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NEUROPHARMACOLOGY

The Importance of Brainstem Cholinergic Neurons in the Pressor Response to Cocaine

Alzheimer's Research Center, Departments of Pharmacology and Toxicology (J.J.B., L.C.S., C.J.B., M.G.) and Neurosurgery (J.A.D.), Medical College of Georgia, Augusta, Georgia; and Veterans Affairs Medical Center (J.J.B., L.C.S.), Augusta, Georgia

Received July 2, 2004; accepted August 18, 2004.

	Abstract

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After intracisternal injection, 140 nmol (48 µg) of cocaine (but not lidocaine or procaine) evoked an increase in mean arterial pressure (MAP) of 41 mm Hg. The increase in MAP began within 1 min after injection and lasted 10 to 15 min. The pressor response to intracisternal injection of cocaine was not mediated through central -adrenergic receptors, but intracisternal pretreatment with D1 or D2 dopamine receptor antagonists shortened the duration of the response. Pretreatment with intracisternal injection of hemicholinium-3 to deplete medullary acetylcholine produced a dose-dependent inhibition of the pressor and tachycardic responses to intracisternal injection of cocaine. Central pretreatment with hemicholinium-3 also inhibited the pressor response to intravenous injection of 0.5 mg/kg cocaine. Atropine pretreatment was only partly effective in blocking the pressor and tachycardic responses to intracisternal injection of cocaine. However, a single intracisternal injection of the nicotinic ganglionic receptor blocker hexamethonium inhibited the pressor response to cocaine administered intracisternally 24 h later, and on each of the following 4 days. The blocking effect of hexamethonium was not mimicked by the 7 selective antagonist methyllycaconitine or by the 42 subtype-preferring antagonist dihydro--erythroidine. The data suggest that the pressor response to cocaine is mediated by medullary acetylcholine release on to nicotinic receptors of the ganglionic type, enhancing the output of bulbospinal sympathetic premotor neurons. Our results provide new evidence for the prolonged inactivation of relevant central nicotinic receptors by nicotinic receptor antagonists, and suggest that such compounds might be used safely for cocaine overdose, as well as for antiabuse issues without the concern for autonomic side effects.

In addition to the well known central biogenic amine pathways involved in cardiovascular regulation, cholinergic neurons in both rostral (posterior hypothalamic) and caudal (medullary) centers of the brain mediate excitatory sympathetic tone over descending vasomotor pathways. This phenomenon can be readily demonstrated by the marked pressor responses observed in several animal species, including humans, after the administration of centrally-acting cholinergic (muscarinic) agonists (for review, see Brezenoff and Giuliano, 1982; Buccafusco and Brezenoff, 1986; Buccafusco, 1992, 1996). Central cholinergic neurons also have been reported to participate in mediating the pressor action to central administration of nicotine (Buccafusco and Yang, 1993), and certain noncholinergic neurotransmitter substances such as angiotensin II, substance P, vasopressin, and bradykinin (Buccafusco and Serra, 1985; Trimarchi et al., 1986). These substances have been shown to provoke the release of acetylcholine from neuronal stores, with the neurotransmitter subsequently acting upon muscarinic receptors mediating a sympathoexcitatory response.

Cocaine also possesses the ability to induce the release of acetylcholine in cerebral cortex (Day et al., 1997), striatum (Zocchi and Pert, 1994), nucleus accumbens (Mark et al., 1999), and hippocampus (Imperato et al., 1992). These stimulatory effects of cocaine on cholinergic function may be mediated through facilitation of D1 dopaminergic pathways (Imperato et al., 1992, 1993). Cocaine, like acetylcholine, is an ester that is hydrolyzed by acetylcholinesterase, and cocaine has been reported to interact with muscarinic (Sharkey et al., 1988; Tan and Costa, 1994) and with nicotinic receptors (Niu et al., 1995), although the interaction is one of an antagonist. Moreover, antimuscarinic agents have been reported to reduce cocaine-induced lethality in mice (Ritz and George, 1997) and to prevent the chronic sensitization to cocaine's locomotor-activating effects (Heidbreder and Shippenberg, 1996).

The purpose of this study was to determine whether cocaine could produce a reliable and dose-dependent pressor response in unanesthetized and freely moving rats. We also sought to determine, by pharmacological and neurochemical means, the role of central cholinergic neurons in mediating the pressor response to central administration of cocaine.

	Materials and Methods

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Animal Subjects. Male Wistar Rats, weighing 350 to 450 g, were obtained from Harlan (Indianapolis, IN). The animals were housed in our animal care facilities for at least 5 days before experiments. Standard rat chow and tap water were supplied on an unlimited basis. A 12-h light/dark cycle was maintained. Procedures involving animals were approved by the Institutional Committee for Animal Use in Research and Education.

Preparation for the Central Administration of Drug Solutions. For lateral cerebroventricular (l.c.v.) injection, rats were anesthetized with methohexital (Brevital; 65 mg/kg i.p.) and placed in a flat skull orientation in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA). A small hole was drilled in the skull 0.4 mm posterior and 2.5 mm lateral to bregma. A cannula guide constructed from 23-gauge stainless steel tubing was inserted 3 mm below the surface of the skull and fixed at the base with acrylic cement, which anchored the guide to three small screws placed nearby. The cannula guide was protected by a screw-on cap. For intracisternal injection, rats were anesthetized and the animal's head was positioned in a stereotaxic frame with the skull in a horizontal position. A burr hole (about 1 mm) was placed at the sagital midline approximately 0.5 mm rostral to the suture between the intraparietal and supraoccipital bones. A loose knot was placed in a length of PE-10 tubing 6 mm from the distal end. The distal end of the tubing was inserted by hand until the knot in the tubing was flush with the surface of the skull. This allowed the tip of the catheter to reside in the intracisternal space between the cerebellum and the dura mater juxtaposed to the occipital bone. The proximal end of the tubing was stabilized using acrylic cement to a stainless steel screw. The sterile saline-filled tubing was plugged and tunneled subcutaneously to emerge for about 2 cm at the nape of the neck. For intrathecal injection, rats were anesthetized and the animal's head positioned in a stereotaxic frame. A small incision was made over the caudal-medial skull into which a screw was introduced. A 3-mm nick was made in the atlantooccipital membrane, and a saline-filled catheter (PE-10) was introduced into the subarachnoid space 5.5 cm from the nick to terminate at the T7-T8 region of the spinal cord. The catheter was passed subcutaneously and looped around the skull screw where it was fixed in place with acrylic cement.

Installation of a Chronic Intra-Arterial Catheter. Five days after implanting one of the ventricular cannulas, rats were anesthetized, and a catheter (PE-50) filled with heparinized (30 units/ml) saline was inserted into the iliac artery so that the tip of the catheter terminated in the base of the abdominal aorta below the origin of the renal arteries. The opposite end of the catheter was directed subcutaneously to emerge at the back of the neck where it was stabilized to an anchoring button. After surgery, the animals were returned to plastic cages (45 x 25 x 20 cm), and the catheter was passed through a spring support, and it was connected to a watertight swivel cannula that was mounted 30 cm above the cage floor. A constant infusion (8 ml/day) of heparinized saline maintained the patency of the catheter. In a few cases, an i.v. line filled with heparinized saline was inserted into the left jugular vein sutured in place and exteriorized at the back of the neck. Just before the experiment, the exteriorized araterial catheter was connected to a pressure transducer, and the analog signals were amplified and digitized on a Buxco Electronics LS-14 logging analyzer. Stable levels of mean arterial pressure (MAP) and heart rate (HR) were recorded for at least 15 min before the start of the experiment. The analyzer provided 10-s cumulative averages of MAP and HR over the first 5 min after test drug injection. Thereafter, cumulative averages of these parameters were recorded every 30 s until 15 min after injection. Between 15 and 30 min after injection, cumulative averages were recorded every 5 min.

Central Administration of Drug Solutions in Unrestrained Rats. Animals were allowed to recover from the surgical procedure for installing the chronic arterial line for at least 24 h before use in an experimental protocol. For l.c.v. injections, a 28-gauge stainless steel cannula was connected via PE tubing to a microsyringe pump. The cap and plug were removed from the guide cannula, and the injection cannula (length 12.5 mm, which is 4.5 mm below skull) was inserted through the guide so that the tip rested in the ventricular space. Drugs were dissolved in 5 µl of sterile saline and infused over a 20-s period. The cannula was left in place for at least 2 min after injection to prevent drug from escaping up the guide. After the cannula was removed, the pin and screw cap was replaced. For intrathecal or intracisternal injection, the microsyringe was connected to the distal end of the catheter, and drug solutions were administered followed by an additional 5 µl of saline to clear the contents of the catheter.

Brain Cholinesterase Assay. Rats were sacrificed by decapitation and brains were removed, frozen in liquid nitrogen, and stored at –70°C until assayed. Brains were removed and assayed spectrophotometrically in phosphate buffer at pH 7.9 according to previously published methods (Buccafusco and Smith, 1990). Briefly, brain tissues were dissected and homogenized [25% (w/v) in buffer] for 1 min, and homogenate was then centrifuged at 40,000g for 30 min at 4°C. The reaction mixture included supernatant (100 µl), 7.5 µM acetylthiocholine iodide substrate, and 10 mM dithiobisnitrobenzoic acid. Absorbance at 412 nm was recorded at 25°C for 4 min. Enzyme velocities were expressed as micromoles of substrate hydrolyzed per minute per milligram of protein, and the percentage of inhibition of enzyme activity test compound (relative to control levels) was determined.

Receptor Binding. Animals were sacrificed by decapitation, and the brains were quickly removed and placed on ice. Brain tissue was homogenized (Teflon glass) in 50 mM Tris-HCl buffer, pH 7.0. The tissue homogenate was centrifuged at 49,000g for 20 min at 4°C. The membranes were washed twice by centrifugation and then resuspended in fresh buffer. Next, they were resuspended in fresh Tris/HCl buffer to a concentration representing 0.25 mg of protein per milliliter. Homogenate aliquots were incubated for 90 min with 5 nM [³H]N-methylscopolamine (PerkinElmer Life and Analytical Sciences, Boston, MA) in Tris/HCl buffer, in a final volume 0.25 ml and with concentrations of cocaine or carbachol ranging from 0.032 nM to 1 mM. Atropine sulfate (10 µM) was used to control for nonspecific binding. Separation of bound from free ligand was accomplished by rapid filtration through a Millipore 96-well (type B) filter plate, and each filter was punched into scintillation fluid and the radioactivity was quantified in a liquid scintillation counter. The fractional specific binding of [³H]N-methylscopolamine in the presence of unlabeled ligand was analyzed using a nonlinear curve-fitting program (Table Curve; SPSS Inc., Chicago, IL) that fits the data to either one or two classes of binding sites.

Statistics. All averaged data sets are expressed as the mean ± S.E.M. Cardiovascular data were analyzed by using a two-way analysis of variance with a repeated measures design. An orthogonal t test used for post hoc analyses. Differences between means were considered significant at the P < 0.05 level. For receptor binding studies, the fractional specific binding of ³H ligand in the presence of unlabeled ligand was analyzed using a nonlinear curve fitting program (Table Curve; SPSS Inc.).

Drugs. Cocaine hydrochloride, atropine methylbromide, dihydro--erythroidine hydrobromide, hemicholinium-3 (HC-3) bromide, hexamethonium bromide, methyllycaconitine, SCH-23390 [(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride], S-(–)-eticlopride hydrochloride, mecamylamine hydrochloride, tolazoline hydrochloride, lidocaine hydrochloride, and procaine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). All drugs were dissolved in sterile saline on the day of administration. [³H]N-methylscopolamine was purchased from PerkinElmer Life and Analytical Sciences.

	Results

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Cocaine (70, 140, and 280 nmol) was administered directly into the cerebrospinal fluid of freely moving rats through one of three routes: into the left lateral cerebral ventricle (l.c.v.), into the cisterna magna (intracisternal), and into the spinal intrathecal space (intrathecal). Arterial blood pressure and heart rate were measured over a 30-min observation period. The data for mean arterial pressure (MAP) are presented in Fig. 1. Cocaine induced a rapid, dose-dependent increase in MAP, beginning within the first minute after injection via the intracisternal route. The response peaked at 2 min (increase of 41 mm Hg) and returned to preinjection levels by 15 to 20 min after injection. Injection of cocaine via the l.c.v. route also resulted in a dose-dependent increase in MAP, although the responses were delayed in onset, reduced in magnitude (24 mm Hg), and shorter in duration compared with intracisternal administration. Injection of cocaine into the intrathecal space did not significantly increase MAP. Likewise, i.v. administration of the highest (280 nmol) dose of cocaine (Fig. 1, inset) resulted only in a brief, approximately 10 mm Hg increase in MAP. Therefore, the responses to central administration of cocaine could not be explained by redistribution of the drug from the cerebral spinal fluid to the peripheral circulation. The data for heart rate are presented in Fig. 2. For all routes of administration, the changes in heart rate to cocaine were more variable and less obviously dose-dependent than were the pressor responses. For l.c.v. and intracisternal injection, the heart rate response to cocaine was biphasic for most animals. The response to intracisternal injection of the 140-nmol dose (Fig. 3) was often characterized by an increase that was initially concomitant with the increase in MAP. However, within 5 to10 min, heart rate generally returned to baseline levels, and in most cases continued to decrease below preinjection levels for at least an additional 10 min. Although the magnitudes of the responses were similar for the intracisternal and l.c.v. routes, the duration of the tachycardic response to cocaine was greater after intracisternal injection. Intrathecal injection of cocaine produced a brief increase in heart rate with no apparent subsequent decrease. Intravenous administration of the 280-nmol dose (Fig. 2, inset) produced no immediate effect, but there seemed to be a delayed increase in heart rate 25 to 30 min after injection. The prolonged tachycardic response to a higher i.v. dose of cocaine (0.5 mg/kg equivalent to about 590 nmol) is also depicted in Fig. 8. From these studies, we concluded that the pressor response to central administration of cocaine was best evoked through the intracisternal route and so most of the remaining experiments were performed by administering all drugs in this manner. Also, the 140-nmol (48-µg) dose of cocaine was used as the standard test dose for experiments in which potential blocking drugs were used. It should be noted that central administration of cocaine typically elicited mild and transient increase in activity occasionally with ataxia over about the first 5 min after injection. Thereafter, rats tended to lie quietly, sometimes splayed in position through the end of the observation period. Although this behavior was not quantified, the initial active stage and the secondary inactive stage tended to occur concomitantly with the initial increase and subsequent decrease in heart rate to cocaine.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Effect on MAP produced after injection of cocaine directly into the cerebrospinal fluid of freely moving rats. Cocaine (70, 140, or 280 nmol) was administered at time 0 by microinjection. Each value represents the mean ± S.E.M. derived from six to seven subjects. LCV, lateral cerebroventricular; IT, intrathecal; IC, intracisternal. Inset, pressor response to i.v. bolus injection of 280 nmol of cocaine (N = 5).

View larger version (26K): [in this window] [in a new window]   Fig. 2. HR produced after injection of cocaine directly into the cerebrospinal fluid of freely moving rats. Cocaine (70, 140, or 280 nmol) was administered at time 0 by microinjection. Each value represents the mean ± S.E.M. derived from six to seven subjects. LCV, lateral cerebroventricular; IT, intrathecal; IC, intracisternal. Inset, pressor response to i.v. bolus injection of 280 nmol of cocaine (N = 5).

View larger version (31K): [in this window] [in a new window]   Fig. 3. Change in MAP (top) and HR (bottom) produced by intracisternal injection of 140 nmol of cocaine at time 0. Average resting MAP and HR and the numbers of experiments for each drug are presented in Table 1. Cocaine's effect on MAP and HR was significantly different (P < 0.0001 and P = 0.043, respectively) from those to procaine and lidocaine.

View larger version (33K): [in this window] [in a new window]   Fig. 8. Top, change in HR produced by intracisternal injection of 140 nmol of cocaine at time 0 in animals pretreated by intracisternal injection 60 min earlier with vehicle or with 3.5 or with 35 nmol of HC-3. Average resting HR, and the numbers of experiments for each dose are presented in Table 1. Hemicholinium-3 pretreatment inhibited the magnitude of the initial tachycardic response to cocaine, but it seemed to accentuate the secondary bradycardic response (P = 0.0007). Bottom, change in HR produced by i.v. injection of cocaine (0.5 mg/kg) at time 0 in animals pretreated by intracisternal injection 60 min earlier with vehicle or with 35 nmol of hemicholinium-3. In hemicholinium-3-pretreated rats, the tachycardic response to i.v. cocaine over the first 5 min after injection was undiminished, but the response was not maintained. The differences between the two data sets were nearly significantly different (P = 0.063).

Comparison with Local Anesthetics. Because of its known local anesthetic properties the cardiovascular effects of intracisternally administered cocaine were compared with those to either procaine or lidocaine to ascertain the role of this property in the responses to cocaine. The data are presented in Fig. 3. Note the baseline levels of MAP and heart rate for these and the other agents used in Figs. 3, 4, 5, 7, 8, and 10 are provided in Table 1.) Of the three agents, only cocaine evoked a significant pressor response with the effects significantly different from both procaine and lidocaine. The differences were dependent on time after injection, F(60,867) = 2.21, P < 0.0001. The effects of procaine and lidocaine also differed with respect to heart rate, F(60,867) = 1.35, P = 0.043. Again, only cocaine evoked the initial tachycardic response; whereas the other two agents induced a decrease in heart rate soon after injection that tended to return toward baseline levels within about 5 to 7 min. Thereafter, the responses were variable but generally lower on average than baseline and cocaine-induced levels. Thus, whereas it is unlikely that local anesthetic action plays a role in the pressor and initial tachycardic responses to cocaine, it is not possible to rule out a contribution of this property to the secondary decrease in heart rate to the drug.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Change in MAP produced by intracisternal injection of 140 nmol of cocaine at time 0 in animals pretreated by intracisternal injection 15 min earlier with vehicle, 280 nmol of tolazoline, 140 nmol of SCH-23390, or 140 nmol of eticlopride. Average resting MAP and the numbers of experiments for each drug are presented in Table 1. Pretreatment with tolazoline was ineffective in blocking the pressor response to cocaine (P = 0.28). Pretreatment with SCH-23390 did not alter the magnitude of the pressor response to cocaine, but it significantly shortened the duration of the response (P < 0.0001). Pretreatment with eticlopride did not alter the magnitude of the pressor response to cocaine, but it significantly shortened the duration of the response (P < 0.0001).

View larger version (34K): [in this window] [in a new window]   Fig. 5. The change in HR produced by intracisternal injection of 140 nmol of cocaine at time 0 in animals pretreated by intracisternal injection 15 min earlier with vehicle, 280 nmol of tolazoline, 140 nmol of SCH-23390, or 140 nmol of eticlopride. Average resting HR and the numbers of experiments for each drug are presented in Table 1. Pretreatment with tolazoline resulted in a nearly significant inhibition of the secondary fall in heart rate to cocaine (P = 0.058). Pretreatment with SCH-23390 inhibited the initial tachycardic response to cocaine and accentuated the secondary bradycardic response (P < 0.0001). Eticlopride significantly enhanced the secondary bradycardic response to cocaine (P < 0.0001).

View larger version (33K): [in this window] [in a new window]   Fig. 7. Top, change in MAP produced by intracisternal injection of 140 nmol of cocaine at time 0 in animals pretreated by intracisternal injection 60 min earlier with vehicle or 3.5 or 35 nmol of HC-3. Average resting MAP and the numbers of experiments for each dose are presented in Table 1. Hemicholinium-3 pretreatment significantly inhibited the pressor response to intracisternal injection of cocaine (P < 0.0001). Bottom, change in MAP produced by i.v. injection of cocaine (0.5 mg/kg) at time 0 in animals pretreated by intracisternal injection 60 min earlier with vehicle or with 35 nmol of hemicholinium-3. Hemicholinium-3 pretreatment significantly inhibited the pressor response to i.v. injection of cocaine (P < 0.0001).

View larger version (34K): [in this window] [in a new window]   Fig. 10. Top, change in MAP produced by intracisternal injection of 140 nmol of cocaine at time 0 in animals pretreated by intracisternal injection 15 min earlier with vehicle or 50 or 125 nmol of atropine. Average resting MAP and the numbers of experiments for each dose are presented in Table 1. Pretreatment with atropine partly (but significantly) reduced the expression of the pressor response to cocaine (P < 0.0001), and both doses produced effective antagonism of the cocaine response (P < 0.001). Bottom, pretreatment with atropine reduced the magnitude of the initial tachycardic response to cocaine, but only the lower dose (50 nmol) of atropine seemed to accentuate the secondary fall in heart rate. The effects of atropine were statistically significant (P < 0.0001); the effects were primarily relegated to the 50-nmol dose, t = 10.3, P < 0.001 (for the 125-nmol dose, t = 1.7, P = 0.08).

Role of Noradrenergic and Dopaminergic Receptors. In the next series, rats were pretreated with one of three receptor antagonists: tolazoline (-adrenergic), SCH-23390 (D1 dopaminergic), or eticlopride (D2 dopaminergic) by the intracisternal route. Of the three adrenergic antagonists, only eticlopride seemed to affect resting parameters, significantly decreasing heart rate before cocaine injection (Table 1). However, this effect may have been more a reflection of the higher than usual resting heart rate values for this study group (averaging 455 beats/min), and it is unlikely that the effect on precocaine heart rate impacted the responses to cocaine since the heart rate values immediately before the cocaine injection were typical of the other resting values (averaging 399 beats/min). A comparison of the three antagonists is presented in Fig. 4. The dose of tolazoline (280 nmol) was chosen based on preliminary experiments in which this dose was shown to completely block the hypotensive and bradycardic responses to intracisternal injection of 20 nmol of the ₂-adrenergic agonist clonidine (data not shown). The dose (140 nmol) for SCH-23390 and eticlopride was chosen based on previous studies in which the drugs were shown to block the effects of dopamine on their respective receptors (Sutoo and Akiyama, 1999). Pretreatment with an effective -adrenergic blocking dose of tolazoline was ineffective in blocking the pressor response to cocaine, F(1,1104) = 1.17, P = 0.28 (Fig. 4), although the drug seemed to eliminate the secondary fall in heart rate (Fig. 5); here, the effect of tolazoline was nearly significant, F(1,1104) = 3.59, P = 0.058. Pretreatment with SCH-23390 did not alter the magnitude of the pressor response to cocaine, but the D1 antagonist significantly shortened the duration of the response, F(1,1104) = 62.5, P < 0.0001. Pretreatment with SCH-23390 also seemed to eliminate the initial tachycardic response to cocaine and to accentuate the secondary bradycardic response (the effect of the drug was dependent on the time after injection, F(45,1104) = 2.12, P < 0.0001). The effects of eticopride were nearly identical to those of SCH-23390. Again, the antagonist affected primarily the duration of the pressor response (Fig. 4), F(1,1104) = 73.6, P < 0.0001, whereas it dramatically enhanced the secondary bradycardic response to cocaine (Fig. 5), F(1,1104) = 153.9, P < 0.0001.

Effect Cocaine on Brain Cholinesterase. Since cocaine is metabolized in large part by plasma esterases, the next series of experiments examined the ability of cocaine, procaine, and lidocaine to inhibit brain cholinesterase in vitro. Because inhibition of brain cholinesterase is known to increase systemic blood pressure (see Introduction), these experiments were undertaken to ascertain the potential role of brain cholinesterase inhibition in cocaine's cardiovascular responses. As indicated in Fig. 6, cocaine and procaine (both esters) inhibited the cholinesterase activity of rat brain homogenates. Cocaine was much less potent (IC₅₀ approximately 0.6 mM) than the classical cholinesterase inhibitor physostigmine (K_i = 0.07 µM; Brezenoff et al., 1982), which does increase MAP after either peripheral or central administration. Unlike physostigmine, cocaine's anticholinesterase activity was not time-dependent (Fig. 6, inset).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effect of cocaine and procaine on the cholinesterase activity of rat brain homogenates. Cocaine's anticholinesterase activity was not time-dependent (inset). Each value represents the mean of two complete experiments, each performed in triplicate.

Effect of HC-3 on the Cardiovascular Response to Cocaine. The participation of brain cholinergic neurons in the cardiovascular response to cocaine was examined by pretreating rats by intracisternal injection of HC-3 to deplete endogenous acetylcholine. The doses chosen were previously shown to significantly decrease brainstem acetylcholine content (Finberg et al., 1979; Buccafusco and Spector, 1980) and to block the pressor response to cholinesterase inhibitors (Makari et al., 1989). Pretreatment with HC-3 did not alter resting MAP or HR before cocaine administration (Table 1). Cocaine was injected 60 min after hemicholinium-3 to allow for complete acetylcholine depletion. Hemicholinium-3 pretreatment resulted in a significant dose-dependent decrease in the magnitude of the pressor response to intracisternal injection of cocaine (Fig. 7), F(2,378) = 16.6, P < 0.0001. Hemicholinium-3 pretreatment inhibited the magnitude of the initial tachycardic response to cocaine, but it seemed to accentuate the secondary bradycardic response (Fig. 8), F(2,378) = 7.50, P = 0.0007. Next, we sought to determine whether depletion of medullary acetylcholine levels could alter the pressor response to cocaine administered by the i.v. route. In these experiments, rats were pretreated either with vehicle (sterile saline) or with 35 nmol of hemicholinium-3 by the intracisternal route. After 60 min, 0.5 mg/kg cocaine (this dose was chosen based on preliminary experiments to evoke a pressor response of similar magnitude to that produced by intracisternal injection) was administered by i.v. bolus injection (Fig. 7, bottom). In vehicle-pretreated animals, i.v. cocaine induced a rapid increase in MAP of up to 40 mm Hg, which decayed to baseline levels within about 10 min. In hemicholinium-3-pretreated rats, the same dose of cocaine evoked a more transient pressor response of less than half the magnitude of the control cocaine response. The differences between the two data sets were significant, F(1,644) = 59.9, P < 0.0001. In vehicle-pretreated rats, i.v. cocaine induced a rapid increase in heart rate that was maintained throughout the observation period (Fig. 8, bottom). In hemicholinium-3-pretreated rats, the tachycardic response to i.v. cocaine over the first 5 min after injection was undiminished, but the response was not maintained, falling to baseline levels by about 14 min after cocaine injection. The differences between the two data sets were nearly significantly different, F(1,644) = 3.46, P = 0.063.

Effect of Cocaine on Muscarinic Receptors. As presented in the Introduction, cocaine shares structural characteristics with the muscarinic antagonist atropine. The next series tested the possibility that cocaine's cardiovascular actions might be mediated partly through interaction with medullary muscarinic receptors, activation of which is known to induce an increase in MAP (Buccafusco, 1996). Cocaine's ability to bind to muscarinic receptors was inferred from the drug's ability to displace the specific binding of [³H]methylscopolamine to rat brain membranes (Fig. 9). The cocaine binding data were compared with those associated with the cholinergic agonist carbachol and the muscarinic antagonist atropine. The atropine binding data were well fit to a single-site model. In contrast, carbachol displacement of [³H]methylscopolamine binding exhibited both a low-affinity and a high-affinity component. The cocaine displacement curve was more similar to the carbachol curve, again exhibiting low- and high-affinity components. In fact, there was little difference in affinity between carbachol and cocaine for both components of the binding curve.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Ability of cocaine to bind to muscarinic receptors as inferred from the drug's ability to displace the specific binding of [3H]N-methylscopolamine to rat brain membranes. The cocaine binding data were compared with those associated with carbachol and atropine. Each value represents the mean of two complete experiments, each performed in triplicate.

This result prompted the next series in which rats were injected by the intracisternal route either with vehicle or with atropine before cocaine (140 nmol intracisternal). These data are presented in Fig. 10. Surprisingly, relatively high doses of atropine were only partly (but significantly) effective in reducing the expression of the pressor response to cocaine, F(2,1334) = 37.6, P < 0.0001; and both doses produced significant effects (P < 0.001). The higher (125 nmol) dose of atropine elevated heart rate at the time of cocaine administration (Table 1). This may have been due to some redistribution of the antagonist to the peripheral circulation. Nevertheless, after atropine pretreatment, the magnitude of the initial tachycardic response to cocaine was reduced, but only the lower dose (50 nmol) of atropine seemed to accentuate the secondary fall in heart rate. The effects of atropine were statistically significant, F(2,1334) = 53.1, P < 0.0001; the effects were primarily relegated to the 50-nmol dose, t = 10.3, P < 0.001 (for the 125-nmol dose, t = 1.7, P = 0.08).

Role of Nicotinic Receptors. The data thus far are consistent with a role for endogenous acetylcholine in the cardiovascular response to intracisternally injected cocaine; however, muscarinic receptor-mediated neurotransmission could not fully account for cocaine's effectiveness. In this series, the aim was to study the effect of nicotinic acetylcholine receptor antagonists on the cocaine response. During preliminary experiments with the first compound, hexamethonium, we were unimpressed with the effectiveness of the drug given just before cocaine. However, in reassessing the effects of cocaine on the following day in the absence of pretreatment drug, we found the response to cocaine to be diminished. This caused us to follow the response to cocaine longitudinally in each subject over a course of six daily injections. Baseline values for MAP and heart rate for the various pretreatment drugs used in the data documented in Figs. 11 and 12 are presented in Table 2. There were no significant differences in the baseline levels among the individual drug studies. The time point of maximal cocaine response was used for comparison. Across the individual drug studies these ranged from 1.83 to 2.5 min after injection. As indicated in Fig. 11, pretreatment with vehicle failed to significantly alter the maximal pressor, F(5,54) = 0.48, P = 0.79, or tachycardic, F(5,54) = 0.77, P = 0.57) responses to intracisternal cocaine over the 6-day daily dosing schedule. A single intracisternal dose (140 nmol) of hexamethonium administered on day 0 failed to significantly inhibit the maximal pressor response to intracisternal injection of cocaine administered 15 min after the blocking drug. However, when cocaine was administered as a single injection on each of the four following days, the pressor responses were significantly inhibited, F(5,43) = 4.54, P = 0.0022. On day 5, the cocaine response returned nearly to baseline levels. The lower dose (14 nmol) of hexamethonium tended to reduce the cocaine response on days 1 and 2, but the effect was not significant. Although the day-to-day effect was somewhat more variable, hexamethonium treatment on day 0 inhibited the expression of the tachycardic response to cocaine when it was administered on days 1, 3, and 4, F(5,43) = 2.48, P = 0.047.

View larger version (30K): [in this window] [in a new window]   Fig. 11. Ability of intracisternal pretreatment with vehicle or one of four nicotinic receptor antagonists administered on day 0 to alter the cardiovascular response to intracisternal injection of cocaine (140 nmol) administered 15 min after the pretreatment. On experimental days 1 to 5, only cocaine (140 nmol intracisternal) was administered at time 0. In the left-hand series of graphs, data are presented for the maximal increase in MAP (measured at about 2 min after cocaine injection) relative to the precocaine baseline levels (as indicated in Table 2). In the right-hand series of graphs, data are presented for the accompanying changes in HR. HEX 14, 14 nmol of hexamethonium; HEX 140, 140 nmol of hexamethonium; MEC, 50 nmol of mecamylamine; MLA, 50 nmol of methyllycaconitine; DHE 80, 80 nmol of dihydro--erythroidin; DHE 160, 160 nmol of dihydro--erythroidin. *, significantly different from day 0 mean (P < 0.04); +, analysis of variance just missed statistical significance, but the individual t values = P < 0.05.

View larger version (41K): [in this window] [in a new window]   Fig. 12. Top, change in MAP produced by intracisternal injection of 140 nmol of cocaine at time 0 in animals pretreated by intracisternal injection 3 days earlier with vehicle, 140 nmol of hexamethonium (HEX), or 50 nmol of mecamylamine (MEC). Average resting MAP and the numbers of experiments for each dose are presented in Table 2. Both antagonists reduced the pressor response to cocaine (P < 0.0001) (hexamthonium: t = 12.3, P < 0.001; mecamylamine: t = 6.0, P < 0.001). Bottom, both antagonists nearly eliminated the initial tachycardic response to cocaine while accentuating the secondary fall in heart rate (P < 0.0001; hexamethonium: t = 26.7, P < 0.001; mecamylamine: t = 18.1, P < 0.001).

Using the same single-dose regimen, three other nicotinic antagonists were evaluated. Treatment with the ganglion blocker ( subunit-preferring) mecamylamine (50 nmol) resulted in subsequent inhibition of the maximal pressor response to intracisternally injected cocaine; however, the overall effect just missed the required level of significance, F(5,30) = 2.25, P = 0.075. The effect was relegated primarily to days 2, 4, and 5. Mecamylamine treatment also tended to reduce the tachycardic response to cocaine, but the effect was not statistically significant, F(5,48) = 1.67, P = 0.16. The 7 subunit-selective antagonist MLA (50 nmol) failed to alter the pressor responses to intracisternally injected cocaine, F(5,20) = 0.11, P = 0.98, although the drug did inhibit the cocaine-induced increase in heart rate on days 2 and 3, F(5,20) = 2.88, P = 0.041. Treatment with the 2 subunit-preferring nicotinic receptor antagonist DHE also failed to inhibit the pressor response to intracisternally injected cocaine, either acutely or over the five following days, even when the dose was increased to 160 nmol, F(5,42) = 0.28, P = 0.92. The drug decreased the cocaine-induced tachycardic response on days 2 and 3, although the mean difference did not achieve the required level of significance, F(5,42) = 1.98, P = 0.10.

The data presented in Fig. 12 provide a better appreciation of the time course of the responses evoked by intracisternally administered cocaine during a single day's session in the hexamethonium and mecamylamine series. The data represent the cocaine responses (140 nmol intracisternal) registered on experimental day 3 after the single intracisternal injection of 140 nmol of hexamethonium or of 50 nmol of mecamylamine on experimental day 0. Both antagonists reduced the maximum pressor response as well as the duration of the effect, F(2,1715) = 76.5, P < 0.0001 (hexamethonium: t = 12.3, P < 0.001; mecamylamine: t = 6.0, P < 0.001). Both antagonists nearly eliminated the initial tachycardic response to cocaine, whereas accentuating the secondary fall in heart rate, F(2,1715) = 53.6, P < 0.0001 (hexamethonium: t = 26.7, P < 0.001; mecamylamine: t = 18.1, P < 0.001).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Perhaps one of the most intriguing aspects of cocaine's centrally mediated pressor action is that the effect does not seem to be mediated by brain -adrenergic receptors. Most other direct- and indirect-acting adrenergic drugs decrease MAP after direct central administration. This includes agonists such as norepinephrine, dopamine, and clonidine, as well as drugs that act presynaptically such as -methyldopa, phentermine, and amphetamine (Kobinger, 1978). In each case, the hypotensive action of these drugs was shown to be mediated primarily through central -adrenergic receptors. In fact, the concept that activation of brain adrenergic pathways involved in central cardiovascular regulation leads to sympathoinhibition and a fall in blood pressure dominated the literature for several decades. Cocaine, however, is not the first sympathomimetic amine to induce an increase in MAP upon direct central administration (King and Pang, 1988; Colombari et al., 1992; Ebihara et al., 1993; Correa and Peres-Polon, 1995). The potential for cocaine to enhance dopaminergic neurotransmission also did not seem to explain the central sympathoexcitatory action of the drug. This is because dopamine and other dopaminergic agonists such as bromocriptine decrease blood pressure upon central administration (Kimura et al., 1981; Park et al., 1991; Tangri et al., 1993). Moreover, the results of the present studies were not consistent with a major role for dopamine receptors in mediating the pressor action of cocaine. In fact, the dopamine blockers merely reduced the duration of the response without affecting the maximal change in MAP. The effect of the antagonists on the duration of the pressor response might have been a consequence of their effects on heart rate. Both SCH-23390 and eticlopride accentuated the secondary bradycardic response to cocaine. From a cardiovascular standpoint, the enhanced bradycardia would oppose cocaine's ability to sustain the pressor response. This possibility is suggested by the observation that eticlopride, which more dramatically enhanced the secondary bradycardia to cocaine, also was more effective than SCH-23390 in reducing the duration of the cocaine pressor response. It is likely, therefore, that the pressor response to intracisternal administration of cocaine is initiated over a different neural pathway than then is the heart rate response. In addition to reducing the duration of the pressor response to cocaine, the dopamine antagonists blocked the expression of the initial tachycardic response to cocaine, whereas they enhanced the decrease in heart rate. Tolazoline was the only agent to completely eliminate the secondary bradycardic response to cocaine, suggesting that this response was mediated by -adrenergic receptors. In this respect, cocaine's actions may be considered clonidine-like. Indeed the late-occurring sedative-like action of cocaine in the animals is reminiscent of clonidine. These findings also concur with those of Abrahams et al. (1996) who demonstrated that bilateral injection of ₂-adrenergic receptor antagonists into the rostral ventrolateral medulla attenuated the decrease in sympathetic nerve activity induced after i.v. injection of cocaine in anesthetized rats.

Another unexpected finding was the localization of the pressor response to medullary regions. Cocaine's behavioral actions, including its psychotomimetic effects and its rewarding properties are considered to originate in cortical regions. Although a pressor response could be elicited from injections made into the lateral ventricle, the effect was not as robust as the responses after intracisternal injection. The virtual absence of responses elicited after intrathecal and intravenous administration ruled out the possibility that significant drug was redistributed to a potentially active spinal site or to the peripheral circulation. Biphasic heart rate responses of similar magnitude could be elicited from either l.c.v. or intracisternal routes of cocaine administration, although for the 280-nmol dose, the intracisternal route provided a more prolonged tachycardic phase.

Although the actual site(s) of action for the pressor and tachycardic responses to cocaine within medullary regions has not been identified, they seem to be mediated though local cholinergic pathways. This conclusion is based on the effectiveness of pretreatment with hemicholinium-3 to nearly eliminate both responses. The presynaptic nature of hemicholinium-3-induced blockade of cholinergic cardiovascular pathways has been demonstrated by the ability of the drug to decrease brain medullary acetylcholine levels (Finberg et al., 1979; Buccafusco and Spector, 1980) and to inhibit the pressor response to central injection of cholinesterase inhibitors, but not to direct receptor agonists such as carbachol (Buccafusco and Brezenoff, 1979; Magri and Buccafusco, 1988; Makari et al., 1989). Therefore, cocaine's sympathoexcitatory actions after intracisternal injection require the participation of a functioning cholinergic synapse. That cocaine might be acting as a cholinesterase inhibitor was ruled out by the very low potency exhibited by the drug on the brain-derived enzyme. Notwithstanding the observation that cocaine interacted in vitro with rat brain muscarinic receptors with equal potency to carbachol, the hemicholinium-3-sensitive nature of the response and the only partial degree of inhibition of the pressor response in atropine-treated rats (even in the presence of relatively high doses of the antagonist) seem to limit support for cocaine acting directly as a muscarinic receptor agonist. One issue to address here is the use of the quaternary methylbromide salt of atropine in these studies. We used this derivative of atropine in the anticipation that we would limit diffusion relative to the more lipid-soluble and highly diffusible tertiary derivative (Brezenoff et al., 1988). In retrospect, and in knowing of the role of nicotinic receptors in the cocaine response (see below), it is possible that the quaternary methylbromide compound may have produced a partial blockade of local nicotinic receptors as has been suggested for peripheral nicotinic sites (King and Ryall, 1981).

The third unexpected finding was that of the delayed onset and the very prolonged (up to 4 days) duration of the blockade of the pressor and tachycardic responses to cocaine by intracisternal-preadministered hexamethonium. The bisquaternary nicotinic antagonist chlorisondamine can produce functional central nicotinic receptor blockade for up to several weeks after administration of a single dose under certain experimental conditions (see Chadman and Woods, 2004); though the reason for the enhanced duration of the blockade is unknown. Chlorisondamine, hexamethonium, and mecamylamine share the ability to block autonomic ganglionic transmission. With neuronal nicotinic receptors they exhibit low or nonexistent affinity for the agonist binding site, inhibiting receptor function through allosteric interactions and/or channel blockade. The prolonged onset and duration of functional nicotinic blockade after central administration of either hexamethonium or mecamylamine (to our knowledge) has not been reported. However, the prolonged duration of the blocking effect produced by these nicotinic antagonists was not due to cholinergic blockade per se, since pretreatment with hemicholinium-3 was only effective on an acute basis in its ability to block response to cocaine (data not shown). The partial subtype specificity of the antagonists that were used in this study provides some indication as to the relevant subtype of nicotinic receptor involved in the cocaine-induced pressor response. The lack of effectiveness of MLA and DHE tends to rule out significant contributions of either, respectively, 7 or 42 subtypes. Mecamylamine does exhibit some selectivity for the 32 subtype (Levin and Rezvani, 2002), pointing to the latter as a potentially important target receptor.

The role of central nicotinic receptors in the cardiovascular response to cocaine is perhaps not that surprising since the drug can induce the release of endogenous acetylcholine (Imperato et al., 1993; Zocchi and Pert, 1994; Mark et al., 1999), which in turn has the potential of stimulating synaptic nicotinic receptors. In most cases, the acetylcholine-releasing effects of cocaine were assumed or demonstrated to be indirect, i.e., via activation of dopaminergic pathways (see Introduction). This is most likely not the case for the cocaine-induced increase in MAP reported here. Instead, cocaine may interact directly with nicotinic receptors. Evidence exists for both inhibitory (Damaj et al., 1999; Francis et al., 2000) and excitatory (Zernig et al., 1997; Reid et al., 1998) interactions of cocaine with nicotinic receptors. Of particular relevance is the finding that nicotine can activate medullary cardiac parasympathetic cells either directly by binding to somatic nicotinic receptors, or indirectly by binding to nicotinic receptors on glutamatergic neurons that innervate the cardiac vagal cells (Neff et al., 1998). If cocaine mimics nicotine in this respect, enhanced cardiac vagal outflow could partly explain the bradycardic actions of centrally administered cocaine. Similar actions of cocaine with respect to medullary vasomotor neurons may underlie the pressor response to cocaine. Indeed, the pressor response induced by l.c.v. injection of nicotine also was blocked by pretreatment with hemicholinium-3 (Buccafusco and Yang, 1993).

Since the early 1990s, it has been recognized that the sympathomimetic action of systemically administered cocaine may involve a central component (Knuepfer et al., 1993). Although a central mechanism was not always confirmed in certain animal models (Gillis et al., 1995), a more recent study in human subjects seemed to support the role of the CNS (Vongpatanasin et al., 1999). The results of this study support the concept of a central component since pretreatment with central administration of hemicholinium-3 blocked the subsequent pressor response to intravenous administration of cocaine. Whereas this issue was not a main objective of the study, it does serve to underscore the necessity to include central mechanisms when considering approaches to the development of treatment strategies for cocaine overdose.

Although it was not anticipated in the outset of this study, the results continue to underscore the close relationship between nicotine and cocaine abuse. They also support the premise that nicotinic receptors may serve as potential targets in the development of drugs for the treatment of cocaine overdose, as well as for antiabuse issues (Reid et al., 1998; Levin et al., 2000; Young et al., 2001). Our results also provide new evidence for the prolonged inactivation of relevant central nicotinic receptors by certain nicotinic receptor antagonists and suggest the possibility that compounds of this class might be used safely without the concern for autonomic side effects since only one dose per several days may be required in treatment strategies. Finally, the role of brain neuronal systems releasing acetylcholine might be scrutinized more carefully when considering the addictive properties of cocaine since reward as a motivation to drug-seeking behavior may not solely depend upon the release of dopamine (Cannon and Palmiter, 2003).

	Acknowledgements

 
This work was supported by a Merit Review grant from the Office of Research and Development, Medical Research Service of the Department of Veterans Affairs.

	Footnotes

 
doi:10.1124/jpet.104.073619.

ABBREVIATIONS: CNS, central nervous system; l.c.v., lateral cerebroventricular; PE, polyethylene; MAP, mean arterial pressure; HR, heart rate; HC-3, hemicholinium-3; MLA, methyllycaconitine; DHE, dihydro--erythroidin.

Address correspondence to: Dr. Jerry J. Buccafusco, Alzheimer's Research Center, Department of Pharmacology and Toxicology, 1120-15th St., Augusta, GA 30912-2300. E-mail: jbuccafu{at}mcg.edu

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