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
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Abstract |
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Introduction |
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Materials and Methods |
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Male Wistar rats weighing 250300 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 2025°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, 1986), 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 (1012 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: acetylcholineHCl (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.
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Results |
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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. 1). 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 1
. The distribution of the injection sites within the cingulate cortex is presented in Figure 2
.
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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. 3). The distribution of the injection sites within the occipital cortex is presented in Figure 3
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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. 4).
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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. 4).
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. 4).
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Discussion |
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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, 1992). Verberne (Verbene, 1996
) 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, 1990
) 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, 1998
) may be selected as likely candidates involved in the autonomic effects of medial prefrontal cortex stimulation.
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Notes |
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Address correspondence to F.M.A. Corrêa, Department of Pharmacology, School of Medicine of Ribeirão Preto, USP, 14049900, Ribeirão Preto, São Paulo, Brazil. Email: fmdacorr{at}fmrp.usp.br.
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