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NEUROPHARMACOLOGY

Role of 7 Nicotinic Acetylcholine Receptors in the Pressor Response to Intracerebroventricular Injection of Choline: Blockade by Amyloid Peptide A1-42

Department of Pharmacology and Toxicology, Alzheimer's Research Center, Medical College of Georgia, and the Veterans Administration Medical Center, Augusta, Georgia

Received for publication November 20, 2003
Accepted February 19, 2004.

	Abstract

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Systemic blood pressure and cardiac function have long been known to be under the control of central autonomic and hormonal pathways that, in part, use cholinergic neural systems. Recently choline, a precursor and product of acetylcholine metabolism, has been shown to serve as a selective endogenous agonist for the 7 subtype of the nicotinic acetylcholine receptor (7nAChR). This receptor subtype mediates several responses to nicotine in animals, most notably, neuroprotection and enhanced cognition. The purpose of this study was to determine whether the cardiovascular changes induced by central injection of choline in rats also were mediated by 7nAChRs. Moreover, we sought to determine whether these cardiovascular changes to choline could be blocked by central pretreatment with amyloid peptide (1-42) (A1-42), a neurotoxic component of cerebral amyloid that is known to bind with high affinity to 7nAChRs. Central, i.c.v. injection of choline (50, 100, or 150 µg) produced dose-dependent (10-15-min duration) pressor response of up to about 20 mm Hg. The most consistent change in heart rate included a brief increase (up to 40 beats/min) that lasted 2 to 3 min, followed by a prolonged decrease averaging 50 beats/min that lasted up to 30 min. Pretreatment (i.c.v.) with the selective 7nAChR antagonists -bungarotoxin and methyllycaconitine significantly inhibited the pressor and heart rate responses to subsequent injection of choline. Pretreatment with the non-7-preferring antagonist dihydro--erythroidin was not effective. These findings suggested that the cardiovascular response to i.c.v. injection of choline was mediated at least in part through 7nAChRs. Pretreatment (30 min) with low doses (1-100 pmol) of amyloid peptide A1-42 (but not with A40-1) administered by the i.c.v. route significantly inhibited the choline-induced blood pressure increase as well as the choline-induced decrease in heart rate.

The peripheral circulation and cardiac function, to varying degrees and under specific conditions, are continuously regulated through the output of central autonomic and hormonal pathways. Although many important questions remain unanswered, a great level of understanding has been achieved concerning the central control of blood pressure. In this complicated and interconnected regulatory system, central cholinergic neural pathways have been shown to play important roles; and in unanesthetized rats, cholinergic agonists, both muscarinic (Brezenoff and Giuliano, 1982; Buccafusco, 1992) and nicotinic (Brezenoff and Giuliano, 1982; Buccafusco and Yang, 1993; Khan et al., 1994), have been shown to induce increases in arterial blood pressure. Changes in heart rate (HR) often are unpredictable, depending upon the particular agonist, and upon ongoing arousal and activity (Magri and Buccafusco, 1988). Some years ago, Caputi and Brezenoff (1980) demonstrated that central i.c.v. injection of choline induced a short lasting, but significant increase in arterial pressure and a much longer lasting bradycardic response in unanesthetized rats. They failed to inhibit the pressor response through pretreatment of the animals by i.c.v. injection with either the muscarinic antagonist atropine or the nicotinic antagonist mecamylamine. Neither drug alone nor their combination modified the pressor response. However, neither of these compounds is subtype-selective within their respective class, and atropine induces acetylcholine release. In light of the recently discovered ability of choline to serve as a selective agonist at 7nAChRs (Papke et al., 1996; Alkondon et al., 1999; Pereira et al., 2002), it is reasonable to consider that this receptor subtype mediates the choline-induced pressor response. With reasonably subtype-selective nAChR antagonists now available, this possibility can readily be examined. Thus, the first portion of this study was directed at determining whether the cardiovascular response to i.c.v. administration of choline was mediated predominantly through activation of 7nAChRs.

In the AD brain large deposits of amyloid plaques are present that consist chiefly of amyloid peptides. In fact, recently, amyloid peptides containing a crucial binding sequence, including the neurotoxic A1-42, have been shown to bind with high affinity to 7 nAChRs (Wang et al., 1999, 2000). Moreover, A and 7nAChRs seem to colocalize within the AD brain. A peptides have been shown to inhibit the cellular responses to nAChR agonists (Liu et al., 2001; Pettit et al., 2001) and to prevent the cytoprotective action of nicotine in vitro (Li and Buccafusco, 2003). There also exists evidence to suggest that amyloid deposition in AD-related transgenic mouse strains alters the expression of nicotinic receptors in vivo (Bednar et al., 2002). Whereas A peptides have been directly introduced into the cerebrospinal fluid of rodents by prolonged i.c.v. administration to elevate brain amyloid and to produce neurotoxicity, regimens specific for pharmacological interactions between the peptide and 7nAChRs in vivo have not been determined. Therefore, the second portion of this study was designed to assess the ability of A peptides to inhibit the magnitude and expression of the acute pressor response to choline. In addition to the practical application of the study, these results may provide further in vivo evidence for the ability of A peptides to target 7nAChRs, a subtype that is highly expressed on neural cells known to be selectively vulnerable to the toxic consequences of AD (Davies and Feisullin, 1981; Sugaya et al., 1990; Hellstrom-Lindahl et al., 1999).

	Materials and Methods

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Materials. Choline iodide, methyllycaconitine citrate (MLA), atropine sulfate, amyloid peptide (1-42), amyloid peptide (40-1), and -bungarotoxin (BTX) were obtained from Sigma-Aldrich (St. Louis, MO). Dihydro--erythroidine (DHE) was donated by Merck (Rahway, NJ). The doses used in this study for each nicotinic receptor antagonist were derived from previous studies in which they were shown to exert nicotinic receptor blocking actions in vivo with relative selectivity (Buccafusco and Yang, 1993; Jonnala et al., 2002). The amyloid peptides were dissolved in sterile distilled water at 100 µM, and they were stored as aliquots at -20°C. They were incubated at 37°C for 24 h before use to allow for aggregation.

Experimental Animals. Male Wistar rats (Harlan, Indianapolis, IN), weighing 280 to 380 g, were housed in an environmentally controlled room on a 12/12-h day/night cycle, and they were maintained on standard rat chow and tap water (unlimited). All animal protocols were previously approved by the Medical College of Georgia Committee on Animal Use for Research and Education.

Surgical Procedures. Rats were anesthetized initially with methohexital (sodium), 65 mg/kg i.p. and supplemented as needed. The subject's head was positioned in a stereotaxic frame with the skull in a horizontal position. A midline incision was made to expose the skull. Using stereotaxic landmarks a burr hole was placed over the left lateral cerebral ventricle. An additional burr hole was placed to the right of the first hole and a stainless steel anchoring screw was inserted. A sterile stainless steel or cannula guide was attached to a micromanipulator on the stereotaxic frame. Each guide (Plastics One, Roanoke, VA) had a stainless steel shaft equivalent to 22-gauge tubing, which was inserted into a wider threaded region made of Teflon at the top of the guide. The threaded region permits the introduction of a screw cap and dummy cannula to plug the guide when not in use. The cannula guide (11 mm in length) was lowered to a depth of 4.0 mm below the skull through the burr hole over the ventricle. The tip of the guide was placed over, but without penetrating, the ventricular space. While in position, the guide and screw were covered with acrylic cement. After complete hardening of the cement, the manipulator was retracted and the guide plugged and capped. The skin was pulled around (but not over) the guide and sutured closed.

One week later rats again were anesthetized and a surgical incision was made in the midline of the abdomen to expose the lower portion of the abdominal aorta. A small nick was made in the lower portion of the aorta and a telemetry catheter (implant model TA11PA-C40; Data Sciences International, St. Paul, MN) was introduced and advanced until the tip rested below the origin of the renal arteries. The tip of the catheter contains a nonthrombogenic gel to prevent clot formation. The catheter end was fixed in place in the aorta by using Vetbond tissue adhesive (3M Animal Care Products, St. Paul, MN). The transmitter end of the catheter was placed subcutaneously over the abdomen and the wound was sutured closed.

Measurement of Mean Arterial Pressure and Heart Rate.

Blood pressure and heart rate were measured using the DSI PhysioTel telemetry system (Data Sciences International, St. Paul, MN), which monitored the animals while they moved freely within their cages. Each implanted transducer/transceiver transmitted precalibrated digitized blood pressure data via radio frequency signals to a nearby receiver. The data were sampled continuously, and by using the Dataquest data acquisition system (Data Sciences International), which also calculated heart rate continuously from the pressure waveforms. Mean arterial pressure (MAP) and heart rate values were averaged and presented graphically in 30-s bins.

Microinjections. For i.c.v. injections, a 50-µl Hamilton microsyringe (Reno, NV) was connected to a 28-gauge internal cannula (Plastics One), which was inserted into the cranial 22-gauge cannula guide so that the tip rested within the lateral ventricle. All test compounds were dissolved in 10 µl of sterile normal saline (vehicle), and they were delivered by hand over a period of 15 to 20 s. At the completion of experiments each injection site was verified by injecting 10 µl of India ink. The pretreatment drugs (or vehicle) were administrated 10 or 30 min (as indicated) before the choline (or vehicle) injection. Rats were allowed to recover for at least 1 day after each experiment (i.e., one choline microinjection). Each rat was used in no more than five experiments.

Statistical Analysis. The data depicted in the text and figures are presented as mean ± S.E.M. The existence of a significant difference between or among experimental groups was determined by analysis of variance with repeated measures. A modified t test with Bonferroni's correction for multiple comparisons using the error mean-square term from the analysis of variance was used for analysis between groups of data. The criterion for statistical significance was P < 0.05 for all comparisons.

	Results

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Because all of the data derived from this study are presented as the absolute change from resting (baseline) MAP and heart rate, Table 1 provides the baseline means for each of the experimental paradigms. There were no significant differences among the means for MAP [F(10,65) = 1.25; P = 0.28]. There also were no significant differences among means for resting heart rate [(F(10,65) = 0.50; P = 0.89]. Thus, any change noted below in the magnitude or duration of the choline induced cardiovascular responses produced by pretreatment drugs could not be attributed to alterations in baseline (precholine) MAP or heart rate.

Microinjection of 10 µl of vehicle (sterile saline) on average produced only a brief (<3 min) increase in MAP of up to 3 mm Hg. Microinjection of solutions of choline (50, 100, or 150 µg) within the same volume produced immediate and dose-dependent increases in MAP ranging from 10 to 20 mm Hg (Fig. 1). There was a significant difference between dose component of the ANOVA [F(2,10) = 5.58; P = 0.024]. The responses peaked 3 to 4 min after injection and returned to preinjection levels within 10 to 15 min. In some animals, MAP declined below preinjection values toward the end of the observation period. Simultaneously, heart rate initially increased (to about 40 beats/min) and it peaked about the same time as the increase in MAP (Fig. 2). Thereafter, heart rate declined to below preinjection values. The late decrease in heart rate may reflect a decrease in cardiac output driving the late fall in MAP observed in some animals. Although the heart rate changes were not statistically dose-dependent [F(2,10) = 1.67; P = 0.24], the profile of response to choline was similar to that reported 13 years ago by Caputi and Brezenoff (1980), except for the brief initial increase in heart rate, which they did not report observing. It is possible that the increase in heart rate may have reflected animal activity just after the injection because it was not present in all animals. As observed by Caputi and Brezenoff (1980), and in this study, the duration of the decrease in heart rate usually far exceeded (at least up to 30 min) the duration of the increase in MAP. Also, soon after choline administration, an increase in locomotor activity was often observed, with the effect quickly diminishing; and the rats actually remained quiet, possibly sedate for the remainder of the observation period. Although the behavioral effects were not quantified, they were not apparent after i.c.v. injection of vehicle.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Time course for the change in MAP after i.c.v. injection of choline (50, 100, or 150 µg) in freely moving rats. Each point and vertical bar represents the mean ± S.E.M. derived from four to six experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Time course for the changes in HR after i.c.v. injection of choline (50, 100, or 150 µg) in freely moving rats. Each point and vertical bar represents the mean ± S.E.M. derived from four to six experiments.

To determine whether 7nAChRs contribute to the cardiovascular response produced by choline, rats were pretreated by i.c.v. injection either with MLA or -bungarotoxin, both 7nAChR-specific antagonists. In the first series, administration of 10 µg MLA (10 min before 150 µg of choline) resulted in a complete abolition of the pressor response to choline. Likewise, the biphasic heart rate change after choline was essentially normalized by MLA pretreatment (Fig. 3). In contrast, pretreatment with 0.3 µg of atropine failed to significantly modify the pressor response to choline. This i.c.v. dose of atropine was shown previously to completely block the pressor response to central microinjection of muscarinic agonists (Caputi and Brezenoff, 1980). Atropine pretreatment partly, but significantly, reversed the changes in heart rate produced by choline. The administration of 10 µg of -bungarotoxin (30 min before 150 µg of choline), like MLA, significantly inhibited the magnitude of the choline-induced pressor response. These data are presented in Fig. 4. The longer pretreatment time for -bungarotoxin in this series was in consideration of the drug's slow binding kinetics. Toxin pretreatment significantly inhibited the choline-induced initial increase in heart rate, but it had no effect on the subsequent bradycardic response (Fig. 4, inset). To further examine the specificity of the choline response, the 2-subtype preferring antagonist DHE was used as the pretreatment agent. Unlike MLA and -bungarotoxin, pretreatment with 6 µg of DHE failed to significantly inhibit the pressor response to subsequent injection of 150 µgof choline (Fig. 4). The heart rate changes induced by choline also were not affected by DHE (Fig. 4, inset).

View larger version (41K): [in this window] [in a new window]   Fig. 3. Effect of i.c.v. pretreatment with 10 µl of saline (vehicle), 10 µg of MLA, or with 0.3 µg of atropine on the change in MAP to i.c.v. injection of 150 µg of choline. Saline MLA or atropine was injected 10 min before choline. Compared with saline, MLA significantly inhibited the pressor effect induced by choline [F(1,497) = 80.1; P < 0.0001]. Atropine pretreatment was without significant effect on the pressor response to choline [F(1,497) = 0.72; P = 0.40]. Inset, accompanying HR changes to choline. Both MLA pretreatment [F(1,497) = 55.5; P < 0.0001] and atropine pretreatment [F(1,495) = 9.6, P = 0.002] inhibited the changes in HR to choline.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Effect of i.c.v. pretreatment with 10 µl of saline (control), 10 µg of BTX, or 6 µg of DHE on the change in MAP to i.c.v. injection of 150 µg of choline. Saline or the antagonists were injected 30 min before choline. Compared with the saline pretreatment group, -bungarotoxin significantly inhibited the increase in MAP produced by 150 µg of choline [F(1,465) = 19.4; P < 0.0001]. Under the same conditions, 6 µg of DHE failed to significantly alter choline induced pressor response [F(1,350) = 0.67; P = 0.41]. There also was a significant difference between the BTX and the DHE-treated groups [F(1,350) = 51.6; P < 0.0001]. Inset, accompanying HR changes to choline. As with the effects on MAP, significant differences were found between the BTX versus saline pretreatment groups [F(1,469) = 34.2; P < 0.0001] and between the BTX versus DHE pretreatment groups [F(1,350) = 4.9; P = 0.028]. DHE pretreatment failed to significantly inhibit the HR changes produced by choline [F(1,350) = 2.2; P = 0.14].

With the data suggesting that the initial pressor response to i.c.v. microinjection of choline was mediated predominantly by 7nAChRs, we next sought to determine whether pretreatment with the amyloid peptide (also known to block 7nAChRs) would effectively inhibit the choline response. A1-42 (1, 10, or 100 pmol) was administrated 30 min before choline. The peptide itself elicited no change in resting MAP or heart rate (Table 1). The two higher doses of A1-42 (10 pmol and 100 pmol) significantly inhibited the increases in MAP produced by i.c.v. injection of 150 µg of choline (Fig. 5). Under the same conditions the reverse peptide A40-1 (100 pmol) failed to significantly affect the pressor response to choline (Fig. 6, top). As with the receptor antagonists, the higher dose of the A1-42 (100 pmol) normalized the biphasic heart rate response to i.c.v. injection of choline, whereas the same dose of the reverse peptide did not (Fig. 6, bottom).

View larger version (34K): [in this window] [in a new window]   Fig. 5. Dose effect of A1-42 (1, 10, or 100 pmol i.c.v.) on the choline (150 µg i.c.v.)-induced changes in MAP. Amyloid peptides were administrated 30 min before choline. A1-42 (100 pmol) significantly inhibited the increase in MAP produced by i.c.v. injection of choline [F(1,645) = 208.3; P < 0.0001]. The 10-pmol dose of A1-42 also significantly inhibited the increase in MAP produced by choline relative both to the saline control [F(1,492) = 96.0; P < 0.0001]; and with respect to the 1-pmol dose [F(1,492) = 9.6; P = 0.002]. The 1-pmol dose of A1-42 was not significantly effective in blocking the choline response (P > 0.05). The first time point (0 min) indicates the time of choline administration.

View larger version (38K): [in this window] [in a new window]   Fig. 6. Comparison of the abilities of i.c.v. pretreatment with A1-42 and the reverse sequence peptide A40-1 to block the choline induced changes in MAP and heart rate. The peptides were administrated 30 min before choline. Top, pressor response to choline (150 µg i.c.v.) was significantly greater after pretreatment with A40-1 compared with A1-42 [F(1,497) = 33.3; P < 0.0001]; and A40-1 pretreatment failed to significantly inhibit the pressor response to choline as compared with the data derived from vehicle-pretreated rats [F(1,405) = 0.065; P = 0.8]. Bottom, changes in heart rate to choline were significantly greater after pretreatment with A1-42 compared with saline [F(1,487) = 46.7; P < 0.0001]; and A40-1 pretreatment failed to significantly inhibit the changes in heart rate to choline compared with the data derived from vehicle pretreated rats [F(1,485) = 0.58; P = 0.4].

	Discussion

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7nAChRs have been suggested to participate in important higher brain functions such as working memory (Addy et al., 2003), in cell survival and neuroprotection (Li et al.,2000; Jonnala and Buccafusco, 2001; Shimohama and Kihara, 2001), and to serve as a potential drug target in disease states such as schizophrenia (Marutle et al., 2001; Araki et al., 2002), epilepsy (Gil et al., 2002), Alzheimer's disease (Briggs et al., 1997; Meyer et al., 1997; Kem, 2000), and Parkinson's disease (Burghaus et al., 2003). As new 7nAChR compounds become available it would be advantageous to have an efficient, objective, and experimentally simple model for drug efficacy. In this study, the most technically challenging procedure was the installation of the aortic blood pressure telemetry catheter. However, similar results may be obtained by using a standard catheter inserted into any major artery. In fact, for our very first dose finding experiments we directly catheterized the iliac artery and exteriorized the catheter to a watertight swivel for the measurement of MAP in unanesthetized animals as we have described previously (Buccafusco and Yang, 1993). The use of the telemetry catheter was somewhat more advantageous because there are no exterior catheters to maintain, and the preparation has a longer useful lifetime.

The most reproducible response produced by i.c.v. microinjection of choline was the pressor response that occurred during the first 10 to 15 min after injection. Except for the magnitude of the pressor response (which was greater than that to choline), the cardiovascular changes to i.c.v. administered nicotine as we described them several years ago (Buccafusco and Yang, 1993) were very similar to those produced by i.c.v. administration of choline. The maximal pressor response to nicotine (averaging about 30 mm Hg) was achieved with 50 µg (0.11 µmol) of the drug, compared with 150 µg (0.65 µmol) of choline. This 6-fold difference in potency was somewhat surprising in view of the 50-fold difference in binding potencies (versus ¹²⁵I--bungarotoxin) between the two drugs for the 7nAChR (Jonnala et al., 2003). However, Alkondon et al. (1999, 2000), in studying 7-mediated currents recorded from CA1 interneurons in rat hippocampus, reported that the EC₅₀ values for choline and nicotine were, respectively, 2270 and 158 mM, representing only a 14-fold difference in potencies. Likewise there was no more than a 10-fold difference in potency between choline and nicotine in their ability to increase intracellular Ca²⁺ in cultured porcine superior cervical ganglion cells (Si and Lee, 2002). We had noted similar mismatches between binding affinity and pharmacological response earlier in studying nicotine and choline as cytoprotective agents in vitro (Jonnala et al., 2003), so it is not likely that the differences in drug effectiveness could be attributed to differences in their brain distribution or elimination. Also, choline seems to be slightly less efficacious than nicotine though it has been characterized as a full 7nAChR agonist (Pereira et al., 2002). Again, we had noted this potential discrepancy in our earlier cytoprotection study.

Although the effect of i.c.v. choline on heart rate was more variable than the effect on MAP, the responses seemed to be susceptible to the same antagonists as was the initial increase in MAP. MLA pretreatment almost completely normalized the biphasic change in heart rate to choline; whereas -bungarotoxin pretreatment seemed to inhibit only the initial tachycardic phase. The differential effect may be due to differences in drug distribution between the two antagonists, and it suggests that the two phases of the heart rate response may be mediated in different brain regions. The ability of i.c.v.-injected atropine to inhibit the heart rate changes to choline suggests that both muscarinic and nicotinic receptors play a role within the pathway mediating the bradycardic response to choline. The lack of effectiveness of atropine on the increase in blood pressure to choline indicates that the pressor pathway may not include muscarinic receptors.

This model not only provides a useful means for assessing the effectiveness of 7nAChR agonists and antagonists, but the results suggest that this subtype of nicotinic receptors plays some role in central cardiovascular regulation. For example, we noted earlier that central nicotinic receptors partly mediate the pressor responses to i.c.v. administration of certain neuroactive peptides such as bradykinin, angiotensin II, and substance P (Buccafusco and Serra, 1985; Trimarchi et al., 1986). More recently, we reported that in rat models genetically manipulated to maintain an elevated blood pressure, there is a link between decreased expression of brain 7nAChRs and working memory deficits (Gattu et al., 1997; Terry et al., 2001). In the present study the pressor response to i.c.v.-administered choline was blocked by pretreatment with the 7 subtype-selective antagonists MLA and -bungarotoxin. The lack of effect of pretreatment with DHE, the 2 subtype-preferring antagonist, supported the concept that the initial pressor response to choline was mediated by the 7 subtype. Of course, the participation of other non-7-containing subtypes cannot be ruled out at this time; but the data do fit the known specificity of choline for 7nAChRs (Papke et al., 1996; Pereira et al., 2002; Jonnala et al., 2003). Additional support may be derived from the finding that an analog of choline, CDP-choline, also increased blood pressure and decreased heart rate after i.c.v. microinjection in rats (Savci et al., 2002). The pressor effect was significantly reduced after pretreatment with the nonselective nicotinic antagonist mecamylamine.

Having characterized the specificity of the receptors mediating the pressor response to choline, it was then possible to determine whether i.c.v. administration of A1-42 would inhibit the subsequent pressor response to i.c.v.-injected choline. Cerebroventricular infusion, or repeated i.c.v. injection of A1-42 in rats has been used to assess the various pharmacological responses induced by the peptide, particularly regarding its potential amnestic actions (Maurice et al., 1996; Yamada et al., 1998; Nakamura et al., 2001; Yamaguchi and Kawashima, 2001; Olariu et al., 2002). Moreover, the related peptide A25-35 (15 nmol/injection) has been reported to impair short-term working memory even after a single acute injection (Stepanichev et al., 2003). The dose used in the memory experiment was 150-fold greater than the most effective dose we used to block the pressor response to choline.

The mechanism by which A1-42 interferes with the performance of certain memory-related tasks is not clear, but the peptide has this feature in common with the nicotinic receptor antagonist mecamylamine (Elrod and Buccafusco, 1991; Hiramatsu et al., 1998; Levin and Simon, 1998). Future studies will be required to determine whether the amnestic effects induced after central microinjection of A peptides are related to their ability to bind to 7nAChRs. Nevertheless based on the data presented here, it is clear that A1-42 does produce a functional blockade of 7nAChRs at low pharmacologic doses. The specificity of the A1-42 inhibition of the choline pressor response was supported by the failure of A40-1 peptide (which contains the critical 7nAChR binding sequence in reverse order) to block the choline response. Although A1-42 significantly inhibited the pressor response to choline, the maximal degree of inhibition amounted to about a 60% decrease relative to the control response. Clearly the nicotinic antagonists were more effective, nearly eliminating the choline-induced pressor response. This difference might reflect differences in the nature of the binding kinetics between A1-42 and the antagonists. Alternatively, A1-42 might not be distributed throughout the brain as readily as MLA and -bungarotoxin. Indeed, the potential site of action for choline in eliciting its pressor response is not known. Stimulation of specific hypothalamic sites with cholinergic drugs has been reported to generate a pressor response mediated via nicotinic receptors (Schaeppi, 1967; Day and Roach, 1977). A hypothalamic site of action would explain the rapid onset of the pressor response to choline as the drug would readily redistribute to periventricular hypothalamic regions. To support a hypothalamic site of action, the pressor response to i.c.v. injection of CDP-choline was partly mediated via the release of vasopressin into the circulation (Savci et al., 2002). Also, -bungarotoxin binding sites are relatively highly expressed in periventricular hypothalamic regions encompassing the dorsomedial, ventromedial, posterior hypothalamic nuclei, the dorsal premamillary nucleus, and the arcuate nucleus (Gattu et al., 1997; Tohyama and Takatsuji, 1998).

In summary, these data support the concept that the pressor response to i.c.v. microinjection of choline may be used as an unbiased measure of 7nAChR stimulation in vivo. They also support the ability of acute injection of A1-42 to produce a rapid functional blockade of 7nAChRs at pharmacologically (and likely physiologically in the case of AD) relevant doses. The use of telemetric catheters for monitoring cardiovascular status permits free movement of the animals and could allow for the assessment of various parameters of locomotor activity and other behaviors. Finally, some consideration should be given to the possibility that amyloid A-7nAChR interactions may play a role in the dysautonomia exhibited by some individuals with AD (Giubilei et al., 1998).

	Acknowledgements

 
We are grateful to Laura C. Shuster, who provided excellent technical support.

	Footnotes

 
This study was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.

DOI: 10.1124/jpet.103.063321.

ABBREVIATIONS: AD, Alzheimer's disease; 7nAChR, 7 nicotinic acetylcholine receptor; A1-42, amyloid peptide (1-42); MLA, methyllycaconitine; DHE, dihydro--erythroidin; MAP, mean arterial pressure; GTS-21, 3-(2,4-dimethoxybenzylidene)anabaseine; HR, heart rate; BTX, -bungarotoxin.

Address correspondence to: Dr. Jerry J. Buccafusco, Alzheimer's Research Center, Medical College of Georgia, Augusta, GA 30912-2300. E-mail: jbuccafu{at}mcg.edu

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