Cardiovascular Response to the Injection of Acetylcholine into the Anterior Cingulate Region of the Medial Prefrontal Cortex of Unanesthetized Rats

G.E. Crippa, V.L. Peres-Polon1, R.H. Kuboyama1 and F.M.A. Corrêa

Department of Pharmacology, School of Medicine of Ribeirão Preto, USP, 14049-900 Ribeirão Preto and , 1 Department of Physiology, School of Dentistry of Ribeirão Preto, USP, 14049-903 Ribeirão Preto, São Paulo, Brazil


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Injection of acetylcholine (ACh) (2.5–60 nmol) into the anterior cingulate cortex caused dose-dependent hypotensive responses (Emax = –25.3 mmHg) and no change in the heart rate. The hypotensive response to 30 nmol of ACh was blocked by local pretreatment with atropine (3 nmol) or 4-DAMP (6.7 nmol), a non-tropine muscarinic antagonist. When the same dose of atropine was injected i.v., no changes were observed in the hypotensive response to intracortical ACh. This observation rules out the possible leakage of ACh into the peripheral circulation and favors the idea of a cortical site of action. The injection of the same dose of ACh into the corpus callosum or the occipital cortex did not cause changes in the cardiovascular system. The present results confirm earlier evidence that the cingulate cortex is involved in the control of the autonomic system and indicate that cholinergic muscarinic receptors in the cingulate cortex mediate a hypotensive response without a change in heart rate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is evidence that the cingulate region of the medial prefrontal cortex is involved in the control of the cardiovascular system (Cecheto and Saper, 1990; GoVerberne and Owens, 1998Go). Pressor as well as depressor responses were reported after electrical stimulation of the cingulate cortex (Kaada, 1960Go; Burns and Wyss, 1985Go; Hardy and Holmes, 1988Go). The electrical stimulation of the cingulate cortex elicits blood pressure changes in both anesthetized and awake rats (Burns and Wyss, 1985Go; Hardy and Holmes, 1988Go; Verberne, 1996Go). Similar responses were reported when neuronal cell bodies were stimulated using glutamate (Verberne, 1996Go) or the glutamate agonist D,L-homocysteic acid (Fisk and Wyss, 1997Go), indicating that cortical neurons, and not passing fibers, are capable of influencing the circulation. Acetylcholine (ACh) is abundant in the central nervous system (CNS), including the cortex (Woolf, 1991Go). Central cholinergic mechanisms have been proposed to be involved in cardiovascular control (Buccafusco, 1996; Kubo, 1998Go). Pressor responses were reported after the injection of ACh into the septal area (Peres-Polon and Corrêa, 1994Go). Hypotensive responses and bradycardia were reported after the injection of ACh into the nucleus tractus solitarii (Sundaram et al., 1989Go). Immunohistochemistry studies have demonstrated the presence of cholinergic neurons in the cerebral cortex as well as important cholinergic projections from the hypothalamic preoptic region to the cingulate cortex in the rat (Woolf, 1991Go). In addition, cholinergic binding sites are present throughout cortical areas (Fibiger 1982Go). However, there are no reports on the possible cardiovascular effects of ACh injection into the rat cortex. In the present experiment we studied the cardiovascular effect of the injection of ACh into the cingulate cortex of unanesthetized rats.


    Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Animal Preparation

Male Wistar rats weighing 250–300 g were used (n = 56). Animals were kept in the Animal Care Unit of the Department of Pharmacology of the School of Medicine of Ribeirão Preto, University of São Paulo. Rats were housed individually in plastic cages, in a room at 20–25°C and kept on a 12 h light/dark cycle with free access to water and commercial food.

Rats were anesthetized with tribromoethanol, 250 mg/kg, i.p. After local anesthesia with 2% xylocaine, the skull was surgically exposed and a stainless steel guide cannula (0.7 mm o.d.) was implanted 1 mm above the injection site using a stereotaxic apparatus (Stoelting, USA). Stereotaxic coordinates for cannula implantation into the cingulate cortex were selected from the brain atlas of Paxinos and Watson (Paxinos and Watson, 1986Go), AP = +1.2 mm; L = 0.5 mm from the medial suture and V = –2.7 mm from the skull. Coordinates for cannula implantation into the occipital cortex were AP = –6.3 mm from the bregma, L = 6.0 mm from the medial suture, V = –2.5 mm from the skull. Cannulas were fixed to the skull with dental cement and two metal screws. A tight-fitting mandril was kept inside the guide cannula to avoid its occlusion. After surgery animals were treated with 100 000 units of benzyl penicillin. Three days thereafter, animals were anesthetized with tribromoethanol and a polyethylene catheter was implanted into the femoral artery for blood pressure recording. The arterial catheter consisted of a segment of PE-10 tubing (4.5 cm) heat-bonded to a longer segment of PE-50 tubing (10–12 cm). The catheter was filled with 0.3% heparin (5000 U/ml) in sterile saline (150 mM NaCl). The PE-10 segment was introduced into the femoral artery until the tip reached the aorta. The catheter was secured in position with thread and the PE-50 segment was passed under the skin to be extruded on the dorsum of the animals. After surgery, the animals were allowed to recover for 24 h. Whenever the i.v. route was used for drug injection, another similar catheter was simultaneously inserted into the femoral vein.

Measurement of Cardiovascular Responses

During the experiment the animals were kept in individual cages and the mean arterial blood pressure of conscious, freely moving rats was recorded using an HP-7754A polygraph (Hewlett Packard, USA) at a paper recording speed of 0.25 mm/s. Blood pressure baseline values were calculated as the average of the 3 min recording prior to the injection. Peak responses were calculated on the basis of the average mean blood pressure recordings obtained at the response plateau. Whenever heart rate (b.p.m.) measurements were made, the data were derived from pulse counting at a paper recording speed of 5 mm/s.

Drug Injections

Drugs were dissolved in sterile saline. The different drug concentrations were calculated as free base. No changes in pH were observed when compared to saline alone. A 10 µl syringe (model 705-N, Hamilton Co., USA) and a stainless steel (30 gauge) dental injection needle were used for drug injection into the cortex. Drugs were dissolved in a final volume of 0.5 µl and injected over a period of 30 s. Whenever a smaller volume (50 nl) was injected, a 1 µl syringe (model 7001-KH, Hamilton) was used. One minute was allowed to elapse before the injection needle was removed from the guide cannula to avoid reflux. Control saline injections were performed 30 s before the injection of the first dose of ACh in all animals. After pretreatment with the antagonists, an interval of 20 min was allowed to elapse before the second injection of ACh. For i.v. injections, drugs were dissolved in saline and injected in a volume of 0.1 ml/100 g body wt.

Drugs

The following drugs were used: acetylcholine–HCl (Sigma), atropine– HCl (Sigma) and 4-diphenylacetoxy-N-methylpiperedine methiodide (4-DAMP, RBI).

Experimental Protocols

The first group of animals received injections of increasing doses of ACh (2.5, 5, 10, 20, 30 and 60 nmol, n = 6) into the cingulate cortex, diluted in a volume of 0.5 µl. Each dose was randomly administered at 4 h intervals to avoid tachyphylaxis.

The second group of animals received injections of ACh (30 nmol/ 0.5 µl) into the occipital cortex.

The third group of rats was used to evaluate the involvement of muscarinic receptors in the cardiovascular response to the injection of ACh into the cingulate cortex. Two muscarinic antagonists — atropine (3 nmol) and 4-DAMP (6.7 nmol) — were locally injected in a volume of 0.5 µl 20 min prior to the injection of 30 nmol of ACh.

In the fourth part of the study we determined whether the cardiovascular response was due to a central effect. One group of rats received i.v. the same dose of atropine injected into the cingulate cortex (3 nmol) 20 min prior to the intracortical injection of ACh. Another group of rats was injected with atropine into the cingulate cortex 20 min prior to i.v. injection of an equipotent dose of ACh (1.2 µg/kg) that caused hypotensive responses of a magnitude similar to that of the response to 30 nmol of ACh injected into the cortex.

Histological Procedure

At the end of the experiments, the rats were anesthetized with pentobarbital (40 mg/kg) and 0.5 µl of filtered 1% Evan's blue dye was injected into the brain as a marker of the injection site. The chest was surgically opened, the descending aorta occluded, the right atrium severed and the brain perfused with 10% formalin through the left ventricle. The brains were postfixed for 24 h at 4°C, and 40 µm sections were cut with a cryostat (model 950-C, Reichert, USA). Brain sections were stained with 1% neutral red. The actual placement of the injection needles was verified in serial sections.

Statistical Analysis

When required, statistical analysis was performed using nonlinear regression analysis (GraphPad, USA) or Student's two-tailed t-test, with the level of significance set at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Blood Pressure Response to ACh Injected into the Cingulate Cortex

The injection of ACh into the cingulate cortex caused doserelated blood pressure decreases. Responses to the intracortical injections of ACh were only observed at doses >5 nmol/0.5 µl and the maximal response observed was –25.3 ± 3.3 mmHg (n = 6) with 60 nmol of ACh (Fig. 1Go). The injection of 30 nmol ACh dissolved in a small volume (50 nl) caused hypotensive responses similar to those observed when the compound was dissolved in 0.5 µl, although of lower magnitude (–14.7 ± 2.5 and –23.3 ± 2.7 mmHg respectively). A blood pressure recording showing the pattern of the hypotensive response to intracortical injection of ACh is presented in Figure 1Go. The distribution of the injection sites within the cingulate cortex is presented in Figure 2Go.



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Figure 1.  (Top) Dose–effect curve for the hypotensive response to the injection of acetylcholine (ACh) into the medial prefrontal cortex. Doses of ACh were 2.5, 5, 10, 20, 30, 60 nmol/0.5 µl and S = saline (n = 6). Circles represents means and bars the SEM. The regression was statistically significant, P < 0.05, as determined by nonlinear regression analysis, r2 = 0.86, df = 36. (Bottom) Blood pressure recording (BP) showing the hypotensive response to the injection of 30 nmol/0.5 µl of ACh into the cingulate cortex (i.c.) of an anesthetized rat before or after the local pretreatment with 3 nmol/0.5 µl of atropine (ATR).

 


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Figure 2.  Diagrammatic representation of the sites of injection of acetylcholine (ACh) into the cingulate cortex. The injection sites related to the hypotensive response to ACh are represented by filled circles, whereas unresponsive sites are represented by open circles. Coordinates modified from Paxinos and Watson (Paxinos and Watson, 1986).

 
No significant heart rate changes were observed after the injection of 30 nmol of ACh into the cingulate cortex (heart rate prior to ACh = 342 ± 11 b.p.m. and after ACh = 343 ± 6 b.p.m., n = 6), although a –23.3 ± 2.7 mmHg decrease in mean blood pressure was simultaneously recorded.

No change in blood pressure was observed after ACh injection into the corpus callosum.

Blood Pressure Response to ACh Injected into the Occipital Cortex

The injection of ACh into the occipital cortex had no effect on mean blood pressure (MAP before = 101.3 ± 3.7 mmHg and MAP after = 100.3 ± 3.5 mmHg) or heart rate (Fig. 3Go). The distribution of the injection sites within the occipital cortex is presented in Figure 3Go.



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Figure 3.  Lack of effect of the injection of acetylcholine (30 nmol/0.5 µl, n = 4) into the occipital cortex on mean arterial pressure in awake rats, and diagrammatic representation of the sites of injection of acetylcholine (ACh) into the occipital cortex. Coordinates modified from Paxinos and Watson (Paxinos and Watson, 1986).

 
Effect of Local Pretreatment with Saline, Atropine or 4-DAMP on the Blood Pressure Response to the Injection of ACh into the Cingulate Cortex

Saline locally injected into the cingulate cortex caused no change in basal mean arterial pressure (103 ± 1.2 mmHg, n = 4), and no tachyphylaxis was observed for the hypotensive response to the intracortical injection of ACh (ACh response before = –22.7 ± 4 mmHg and after = –18.7 ± 0.7 mmHg, the treatment with saline, n = 4, Fig. 4Go).



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Figure 4.  Effect of the injection of 30 nmol/0.5 µl of acetylcholine into the cingulate cortex on mean arterial blood pressure ({Delta} blood pressure) before (open column) and after (hatched column) local pretreatment with saline (n = 4), atropine (3 nmol.0.5 µl, n = 15) or 4-DAMP (6.7 nmol/0.5 µl, n = 6) or atropine i.v. (2 nmol/0.5 µl, n = 4). Columns represent the mean ( SEM. *P < 0.05, Student's t-test.

 
The intracortical injection of 3 nmol of atropine caused no change in baseline mean arterial pressure per se (102 ± 2 mmHg, n = 15) and blocked the hypotensive response to the intracortical injection of 30 nmol ACh (hypotensive response to ACh before atropine = –27.5  3.3 and after atropine = –4.8 ± 2 mmHg, n = 15) (Figs 1, 4Go).

The intracortical injection of 6.7 nmol of 4-DAMP caused no change in baseline mean arterial pressure per se (105  3.2 mmHg) and blocked the hypotensive response to the intracortical injection of 30 nmol ACh (hypotensive response to ACh before 4-DAMP = –24.8 2.4 and after 4-DAMP= –6  3.7 mmHg, n = 6) (Fig. 4Go).

Effect of Systemic Pretreatment (i.v.) with Atropine on the Blood Pressure Response to ACh Injected i.v. or into the Cingulate Cortex

The i.v. injection of the same dose of atropine (3 nmol) caused no change in baseline mean arterial pressure (102 ± 2 mmHg, n = 4).

The i.v. pretreatment with atropine did not affect the hypotensive response to the intracortical injection of 30 nmol of ACh (ACh response before = –38.2 ± 5 mmHg and after = –30.2 ± 3.6 mmHg, n = 4, the i.v. pretreatment with atropine) (Fig. 4Go).


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study we report dose-dependent hypotensive responses and no significant changes in heart rate after the injection of ACh into the anterior cingulate cortex. Histological analyses indicated a predominant placement of the injection needles at an area corresponding to the anterior cingulate cortex according to the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986Go). Previous studies indicated that electrical stimulation of this area causes depressor responses accompanied by little or no effect on heart rate (Burns and Wyss, 1985Go; Hardy and Holmes, 1988Go). Chemical stimulation of the cingulate cortex with the glutamate agonist D,L-homocysteic acid caused changes in blood pressure and heart rate in conscious or anesthetized rats (Fisk and Wyss, 1997Go). Although hypotensive responses to ACh were presently observed after injections of small volumes (50 nl), larger volumes (0.5 µl) were more effective in producing the hypotensive response. A similar pattern was reported after injection of D,L-homocysteic acid into the cingulate cortex (Fisk and Wyss, 1997Go), indicating that larger populations of neurons may be activated by the larger injection volume. There were no effects when the cannulas were mistakenly placed in the corpus callosum or implanted into the occipital cortex, further suggesting a specific effect of ACh injected into the anterior cingulate cortex. Many studies have suggested that the medial prefrontal cortex participates in the adjustment of autonomic responses (Burns and Wyss, 1985Go; Hardy and Holmes, 1988Go; Neafsey, 1990Go; Verberne, 1996Go). The depressor responses observed after the injection of ACh were similar to those reported after the electrical stimulation of the cingulate cortex in anesthetized rats (Burns and Wyss, 1985Go; Hardy and Holmes, 1988Go).

The hypotensive response to ACh was blocked by local pretreatment with muscarinic antagonists. Atropine or 4-DAMP were effective in blocking the hypotensive response, suggesting the involvement of muscarinic receptors in the cingulate cortex. Agonists and other specific antagonists should be used in future studies to identify the subtype of muscarinic receptor involved in the hypotensive response to ACh injection into the cortex.

Since ACh is a potent vasodilator, there is a possibility that the hypotensive effects observed after its intracortical injection could be due to the spreading of the drug from its injection site to the systemic circulation. The idea that ACh has a local effect at the cortex is favored by the observation that the hypotensive effect of 30 nmol of ACh was completely abolished by local pretreatment with atropine used at 1/10 molar ratio (3 nmol). Even more relevant is the fact that the i.v. injection of the dose of 3 nmol of atropine did not affect the hypotensive response to ACh injected into the cingulate cortex, confirming that the hypotensive response to the intracortical injection of ACh is not caused by a leakage into the systemic circulation. Additionally, bradycardia is concomitantly observed when ACh is injected i.v., whereas no heart rate changes were observed after the injection of ACh into the cingulate cortex. These observations suggest a direct action of ACh in the cortex.

The observation that the injection of atropine or 4-DAMP into the cingulate cortex failed to cause changes in blood pressure by itself indicates the absence of a tonic cholinergic modulation of blood pressure control in the unanesthetized rat.

The present results reveal a pharmacological effect of ACh in the anterior cingulate cortex, with important reflexes on cardiovascular control. However, the precise nature of this cholinergic system cannot be presently established.

The fact that the hypotensive response to intracortical ACh was neither accompanied by bradycardia nor by reflex tachycardia indicates a possible inhibition of the baroreceptor reflex. However, Verberne et al. (1987) observed that electrical stimulation of the medial prefrontal cortex exerts facilitatory influence on the baroreceptor reflex.

Although there is no clear evidence about the mechanism involved in the hypotensive response to the injection of ACh into the cingulate cortex, this mechanism may be mediated by inhibition of the sympathetic nervous system. Electrophysiological studies demonstrated that the hypotensive response caused by electrical stimulation of the lateral prefrontal cortex is mediated by inhibition of vasomotor neurons in the rostroventrolateral medulla (Sun, 1992Go). Verberne (Verbene, 1996Go) demonstrated that depressor responses evoked by stimulation of the medial prefrontal cortex are accompanied by sympathoinhibitory responses recorded from the splanchnic or lumbar sympathetic nerve trunks. The pathway involved in cortically evoked circulatory responses is still unclear. Hardy and Mack (Hardy and Mack, 1990Go) observed that the injection of lidocaine into the hypothalamus reduced the hypotensive response and bradycardia caused by electrical stimulation of the lateral prefrontal cortex. The possibility that lidoicaine could be also acting on passing fibers cannot be excluded. However, the medial prefrontal cortex projects to a diverse range of cortical and subcortical structures, some of which — among others, the insular cortex, lateral hypothalamus, amygdala, solitary tract nucleus, periaqueductal gray area and rostral ventrolateral medulla (Verberne and Owens, 1998Go) — may be selected as likely candidates involved in the autonomic effects of medial prefrontal cortex stimulation.


    Notes
 
The authors thank I.I.B. Aguiar, I.A.C. Fortunato, S.S. Guilhaume and E. Greene for respectively surgical, histology, graphics and editorial support. G.E.C. is the recipient of a Ph.D. fellowship from FAPESP (95/4351–2). The present research was supported by grants from FAPESP (96/4305–3) and CNPq (AI-52245395–3).

Address correspondence to F.M.A. Corrêa, Department of Pharmacology, School of Medicine of Ribeirão Preto, USP, 14049–900, Ribeirão Preto, São Paulo, Brazil. Email: fmdacorr{at}fmrp.usp.br.


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