicited by a footshock stressor. This suggests that the MFC does not exert a modulatory influence on the autonomic activation elicited by physical stressors [11]. The animals in this study were, however, tested 2 weeks after surgery during which compensatory changes in the circuitry regulating autonomic output may have occurred. The animals had also received many exposures to footshock prior to cardiovascular testing, suggesting the possibility that habituation to the stressor may have obscured any effect of lesion.

Lesions of the MFC decrease the incidence of ulcers of the stomach, an autonomically controlled organ, induced by intense stressors in rats [38]. Although changes in the activity of the parasympathetic division of the ANS have been shown to be the primary mediator of the ulcer response, gastric activity is known to be controlled in a reciprocal manner by the parasympathetic and sympathetic divisions. It is therefore difficult to infer from these data which division of the ANS is affected by MFC lesions and, moreover, whether these lesions are facilitatory or inhibitory [6,21,22].

Electrophysiological studies strongly implicate the catecholaminergic projections in the control of the output of the frontal cortex. Both the NAergic and DAergic projections have been shown to inhibit the spontaneous and stress-evoked firing of neurons in at least the MFC [27,42]. Despite this, only one study has examined the possibility that the catecholaminergic projections function to modulate these influences of the MFC and AIC on the output of the ANS. In rats, injections of NA into the AIC were observed to alter basal energy expenditure, which is controlled in part by the ANS, while injections of NA into the MFC were without effect [29]. Since energy expenditure is controlled by a number of other systems, these results do not make clear the role of frontal cortical NA as a modulator of autonomic output.

Taken together, these findings suggest that the projections originating in the MFC and AIC, two cortical regions innervated by the catecholaminergic systems, are inhibitory on at least the basal output of the sympathetic projections of the ANS controlling cardiovascular activity. The possibility that the catecholaminergic innervation of the frontal cortex acts as a modulator of these influences of the frontal cortex on autonomic output has not been directly addressed.

It may be hypothesized that the release of catecholamines in the frontal cortex serves inhibitory or facilitatory functions in the modulation of autonomic output during basal conditions or when it is activated in response to noxious or stressful stimuli. To help resolve this issue, a series of experiments was carried out examining the effects of injections of agonists or antagonists of NAergic and DAergic receptors into the MFC or AIC, on a response to stress mediated by the ANS, the increase in HR induced by tail pinch, in rats anesthetized with urethane.

2. Materials and Methods

2.1. Subjects

A total of 96 male Wistar rats (Charles River) weighing 300-325 g were housed in an animal room maintained at 22°C (light phase 08.00 h-20.00 h). Rat chow and tap water were available ad libitum. Experiments were carried out between 10.00 h and 18.00 h. All experimental proce- dures used were in compliance with the regulations of the Canadian Council on Animal Care.

2.2. Heart rate recording

Rats were anesthetized with urethane (1.5 g /kg , Sigma) administered intraperitoneally (i.p.). Anesthesia was maintained throughout the experiment with supplemental doses of urethane (250 m g / k g i.p.) as required. Core tempera- ture was continuously monitored and maintained between 37.0 and 37.5°C with a homeothermic heating blanket (Harvard Apparatus).

Electrocardiographic (ECG) electrodes made from safety pins were implanted transcutaneously, one on each flank overlying the ribs and one on the back over the scapulae. The signal was amplified and filtered and routed to a MacLab amplifier connected to a Macintosh computer. HR was derived from the ECG signal with MacLab software and averages were continuously recorded over 1.5-s inter- vals.

2.3. Surgery

Rats were placed in a stereotaxic frame (David Kopf), the scalp was cut and retracted and stereotaxically placed holes were drilled over the right MFC and AIC. Cannulae were stereotaxically aimed at the following coordinates, relative to bregma, with the skull fiat. MFC: anterior/posterior = 3.0 mm, lateral = 0.8 mm, dorsal/ventral = - 5 . 5 mm; AIC: anterior/posterior = 2.2 mm, lateral = 4.2 mm, dorsal/ventral = - 6.3 mm.

2.4. Drugs

Drugs were prepared freshly every day and were dissolved in physiological saline with the exception of phentolamine, which was dissolved in distilled water. The following drugs were used. 13-adrenoceptor agonist: isoproterenol hydrochloride (Sigma); 13-adrenoceptor antagonist: propranolol hydrochloride (ICN); ct-adrenoceptor agonist: phenylephrine hydrochloride (RBI); ot-adrenoceptor antagonist: phentolamine hydrochloride (Sigma); D l agonist: SKF82958 (RBI); D I antagonist: SKF83566 (RBI); D 2 agonist: quinpirole (Lilly); D 2 antagonist: Raclopride tartrate (Astra).

2.5. Choice o f urethane as anesthetic

Urethane was used as it is known to spare autonomically mediated increases in HR and blood pressure induced

222 D. Funk, J. S tewart~Brain Research 741 (1996) 2 2 0 -2 2 9

by noxious or painful stimulation [36]. Neither does urethane strongly depress the basal firing of neurons in the CNS, unlike barbiturate anesthetics [25,26]. This character- istic of urethane was also thought to be important, since the primary electrophysiological actions of NA and DA appear to be inhibition of neuronal firing, at least in the frontal cortex. An anesthetic that depresses the firing of cortical cells might be expected to mask effects of drugs mediated by neuronal inhibition.

2.6. Histology

At the end of each experiment, animals were administered an overdose of sodium pentobarbital (Somnotol) and perfused transcardially with physiological saline followed by 10% formaldehyde. Brains were removed and stored in 10% sucrose-formaldehyde prior to slicing into 30-1xm sections in the coronal plane on a freezing microtome.

The placements of cannulae in the MFC and AIC of each rat were drawn onto representative coronal plates from a brain atlas [40]. Animals whose placements were outside of these regions were excluded from the analyses. The placements of the cannulae in the experiment where the vehicle, saline, was injected into the MFC and AIC are respectively shown in Fig. 1A,B. The cannula placements observed in this experiment are representative of those found in the remaining experiments.

2.7. Procedure

Y •

V ¸

y

\ \

/

\

y

\ J

A MFC B AIC Fig. 1. Cannula placements in the experiment in which saline vehicle was injected into the MFC and AIC are shown, respectively, in panel A and B. These placements are representative of those found in the remaining experiments.

The effects of injections of a given drug or combination of drugs were assessed in the MFC and AIC of each animal. The order that the two sites were tested in was counterbalanced across subjects within a particular drug condition.

Prior to testing, 5 -7 tail pinches, each 10 s in duration, separated by at least 5 min were administered 2 cm from the base of the tail with an adjustable tubing clamp (VWR). The tension of the clamp was adjusted until reproducible increases in HR of 10-30 beats per min were obtained in response to a 10-s tail pinch.

A 28-gauge injection cannula connected to a l-lxl sy- ringe prefilled with drug solution was stereotaxically low- ered to the right MFC or AIC. Five minutes after cannula placement, testing was carried out as follows. During the predrug phase, baseline HR was collected for 1 min. Two 10-s tail pinches separated by 5 min were then administered. Five minutes after the second pinch in the predrug phase, drug (10-40 nmol) was injected over 45 s into the site being tested in a volume of 0.5 txl. Five minutes after the intracranial injections, two 10-s tail pinches separated by 5 min were administered. HR was recorded until 5 min after the second tail pinch.

The injection cannula was then withdrawn and the stability of the HR response to tail pinch was again confirmed. Twenty minutes after withdrawal of the can-

nula from the first site, and at least 10 min after the last tail pinch, the injection cannula was placed in the other cortical site and testing was carried out as described above. The reproducibility of the HR response to pinch was also tested following supplementation of anesthesia.

2.8. Presentation of" data

Average HR was recorded continuously over 1.5-s in- tervals throughout testing. To simplify the analysis of data, samples of HR were taken at representative points from each of the two pinches administered in the predrug and postdrug phases. These were the baseline prior to each pinch (Base), the peak HR induced by each pinch (Peak) and the HR 2.5 min after each pinch, as a measure of recovery (Recov). Fig. 2A shows an example of a trace of raw HR data with the three sample points labeled for the first pinch; these sample points are plotted for each of the pinches in Fig. 2B.

For graphical presentation, these data points from individual animals were converted to change scores from their initial baseline HR recorded prior to the first pinch in the predrug phase (AHR). The group means and standard errors of the mean (S.E.M.) were calculated from these data. Data from the postdrug phase were then superimposed on those from the predrug phase in order to illustrate

D. Funk, J. Stewart~Brain Research 741 (1996) 220-229 223

410 ,

400" c ~ 390'

3so~

i ~ '370-

360-

350-

410

400-

"E 390-

O~ 380-

~" 370- "T

360-

350-

A Peak

Intracortical Recov Inie~ion

Base 5 rnin

Pinch 1 Pinch 2 Pir{ch 1 Pir~ch 2

Predrug Postdrug

+ Predrug B * Postdrug

B ~ P ~ Re~-'ov B ~ Pq~k Re~v B~e P ~ Rely B ~ P~k Rely Pinch 1 Pinch 2 Pinch 1 Pinch 2

50-

40"

c ~ 30.

~ 20"

10'

O'

-10'

- o - - Predrug C - -A - Postdrug

n=9

Base Peak Recov B~se P~ik Recov

Pinch 1 Pinch 2

Fig. 2. An example of a plot of the raw HR data sampled every 1.5 s (A). The three samples of HR from each of the pinches in the predrug and postdrug phases are plotted in panel B. For presentation, these data from individual animals were converted to change scores from their initial baseline HR (AHR), from which means and standard errors of the mean (S.E.M.) were calculated. The resulting means and standard errors from the predrug phase were then superimposed on those from the postdrug phase (C). Panel C also shows that unilateral injections of 0.5 pA of physiological saline into the MFC affected neither basal HR nor the HR responses to tail pinch.

the effects of the intracortical injections on basal HR and on the responses to tail pinch (Fig. 2C).

Fig. 2C also shows that injections of 0.5 ~1 of saline into the MFC did not significantly affect basal HR or the magnitude of the HR response to tail pinch. The data from this control experiment also demonstrate that the two pinches administered prior to the injections did not effect the magnitude of the response to tail pinch administered after the injections.

2.9. Statistics

Analyses of variance (ANOVA) using repeated mea- sures were carried out on the absolute HR values for a given drug or combination of drugs at each site. The

factors and levels of the design were drug phase (Predrug and Postdrug), pinch number (Pinch 1 and Pinch 2), and sample (Base, Peak and Recov). Analyses of simple effects were used to assess the effects of intracortical injections of drugs on basal HR and on the response to tail pinch.

3. Results

3.1. Drugs acting at NAergic receptors

3.1.1. Drugs acting at fl-adrenoceptors Unilateral injections of the ~-adrenoceptor agonist, iso-

proterenol (10 nmol), into either the MFC (Fig. 3A) or AIC (Fig. 3B) caused increases in basal HR. These effects are reflected in the significant main effects for drug (MFC:

FI, 9 --- 14.78, P < 0.005; AIC: F~,~0 = 15.13, P < 0.005) and in the simple effects of drug at the baseline prior to the

first pinch (MFC: FI,54 = 15.20, P < 0.005; AIC: Fl,60 = 9.36, P < 0.01). Injections of isoproterenol into either the

5u

40"

e- • -- 30" E

20

n- • 1- 10.

-10

A

~-adrenoceptor Agonist, Isoproterenol

M F C - - -o - Predrug Postdrug

50

B AIC n=l 1

¢- • ~ 30'

~ 20

r r "1- 10

0"

-10-

Base Peak Recov Base ~ Recov Pinch 1 Pinch 2

Fig. 3. Mean AHR (5:1 S.E.M.) shown in response to two 10 s tail pinches separated by 5 min, administered prior to (Predrug), and following (Postdrug) unilateral injections of 10 nmol of an agonist of ~3-adrenoceptors, isoproterenol, into the MFC (A) or AIC (B).

224 D. Funk, J. Sten'art / Brain Research 741 (1996) 220-229

MFC or AIC did not, however, significantly affect the magnitude of the response to tail pinch.

Injections of the [3-adrenoceptor antagonist, propranolol (40 nmol), into the MFC (Fig. 4A) decreased baseline HR significantly, as shown by the test for the main effect of drug (Fl., ~ = 16.76, P < 0.005). This is accounted for primarily by its effects prior to the second pinch. These injections also reduced the magnitude of the HR response to pinch, as shown by the significant drug-sample interaction (F2.~ = 7.31, P < 0.01). Tests of the simple effects of this interaction revealed that the drug significantly reduced the magnitude of the pinch response during both the first (F2,3~ , = 4.46, P < 0.05) and the second pinches (F=,~, = 5.68, P < 0.01). The decreases in the magnitude of the pinch response were characterized by reductions in the magnitude of the peak increases and in the more rapid return of HR to prepinch levels after each pinch.

Injections of propranolol into the AIC reduced basal HR, but not to a statistically significant degree (Fig. 4B). These injections did, however, reduce the magnitude of the HR response to tail pinch. Although the drug-sample interaction only approached statistical significance (F=.l~ = 3.42, P = 0.05), the test of the simple effect of this

50

40"

• -- 30" E

20"

r r "T" 10-

-10-

[3-adrenoceptor Antagonist, Propranotol

A MFC + Predrug

Postdrug

n=10

5Q

40-

"~" 30-

20-

rc 10- 1-

O-

- I0 -

B AIC n=10

Ba~se Peak Recov Base Peak Recov

Pinch 1 Pinch 2

Fig. 4. Mean AHR (±1 S.E.M,) shown in response to two 10-s tail pinches separated by 5 rain, administered prior to (Predrug), and following (Postdrug) unilateral injections of 40 nmol of an antagonist of [3-adrenoceptors, propranolol, into the MFC (A) or A1C (B).

50-

40-

• ~ 3o- E

"~ 2c- & rr 'r- lO-

-10-

[3-adrenoceptor Agonist, Isoproterenol + I~-adrenoceptor Antagonist, Propranolol

A MFC - - -=-- Predrug

+ Postdrug

n=5

-ff E

a3

rr" -1- -"4

50

40-

30-

20-

10-

0-

-10-

B AIC n=5

Base Peak Recov 13&se Peak Recov

Pinch 1 Pinch 2

Fig. 5. Mean ,SHR (±1 S.E.M.) shown in response to two 10 s tail pinches separated by 5 min, administered prior to (Predrug)+ and follow- mg (Postdrug) unilateral injections of a combination of I0 nmol of an agonist of B-adrenoceptors, isoproterenol and 40 nmol of an antagonist of ~-adrenoceptors, propranolol, into the MFC (A) or AIC (B).

interaction revealed statistical significance at the time of the second pinch (F2,36 = 4.63, P < 0.05). The magnitude of the peak increase in HR was reduced, and the recovery of HR was more rapid after injections of propranolol into the AIC.

The antagonist of the [3-adrenoceptor, propranolol (40 nmol), was able to block the effects of the agonist, isoproterenol (10 nmol), on basal HR when both drugs were injected into the MFC (Fig. 5A). This effect was not immediate, however; isoproterenol led to an elevation of basal HR prior to the first pinch. Similarly, propranolol did not completely block the initial increases in basal HR induced by isoproterenol when both drugs were injected into the AIC (Fig. 5B), but by the time of the baseline sample before the second pinch, propranolol decreased basal HR to levels below the preinjection baseline.

3.1.2. Drugs acting at cg-adrenoceptors Injections of 20 nmol of an agonist or an antagonist of

e~-adrenoceptors, phenylephrine and phentolamine, respectively, into the MFC or A1C did not significantly affect basal HR or the magnitude of the HR response to tail

D. Funk, J. Stewart~Brain Research 741 (1996)220-229 225

pinch. The effects of injections of a combination of an agonist of 13-adrenoceptors, isoproterenol (10 nmol), and an agonist of oL-adrenoceptors, phenylephrine (20 nmol), were not different from those seen when isoproterenol was injected alone.

3.2. Drugs acting at DAergic receptors

The D1 receptor agonist, SKF82958 (20 nmol), did not significantly affect basal HR or the magnitude of the HR response to pinch when injected into the MFC or AIC. Neither did the D 1 receptor antagonist, SKF83566 (20 nmol) affect these parameters when injected into the MFC or AIC.

Injections of 20 nmol of the D 2 agonist quinpirole or 20 nmol of the D 2 antagonist raclopride into the MFC or AIC did not significantly affect basal HR or the magnitude of the HR response to pinch. There was, however, a slight tendency for quinpirole to reduce basal HR when it was injected into either the MFC or AIC (Fig. 6A,B). Raclo- pride, in contrast, caused slight increases in basal HR when injected into either cortical site (Fig. 7A,B).

50

40"

30"

20"

n- -r- 10-

,<1

-10.

A MFC

D2 Agonist, Quinpirole

--m-- Predrug ---,lk--- Postdrug

n=5

5u

4O

t- 30

~ zo & n- -1- 1

-10

A MFC

132 Antagonist, Raclopride

---m.-- Predrug

Postdrug

n=5

5u

40'

.-- 30 E

~ 20 & n'- I 10-

-10-

B AIC n=4

Base Pe~k Recov Base Peak R~=cov

Pinch 1 Pinch 2

Fig. 7. Mean AHR (_+I S.E.M.) shown in response to two 10-s tail pinches separated by 5 rain, administered prior to (Predrug), and following (Postdrug) unilateral injections of 20 nmol of the D e receptor antagonist raclopride into the MFC (A) or AIC (B).

Injections of combinations of agonists and antagonists of D 1 and D 2 receptors into the MFC or AIC affected neither basal HR nor the magnitude of the HR response to tail pinch significantly.

50

46.

.¢:: 30

f f l

N 2o-

"1- 10-

-10

B AIC n=8

/% Base Peak Recov B~tse Peak Recov

Pinch 1 Pinch 2

Fig. 6. Mean AHR (_+ 1 S.E.M.) shown in response to two 10-s tail pinches separated by 5 rain, administered prior to (Predrug), and following (Postdrug) unilateral injections of 20 nmol of the D 2 receptor agonist quinpirole into the MFC (A) or AIC (B).

3.3. Combinations of drugs acting at DAergic and NAergic receptors

3.3.1. fl-Adrenoceptor agonist and a D 2 receptor antagonist

The intra-MFC injection of a combination of an agonist of [3-adrenoceptors, isoproterenol (10 nmol) and an antagonist of D 2 receptors, raclopride (20 nmol), increased basal HR, as reflected in the significant main effect of drug ( E l , 7 = 34.80, P < 0.001) (Fig. 8A). Injections of this combination of drugs into the MFC did not affect the magnitude of the response to tail pinch, as shown by the results of the test for the drug-sample interaction (F2,14 = 0.98, P > 0.39).

Injections of the combination of isoproterenol and raclopride into the AIC increased baseline HR (Fig. 8B). This is reflected in the significant main effect of drug (F1, s = 1,22, P < 0.05) and in the significant simple effect

226 D. Funk, d. Stewart~Brain Research 741 (1996) 220-229

E

rr "r"

5 O

A

40-

30"

20-

10-

0"

-10-

[}-adrenoceptor Agonist, Isoproterenol + D2 Antagonist, Raclopride

---c3-- Predrug os o,u0

5 0

4 0 -

~ " 3O-

E 2O-

r r 10- "1- <1

O-

- 1 0 -

AIC n=6

Base Peak Re'cov Base P e a k Recov

Pinch 1 Pinch 2

Fig. 8. Mean AHR (_+1 S.E.M.) shown in response to iwo 10-s tail pinches separated by 5 rain, administered prior to (Predrug), and tbllow- ing (Postdrug) unilateral injections of a combination of 10 nmol of an agonist of [3-adrenoceptors, isoproterenol, and 20 nmol of the D 2 receptor antagonist raclopride into the MFC (A) or AIC (B).

of drug at the baseline prior to the first pinch ( F t..~0 = 8.28, P < 0.05). These injections also increased the magnitude of the response to tail pinch, which is reflected in the significant d rug-sample interaction (F2,~o=7.43, P < 0.05). Analysis of the simple effects of this interaction showed that it was significant during both the first pinch (F2,20 =4 .37 , P < 0.05) and the second pinch (F~2, = 4.21, P < 0 . 0 5 ) . The change in the response to pinch caused by injections of this drug combination into the AIC was characterized by increases in the magnitude of the peak response.

These injections of the combination of an agonist of [3-adrenoceptors, isoproterenol, and a D~ antagonist, raclopride, into either the MFC or AIC appeared to result in larger increases in basal HR than occurred when isoproterenol was injected alone (cf. Fig. 3A,B).

3.3.2. [3-Adrenoceptor antagonist and a D 2 receptor agonist

Injections of a combination of an antagonist of [3-adrenoceptors, propranolol (40 nmol) and an agonist of D~

receptors, quinpirole (20 nmol) significantly reduced basal HR when made into the MFC, as revealed by the test for the main effect of drug (El, 7 = 7.80, P < 0.05) (Fig. 9A). These injections also significantly reduced the magnitude of the response to tail pinch, an effect reflected in the d rug-sample interaction (F2.j4 = 5.72, P < 0.05). The drug-induced changes in the response to pinch were characterized by reductions in the magnitude of the increase in HR caused by pinch, and a more rapid recovery of HR following each pinch,

Injections of propranolol and quinpirole into the AIC strongly decreased basal HR (Fig. 9B). This is reflected in the significant main effect of drug (El, 6 = 16.37, P < 0.01) and in the significant simple effect of drug at the baseline prior to the first pinch (FL36 = 11.77, P < 0 . 0 1 ) . The injection of the combination of propranolol and quinpirole into the AIC appeared to decrease basal HR to a degree greater than was observed following injections of propranolol alone (cf. Fig. 4B).

J]-adrenoceptor Antagonist, Propranolol + D2 Agonist, Quinpirole

50"

40"

C ~ 30"

03 20 -

rr I lO- <

-10-

A MFC Predrug Postdrug

n=8

50

40-

.-- 30-

20-

rr "1- 10" ,,d

-10"

B AIC n=7

Base P e a k Recov B a s e P e a k Recov

Pinch 1 Pinch 2

Fig 9. Mean AHR (_+1 S.E.M.) shown in response to two 10-s tail pinches separated by 5 min, administered prior to (Predrug), and following (Postdrug) unilateral injections of a combination of 20 nmol of the D 2

receptor agonist quinpirole and 40 nmol of an antagonist of 13-adrenoceptors. propranolol, into the MFC (A) or AIC (B).


Injections of this combination of drugs into the AIC also decreased the magnitude of the HR response to tail pinch. Although the drug-sample interaction was not statistically significant, subsequent analyses of the simple effect of the interaction revealed that the injections significantly altered the HR response to the second pinch (F2.z4 = 3.89, P < 0.05), which resulted from a reduction in the magnitude of the peak increase in HR. Injections of this drug combination into the AIC did not appear to result in larger decreases in the magnitude of the pinch response when compared to the case when propranolol was injected alone (cf. Fig. 4B), although HR did tend to recover more rapidly following injections of the combination.

4. Discussion

4.1. Functions of the NAergic innervation of the frontal cortex

The results of this investigation provide the first clear evidence that the release of NA in the frontal cortex exerts an important modulatory influence on the basal and stress- induced output of the ANS. Unilateral injections of an antagonist of [3-adrenoceptors into the MFC or AIC reduced basal HR and the magnitude of the increase in HR induced by tail pinch in rats anesthetized with urethane. Injections of an agonist of [3-adrenoceptors into these regions increased basal HR but did not affect the magnitude of the pinch response. The intracortical injection of drugs acting at ct-adrenoceptors did not significantly affect basal HR or the magnitude of the HR response to pinch.

These results suggest that the release of NA in discrete regions of the frontal cortex has an important facilitatory action in the modulation of autonomic output during basal conditions and when autonomic output is increased in response to noxious or stressful stimuli. These actions of NA appear to be primarily mediated via the stimulation of [3-adrenoceptors. The fact that such large effects were noted with unilateral microinjections of [3-adrenergic drugs serves to underscore the importance of the release of NA in the frontal cortex as a modulator of basal autonomic output and the autonomic activation induced by stressful stimuli.

Agonists and antagonists of [3-adrenoceptors also act in the periphery to produce their cardiovascular effects. With this in mind, it is possible that the changes in HR observed in the present study after the microinjection of isoproterenol or propranolol into the frontal cortex resulted from diffusion of the drugs to peripheral sites. It is known, however, that the doses of these drugs required to elicit significant changes in HR and blood pressure when injected intracerebroventricularly are much higher than those used in the present study [30,32]. This suggests that the effects of these drugs observed in the present study were

mediated by actions on neurons in the central sites of injection. This is further supported by our observation that microinjections of the same dose of isoproterenol that increased basal HR when made into the frontal cortex did not alter HR when injected into the central nucleus of the amygdala (data not shown).

The present findings are in agreement with the results of anatomical and functional studies implicating the projections descending from the frontal cortex in the control of autonomic output. In the rat, the MFC and AIC project directly to a number of regions known to mediate autonomic reflexes, including the NTS, the RVLM, the dorsal motor nucleus of the vagus, the nucleus ambiguus, the parabrachial nucleus and the preganglionic neurons of the sympathetic nervous system in the spinal cord [16,33,41]. The electrical or chemical stimulation of the MFC or AIC has been consistently shown to decrease basal HR and blood pressure by inhibiting sympathetic output through actions on these centers, most importantly the NTS [3,4,13,14,39]. The present results extend these findings in demonstrating that the NAergic pathways innervating the frontal cortex strongly modulate, in turn, the influence of these descending projections on autonomic centers in the brainstem.

4.2. Functions of the DAergic innervation of the frontal cortex

It was found that the DAergic innervation of the frontal cortex, in contrast, plays a less salient role as a modulator of autonomic output. Unilateral intracortical injections of agonists or antagonists of D 1 or D 2 receptors did not significantly affect basal HR or the increases in HR induced by tail pinch. Evidence was obtained, however, suggesting that DA may be a modulator of the autonomic effects of the stimulation of [3-adrenoceptors in the frontal cortex. When an antagonist of D 2 receptors was coinjected with an agonist of [3-adrenoceptors into the AIC, the magnitude of the elevations in HR seen in response to tail pinch was increased, an effect not seen when either drug was injected alone. These results suggest that, in at least the AIC, the stimulation of D 2 receptors acts to modulate, in turn, the [3-adrenergic influence on autonomic output. The operation of this interaction is supported by the observation that injections of a combination of a D 2 agonist and an antagonist of [3-adrenoceptors into the AIC led to decreases in basal HR that were of greater magnitude than those observed when either drug was injected alone. Since noxious or stressful stimuli such as tail pinch evoke large increases in the release of both DA and NA in the frontal cortex, the present results suggest that the influence of this [3-adrenoceptor-D 2 receptor interaction on autonomic output may be especially important during conditions of stress when the occupation of DAergic and NAergic receptors is high.

228 D. Funk. J. Stewart~Brain Researeh 741 (1996) 220-229

4.3. Circuitry underlying catecholaminergic modulation qf autonomic output in the frontal cortex

eptors, leading to the observed increases in the magnitude of the pinch response.

The synaptic mechanisms and circuitry through which injections of drugs acting at [3-adrenergic and D 2 receptors exerted the effects observed in the present experiments are not known. The injected drugs may have affected receptors located on projection neurons, interneurons or on glia in the MFC and AIC [34,35]. Despite this uncertainty, it is clear that the intracortical injections of agonists or antagonists of [3-adrenoceptors strongly influenced the activity of neurons in the MFC and AIC furnishing projections to centers involved in the regulation of autonomic output.

Noxious or stressful stimuli such as tail pinch are known to elicit the firing of neurons in a number of cortical regions, including the MFC [27]. Since the stimulation of the MFC or AIC is known to decrease HR and blood pressure, it may be hypothesized that the function of the stress-induced increases in neuronal firing in the frontal cortex is to dampen the accompanying increases in cardiovascular output elicited by stressful stimuli.

In this light, a mechanism to explain the effects of injections of p-adrenergic drugs on HR noted the present study may be hypothesized. It was observed that intracortical injections of an agonist of [3-adrenoceptors increased basal HR. Since the stimulation of [3-adrenoceptors has been shown to reduce the firing of pyramidal cells in the MFC and other cortical regions [27,43], the injections of an agonist of [3-adrenoceptors may have reduced the activity of projections descending from the MFC and AIC that are known to exert an inhibitory influence on cardiovascular output. Injections of the antagonist of [3-adrenoceptors may have, on the other hand, increased neurotrans- mission in these descending projections, leading to the observed decreases in basal HR. In the case of the decreases in the magnitude of the HR response to tail pinch induced by intracortical injections of an antagonist of p-adrenoceptors, the drug may have blocked the inhibitory effects of endogenous NA on the activity of these descending projections, leading to a net increase in their inhibitory influence on cardiovascular output. Additional support for this proposed mechanism comes from the observation that the local activation of NAergic receptors reduces the firing of MFC neurons evoked by tail pinch [27,42].

The proposed circuitry can also account for the autonomic influences of the p-adrenoceptor-D~ receptor interaction observed in the AIC. A combination of an agonist of [3-adrenoceptors and an antagonist of Do receptors increased the magnitude of the HR response to pinch when injected into this cortical region. The stimulation of Do receptors has been shown to reduce the production of cyclic adenosine monophosphate, the primary intraneu- ronal messenger induced by the stimulation of [3-adrenoceptors [23,37]. Blockade of the D 2 receptors may theretbre have potentiated the inhibition of the projections descending from the AIC caused by the stimulation of [3-adrenoc-

4.4. Conclusion

Taken together, these findings are in agreement with the demonstrated role of the NAergic projections to other areas of the forebrain in the modulation of autonomic and neuroendocrine output. Most notably, the NAergic projections to the paraventricular nucleus of the hypothalamus are known to serve in the activation of cardiovascular and neuroendocrine responses to stress [12,15,19]. The present results extend these findings in suggesting that the release of NA in the frontal cortex induced by stress exerts an important facilitatory influence on the activation of autonomic output elicited by such stimuli.

Acknowledgements

Supported by a grants from the Medical Research Council of Canada and the Fonds pour la Formation de Chercheurs et l'Aide h la Recherche (FCAR, Qu6bec) to Dr. Jane Stewart. This work was conducted as part of a dissertation submitted by Douglas Funk to Concordia Uni- versity.

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