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Endocrinology Vol. 138, No. 5 1935-1943
Copyright © 1997 by The Endocrine Society

Articles

Adrenocorticotropin Response and Nicotine-Induced Norepinephrine Secretion in the Rat Paraventricular Nucleus Are Mediated through Brainstem Receptors1

Yitong Fu, Shannon G. Matta, James D. Valentine and Burt M. Sharp

Endocrine-Neuroscience Laboratories (Y.F., S.G.M., J.D.V., B.M.S.), Minneapolis Medical Research Foundation; and the Departments of Medicine (S.G.M., B.M.S.), Hennepin County Medical Center and the University of Minnesota, Minneapolis, Minnesota 55404

Address all correspondence and requests for reprints to: Burt M. Sharp, M.D., Endocrine-Neuroscience Laboratories, Minneapolis Medical Research Foundation, 914 South Eighth Street, Minneapolis, Minnesota 55404. E-mail: sharp002{at}maroon.tc.umn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nicotine is a potent stimulus for the secretion of ACTH, and norepinephrinergic neurons originating in the brainstem are involved. Prior reports using in vivo microdialysis in alert rats have shown that nicotine, administered ip or into the fourth ventricle, stimulated the release of norepinephrine (NE) into the hypothalamic paraventricular nucleus (PVN), the site of neurons containing CRH. In the present studies, rats received an iv infusion of nicotine into the jugular vein on alternate days during their active (dark) phase; therefore, direct correlations between the levels of NE microdialyzed from the PVN and plasma ACTH could be made in each animal. Nicotine administered iv (0.045–0.135 mg/kg) elicited dose-dependent increases in both NE and ACTH (P < 0.01). A significant correlation was found between nicotine-stimulated NE release in the PVN and ACTH secretion (r = 0.91, P < 0.01). To address whether the site(s) of action of nicotine was on presynaptic receptors on NE terminals in the PVN or on receptors on neurons in brainstem regions accessible from the fourth ventricle, the nicotinic cholinergic antagonist, mecamylamine (0.1–4.8 µg), was microinjected directly into the PVN or into the fourth ventricle before nicotine infusion. Fourth-ventricular administration of mecamylamine (1.6 µg) or higher, before iv nicotine (0.09 mg/kg), completely blocked both NE release in the PVN (IC50 = 0.64 µg) and ACTH secretion (IC50 = 0.40 µg) (P < 0.01, compared with vehicle before nicotine), whereas it was ineffective when injected directly into the PVN. The results demonstrate that the nicotinic cholinergic receptors in the brainstem, rather than presynaptic receptors within the PVN itself, mediate nicotine-stimulated PVN NE release and ACTH secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMIC paraventricular nucleus (PVN) plays an important role in the control of various endocrine, physiological, and behavioral responses. Peptidergic neurons in the PVN initiate the release of ACTH from the anterior pituitary (1), activate brainstem catecholaminergic neurons involved in cardiovascular and respiratory responses (2, 3), and modulate stress-induced freezing behavior (4, 5). One of the primary neurotransmitters involved in these functions seems to be norepinephrine (NE) (6, 7, 8). Extensive research has provided evidence that NE activates parvocellular CRH-containing neurons in the PVN, thus elevating plasma ACTH levels (1, 9, 10, 11, 12, 13).

Previous studies showed that ip administration or intracerebral ventricular (icv) injections of nicotine into the fourth ventricle (adjacent to the brainstem catecholaminergic regions) induced NE release in the PVN in conscious rats (14, 15). Nicotine-induced ACTH secretion also occurred when nicotine was administered by these same routes (12, 16, 17). Although the NE release was blocked by the nicotinic cholinergic antagonist, mecamylamine, the site of action of systemic nicotine (i.e. at the level of the catecholaminergic nerve terminals in the PVN or at the catecholaminergic cell bodies accessible from the fourth ventricle) is unknown.

The noradrenergic terminals in the PVN arise predominantly from three sites: the nucleus tractus solitarius (NTS)-A2, the A1 ventromedullary region, and (to a lesser extent) locus coeruleus (18, 19, 20). Nicotinic cholinergic receptors (NAchRs) are located both in regions containing NE bodies and in regions receiving NE terminal projections (21, 22, 23, 24, 25). In the NTS specifically, binding studies with 3H-nicotine and 125I-{alpha}-bungarotoxin demonstrated the presence of NAchRs (22, 23), and the mRNA for some of the receptor subunits has been shown in this region (24). Using high resolution autoradiography, Sharp et al. (25) demonstrated 3H-nicotine binding in the neuropil surrounding the PVN, in the regions of incoming NE terminals. Thus, nicotine may activate both sites, resulting in NE release.

In vivo studies indicate that sites accessible from the fourth ventricle contain NAchRs that mediate the activation of NE neurons in the brainstem in response to nicotine (15, 17, 26). Fourth ventricular injections of nicotine resulted in NE release in the PVN, and this action was blocked by mecamylamine (15). Investigations also have shown that ACTH release, caused by the administration of nicotine in the fourth ventricle or intraparenchymally into NTS, was blocked by mecamylamine (17, 26). In addition, infusion of mecamylamine into the fourth ventricle blocked the nicotine-induced expression of PVN cFos, a marker of neuronal activation (27). Taken together, these data suggest that NAchRs on brainstem neurons mediate the stimulatory effects of nicotine on both PVN NE and ACTH release. In contrast, in vitro studies have shown that nicotine stimulated NE release from terminals in hypothalamic slices or synaptosomal preparations (28, 29, 30). Therefore, the potential role of presynaptic NAchRs on PVN NE neurons has not been clarified.

In the current studies, initial investigations were performed to assess whether basal and nicotine-stimulated NE secretions were consistent when a rat was microdialyzed every other day during a 5-day interval (i.e. on days 1, 3, and 5). This approach was then used to establish dose-response relationships for both NE and ACTH release in the same rats, in response to nicotine infusions, to assess the correlation between these dependent variables across a range of nicotine doses. The relationship between NE and ACTH secretion was evaluated further by determining the IC50s for blockade of these responses by mecamylamine. Experiments also were performed to determine whether the site(s) of action of nicotine is on presynaptic receptors on NE terminals in the PVN or on postsynaptic receptors on neurons in brainstem regions accessible from the fourth ventricle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Nicotine sulfate (Pfaltz and Bauer, Inc., Waterbury, CT; dosage determined by the weight of the free base) was used for iv injection. NE hydrochloride, mecamylamine hydrochloride, and nomifensine maleate were purchased from RBI (Natick, MA). Sodium dihydrogen phosphate monohydrate (EM Science), 1-octanesulfonic acid sodium salt (J. T. Baker), triethylamine (Aldrich), EDTA (Fisher Scientific), acetonitrile and phosphate acid (EM Science, HPLC grade) were used for mobile phase preparation. The alert-rat microdialysis systems were obtained from CMA/Microdialysis (Acton, MA). Cellulose fiber tubing (SCE fiber; mol wt cutoff, 10K; od, 235 µm) was obtained from Cordis-Dow Medical Inc. (Miami Lakes, FL). Silica tubing (od, 148 µm; id 73 µm, TSP 075150) was made by Polymicron Technologies Inc.

Animals
All procedures were conducted in accordance with NIH Guidelines concerning the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Minneapolis Medical Research Foundation. Adult male Holtzman rats (250–350 g, HSD, Madison, WI) were used in all experiments and had access to standard rat chow and water ad libitum. Rats were individually housed on a 12-h reversed light cycle (lights off at 0900 h, on at 2100 h) for 14 days before the microdialysis experiments. After the rats had been housed under this reversed light/dark cycle for 7 days, they were anesthetized with xylazine-ketamine (5:35 mg/kg BW, im; Parke-Davis, Morris Plains, NJ), and chronic guide cannulae were stereotaxically implanted into the PVN and/or the fourth ventricle, according to the coordinates of Paxinos and Watson (31): PVN coordinates were AP, -2.1 mm; DV, +6.4 mm; ML, 0.8 mm, from bregma with a flat skull. Fourth ventricle coordinates were AP, -2.7 mm; DV, +2.6 mm; ML, 0.0 mm, relative to lambda and interaural line with flat skull. For experiments requiring the injection of mecamylamine directly into ipsilateral PVN, a double-guide cannula (20 gauge for the dialysis probe and 23 gauge for microinjection) was implanted at the above coordinates. Five days later, rats were jugular-cannulated under Innovar Vet anesthesia [droperidol (3.75 mg/kg) plus fentanyl (0.08 mg/kg); im; Far-Vet, St. Paul, MN) and allowed to recover for another 2 days.

In vivo microdialysis
A small, concentric probe (2 mm, constructed in our laboratory) was used in the present study. The recovery rate of individual probes was determined by in vitro dialysis in a solution containing 200 pg NE/16 µl for 40 min at 22 C; triplicate 20-min samples were obtained, and the average recovery rate was 8.4% ± 0.9 (n = 30). On the day of the experiment, the probe was perfused with a solution of Kreb’s Ringer Buffer (147 mM NaCl, 4.0 mM KCl, and 3.4 mM CaCl2 in polished water; 0.2 µm filter sterilized and degassed) containing 5 µM nomifensine (NE uptaker blocker) (32). Although the modifications reported herein (i.e. mobile-phase composition and ESA detector) resulted in detection of NE without the use of nomifensine, its inclusion was necessary as a basis for comparison with previous studies (14, 15).

This microdialysis model differs from that previously reported (14) in that a concentric dialysis probe was inserted acutely on alternate days in rats receiving an infusion of nicotine during their active (dark) phase. On the day of microdialysis, rats were moved into the alert-rat microdialysis chambers in an isolated dark room, lit only with a red safe-light, and all connections were made quickly to minimize stress to the animal. During the stabilization period that followed the insertion of each probe, the perfusion rate was adjusted at 30-min intervals to 4, 2, and 1 µl/min, respectively, and maintained at 1 µl/min. Twenty-minute samples were collected into glass vials containing 1 µl 5% perchloric acid.

At the end of the experiments, the position of the probe was verified by histological examination (see Fig. 1Go); only data obtained from animals with probes identified within the PVN were used for analysis. Histological identification of misplaced probes correlated with baseline NE levels consistently less than 5 pg/16 µl, whereas the normal baseline for PVN was approximately 12 pg/16 µl. If the baseline value of NE in a rat was below 5 pg/16 µl, the rat was eliminated from the study immediately. This was done because preliminary experiments showed a direct correlation of low baseline NE with misplacement of the dialysis probe into irrelevant regions [e.g. arcuate nucleus (posterior and deep to PVN), bed nucleus of the stria terminalis (anterior), or thalamus (shallow)]. The placement of the microdialysis probe and the acute insertion of the injection tubing into the PVN caused no greater damage than the microdialysis probe alone. This was verified histologically with cresyl violet stain and by demonstration of comparable baselines under both conditions: at zero min (before administration of nicotine, mecamylamine, or vehicle by any route), baseline levels of NE from rats with both the microdialysis probe and the injection tubing in place were 10.2 ± 1.0 pg/16 µl (data from experiment presented in Fig. 6Go); and NE levels were 10.3 ± 1.3 pg/16 µl in rats with the microdialysis probe alone (data from experiment presented in Fig. 2AGo). Placement of cannulae in the fourth ventricle was assessed by microinjection of 1 µl trypan blue; data were analyzed only from rats with blue in the ventricle and none in the surrounding tissue.



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  		Figure 1. Photomicrograph of the placement of a representative microdialysis probe in the hypothalamic PVN. The PVN undergoing dialysis is on the right side of the third ventricle (*), whereas the contralateral PVN is on the left. The track of the probe is indicated by the dashed line. The brain was fixed by cardiac perfusion with 4% paraformaldehyde in 0.05 M phosphate buffer, pH 6.8, at 4 C, and 25-µm cryosections were stained with cresyl violet. Magnification bar = 100 µm.

 


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  		Figure 6. Direct blockade of NAchRs in the PVN by mecamylamine had no effect on nicotine-stimulated NE release. NE release in the PVN was unaffected by a direct microinjection of mecamylamine into the PVN undergoing dialysis. n = 5–6 rats per group.

 


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  		Figure 2. Nicotine (Nic) stimulates NE release in the PVN in a dose-dependent manner. Panel A, The time course for NE release in the PVN in response to iv infusions nicotine compared with saline controls; panel B, peak incremental NE values in response to each dose of nicotine (the approximate ED50 was 0.065 mg/kg). **, P < 0.01, compared with saline; +, P < 0.05, compared with nicotine 0.045 mg/kg. n = 6–8 rats per treatment.

 
HPLC-electrochemical analysis
Samples were injected by a CMA 200 refrigerated autosampler onto a BAS 200A HPLC containing a 150 x 3 mm ODS C18 column perfused at 0.5 ml/min with a mobile phase containing 75 mM sodium dihydrogen phosphate monohydrate, 1.7 mM 1-octanesulfonic acid sodium salt, 100 µl/liter triethylamine, 5 nM EDTA, and 10% acetonitrile; pH 3.0. Samples were analyzed by an ESA Coulochem II 5200A electrochemical detector with an ESA 5041 high sensitivity microbore analytical cell and an ESA 5020 guard cell (ESA, Inc., Chelmsford, MA). Electrochemical detection was performed at 220 mV and 1.0 nA with the guard cell at 350 mV. The limit of detection for NE was 0.5 pg/16 µl.

ACTH RIA
Blood samples were collected from rats at 0 min, then 7 and 30 min after administration. Blood volume was normalized by replacement with 0.5 ml saline iv immediately after each sample was drawn. ACTH was measured on unextracted plasma samples by RIA, as previously described (16), using ACTH antibody Rb7 (a generous gift from Dr. William Engeland, University of Minnesota). The intra- and interassay coefficients of variation were 6.3% and 10.4%, respectively.

Experimental protocols
Systemic routes of injection, such as sc or ip, may induce pain or handling stress or activate vagal afferents, which in turn stimulate brainstem NE neurons. In addition, nicotine injected into the jugular vein has been reported to result in rapid presentation to the CNS (33, 34, 35). Therefore, in the present study, the jugular vein was used for administration of nicotine. Nicotine can induce aversive responses, such as respiratory depression and seizure, even at doses as low as 0.10 mg/kg iv in some rats, when the drug is infused over a 10- to 20-sec period (17, 36). However, in a recently published report on nicotine-stimulated cFos expression in the NTS and PVN, a relatively slow infusion rate (i.e. 0.09 mg/kg·60 sec iv) was used (27). Aversive behavioral responses were absent at the lower doses; therefore, these doses were used in the present study.

The initial experiment was performed to determine the stability of both the basal NE levels and the responses to nicotine, with repeated testing of each rat using a single probe. For each day’s experiment, three preinfusion (basal) microdialysis samples were collected over 60 min, then nicotine was infused iv at 0.135 mg/kg over 90 sec, and dialysates were continuously collected for 100 min. This procedure was repeated on day 3 and day 5 in the same cohort of rats. The second experiment was conducted to determine the dose-response relationship for nicotine-induced PVN NE and ACTH secretion. Rats randomly received infusions of saline or one of three doses of nicotine (each delivered at a constant rate of 0.09 mg/kg per 60 sec): 0.045 mg/kg over 30 sec, 0.09 mg/kg over 60 sec, or 0.135 mg/kg over 90 sec.

To determine whether NAchRs in brainstem regions accessible from the fourth ventricle are involved in nicotine-stimulated NE release in the PVN, rats randomly received artificial cerebrospinal fluid (CSF: 300 µg/ml BSA in 0.05 M phosphate buffer, pH 7.2) or mecamylamine (0.1, 0.4, 1.6, and 4.8 µg) in 500 nl over 60 sec icv into the fourth ventricle and 2 min later were infused iv with 0.09 mg/kg nicotine or saline over 60 sec.

To determine if presynaptic NAchRs on NE terminals in the PVN are involved in nicotine-induced NE release at that site, CSF or mecamylamine (0.4, 1.6, and 4.8 µg) was microinjected directly into the ipsilateral PVN in 200 nl over 2 min, then rats were infused iv with 0.09 mg/kg nicotine 2 min later. We have shown previously that the radial spread of 50 nl nicotine injected into brain tissue over 30 sec was 500–700 µm (37). Based on the assumption of comparable spread of these two highly lipophilic compounds of similar molecular weight, mecamylamine should have been available to all PVN neurons on the ipsilateral side of the injection, given that the anterior-posterior dimension of the PVN is approximately 900 µm (38).

Data analysis and statistics
Chromatograms were analyzed as previously reported (14) and expressed as the mean pg/16 µl sample ± SEM. Peak incremental responses were determined by subtracting the average NE concentration contained in the three basal microdialysates from the peak NE level in each rat. The peak incremental ACTH response was derived by subtracting the ACTH value at the zero-min time point from the peak value at 7 min for each rat. The correlation coefficient for the relationship between NE release and ACTH secretion was determined from the NE peak incremental value and the ACTH peak incremental value. Data were analyzed by one-way ANOVA for repeated measures using StatView. Results were considered significant at P less than 0.05. The number in parentheses (n) in the text and graphs is the number of rats within that treatment group for each study; a different cohort of rats was used for each study.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The microdialysis probe can be used at least three times without significant changes in the in vitro recovery rate of NE; the recovery rates were 8.4% ± 0.9 (n = 30) before use and 7.8% ± 0.5 (n = 24) after three uses. In addition, each rat could be tested three times (on days 1, 3, and 5), because neither preinfusion (baseline) levels of NE nor the peak NE response to nicotine (0.135 mg/kg iv) changed significantly with repeated dialysis (Table 1Go).


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  		Table 1. Lack of alteration in preinfusion (baseline) NE levels and in the amount of NE released in response to nicotine (0.135 mg/kg, iv; n = 7) with repeated microdialysis testing

 
The time course for NE release in the PVN in response to nicotine infusions during the active (dark) phase of the light cycle is shown in Fig. 2AGo. The level of NE after saline infusion did not change: 12.0 ± 1.4 pg/16 µl in the first dialysis sample (at 20 min) and 10.7 ± 1.5 pg/16 µl in the last sample, 160 min later. In addition, basal NE levels during the 60 min before infusion of any dose of nicotine were no different from saline controls; e.g. before nicotine, 0.09 mg/kg, the average NE level for the first three basal samples was 12.6 ± 0.2 pg/16 µl; the level was 11.2 ± 0.5 pg/16 µl for saline controls during the same time period.

NE concentrations were maximal within the first 20 min after the end of the nicotine infusions and returned to baseline levels immediately thereafter (Fig. 2AGo). The peak levels of NE in response to all doses of nicotine were significantly greater than saline controls (11.1 ± 1.3 pg/16 µl for saline): 15.9 ± 1.4 pg/16 µl after nicotine 0.045 mg/kg (P < 0.01, compared with saline), 19.1 ± 1.9 pg/16 µl after 0.09 mg/kg nicotine (P < 0.01) and 20.7 ± 1.1 pg/16 µl after nicotine 0.135 mg/kg (P < 0.01). In addition, NE release in response to the highest dose of nicotine was significantly greater than that produced by the lowest dose (P < 0.05).

Figure 2BGo shows the dose-response relationship for nicotine-stimulated PVN NE release; this is expressed as peak incremental values. NE release was linear over the range of doses tested (r = 0.99, P < 0.01). The high correlation (r = 0.99) indicates that peak NE values depended upon the dose of nicotine across the full range of doses tested. The equation for this relationship (y = 0.11 + 70.39x) shows that for each 0.045 mg/kg increase in the dose of nicotine, the expected increase in NE is 3.3 pg/16 µl. The approximate ED50 was 0.065 mg/kg nicotine, although a plateau in the NE response cannot be demonstrated because higher doses could not be tested without eliciting averse behavioral responses: e.g. seizure or prostration syndrome (17, 27, 34).

Figure 3Go illustrates the plasma ACTH levels in the same rats undergoing dialysis for NE. There was no difference in plasma ACTH at any time after saline infusion. In addition, there were no differences in ACTH levels between treatment groups before infusion of saline or nicotine (0 min time point). ACTH levels were elevated significantly within the first 7 min after the infusion of nicotine (0.045 mg/kg, 0.09 mg/kg, or 0.135 mg/kg) and returned toward baseline within 30 min: compared with saline controls (119.9 ± 18.3 pg/ml), ACTH values were 365.4 ± 63.7 pg/ml (P < 0.05), 546.9 ± 71.9 (P < 0.01), and 675.5 ± 86.1 pg/ml (P < 0.01), respectively. In addition, the ACTH response to the highest dose of nicotine was significantly greater than the response to the lowest dose (P < 0.05).



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  		Figure 3. Nicotine elevates plasma ACTH in a dose-dependent manner. The ED50 for ACTH release in response to nicotine infusion was approximately 0.045 mg/kg. **, P < 0.01, compared with saline; +, P < 0.05, compared with nicotine 0.045 mg/kg.

 
The ACTH baselines observed in the present study were higher than those previously reported by this laboratory (39) but were comparable with those that have been measured by others during the same portion of the light cycle (40). NE and ACTH release exhibit circadian rhythms, characterized by a peak of NE and an elevation of ACTH at the onset of the dark phase in nocturnal animals (40, 41). Therefore, the elevated baselines were caused by normal diurnal variation in plasma ACTH levels.

Figure 4Go illustrates the direct correlation between the change in plasma ACTH (peak incremental response) and the change in NE levels in the PVN (peak incremental release) after the infusion of saline or nicotine (r = 0.91, P < 0.01). This linear correlation indicated that a fixed relationship between NE and ACTH secretion held across the full range of nicotine doses. Thus, an NE increment of 2.0 pg/16 µl was associated with an ACTH increment of 100 pg/ml. To more clearly demonstrate a temporal correlation between PVN NE and plasma ACTH, dialysis samples were taken at 10-min intervals in a subset of rats. NE levels before 0.09 mg/kg nicotine were 5.2 ± 0.48 pg/8 µl, peaked at 9.3 ± 1.6 (P < 0.05, compared with baseline) in the sample obtained in the first 10 min after injecting nicotine, and returned to baseline in the 10 min thereafter (5.0 ± 0.5 pg/µl) (n = 5). Therefore, the peak ACTH response occurred within the time of the peak release of NE.



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  		Figure 4. The correlation between the changes in NE release in the PVN and in ACTH secretion in response to nicotine. The peak incremental change in plasma ACTH correlated directly with the peak incremental change in NE release in the PVN in rats infused with nicotine.

 
Figures 5AGo and 6Go demonstrate that the site of action of nicotine, with respect to NE release, is the norepinephrinergic neurons located in the brainstem that project to the PVN (Fig. 5AGo), rather than the presynaptic NAchRs on NE terminals located within the PVN (Fig. 6Go). Figure 5AGo shows that microinjection of the nicotinic cholinergic antagonist, mecamylamine, into the fourth ventricle resulted in a dose-dependent blockade of NE release in response to 0.09 mg/kg nicotine. Pretreatment with mecamylamine 1.6 µg icv reduced nicotine-stimulated NE release (P < 0.01 compared with CSF/nicotine) to levels no different from control values: mecamylamine 1.6 µg/nicotine = 1.6 ± 0.3 pg/16 µl and mecamylamine 1.6/saline (control) = 1.0 ± 0.5 pg/16 µl. The blockade of NE secretion by the higher dose of mecamylamine (4.8 µg; 1.2 ± 0.2 pg/16 µl) was not different from mecamylamine 1.6 µg/nicotine nor from mecamylamine 1.6 µg/saline, whereas mecamylamine 0.4 µg/nicotine partially inhibited NE release (3.4 ± 0.6 pg/16 µl; P < 0.05 compared with CSF/nicotine and P < 0.05, compared with mecamylamine 1.6 µg/saline). The IC50 for mecamylamine blockade of PVN NE release was 0.64 µg.



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  		Figure 5. Dose-dependent blockade of nicotine-induced NE and ACTH release after microinjection of mecamylamine (Mec) into the fourth ventricle. Panel A shows that NE release in the PVN was blocked in a dose-dependent manner by an icv injection Mec before 0.09 mg/kg nicotine iv. Panel B shows that ACTH release in response to nicotine was significantly reduced by mecamylamine. *, P < 0.05; **, P < 0.01 [compared with CSF/nicotine (positive control)]; +, P < 0.05, compared with mecamylamine 1.6/saline (negative control). n = 5 rats per treatment.

 
In addition, as shown in Fig. 5BGo, microinjection of mecamylamine icv 2 min before nicotine infusion resulted in a dose-dependent blockade of ACTH release in these same rats. Pretreatment with mecamylamine 0.1 µg was ineffective in blocking ACTH secretion, compared with CSF/nicotine controls (456.0 ± 56.5 pg/ml and 592.0 ± 54.9 pg/ml, respectively), whereas doses of mecamylamine 0.4 µg or higher significantly reduced nicotine-stimulated ACTH levels: e.g. mecamylamine 1.6 µg/nicotine = 256.1 ± 49.6 pg/ml; P < 0.05). The IC50 for mecamylamine blockade of ACTH secretion in response to nicotine was 0.40 µg. Finally, mecamylamine 1.6 µg icv alone had no effect on ACTH release.

In contrast to the efficacy of mecamylamine blockade of NAchRs in regions accessible from the fourth ventricle, Fig. 6Go demonstrates that when the same doses of mecamylamine were microinjected directly into the PVN undergoing dialysis, there was no blockade of nicotine-stimulated NE release within the ipsilateral PVN. The NE release in rats receiving even the highest dose of mecamylamine (mecamylamine 4.8 µg/nicotine, 7.5 ± 1.3 pg/16 µl) was no different from control rats (CSF/nicotine, 6.9 ± 1.0 pg/16 µl). In addition, this dose of mecamylamine alone (mecamylamine 4.8 µg/saline, 1.4 ± 0.8 pg/16 µl) had no effect on NE secretion, compared with unstimulated controls (CSF/saline, 0.7 ± 0.7 pg/16 µl).

Because the contralateral PVN was intact and fully responsive, it was expected that the ACTH response to iv nicotine would be unaffected by the unilateral injection of mecamylamine. After mecamylamine 4.8 µg/nicotine (503.5 ± 62.0 pg/ml), ACTH levels were no different from CSF/nicotine (539.4 ± 71.1 pg/ml; F (3, 18) = 0.128, P = 0.94). In addition mecamylamine 4.8 µg/saline alone did not alter baseline ACTH levels: 198.6 ± 21.2 pg/ml and 148.8 ± 23.5 pg/ml, respectively; [F (2, 13) = 0.244, P = 0.79).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have implicated NE in nicotine-stimulated ACTH release. The catecholaminergic neurotoxin, 6-hydroxydopamine, abolished the ACTH response to iv or fourth-ventricular nicotine (12), and depletion of the hypothalamic NE content reduced CRH release (13). It also was shown that administration of {alpha}1 and {alpha}2 adrenergic antagonists into the hypothalamic region of the third ventricle (adjacent to the PVN) blocked the ACTH response to nicotine (12). The current report substantiates these findings by demonstrating a strong correlation between the effect of nicotine on NE release in the PVN and subsequent ACTH release from the pituitary (Fig. 4Go). Furthermore, iv nicotine was shown to stimulate the secretion of both NE and ACTH by acting on NAchRs accessible from the fourth ventricle and not directly at the PVN (Figs. 5Go and 6Go). Taken together, these data demonstrate that brainstem norepinephrinergic neurons, activated by nicotine are involved in ACTH release.

The NTS is most likely the site for these norepinephrinergic neurons. NAchRs have been localized within the NTS (22, 24), although the identity of the neurons containing the receptors and whether the receptors are pre- or postsynaptically located are undetermined. In addition, iv nicotine stimulated neuronal activation (as measured by cFos expression) in NTS neurons containing the catecholaminergic biosynthetic enzyme, tyrosine hydroxylase, and this activation was blocked by an injection of mecamylamine into the fourth ventricle (27). Direct microinjection (50 nl) of nicotine into the NTS resulted in a dose-dependent elevation in plasma ACTH that was blocked by a prior injection of mecamylamine (26). The current study demonstrated that both NE release in the PVN and ACTH secretion were blocked by a fourth-ventricular injection of mecamylamine (Fig. 5Go). Therefore, NAchRs on neurons in the NTS seem to be activated by systemic nicotine, leading to both NE release in the PVN and ACTH secretion.

The effect of iv nicotine on NE release in the PVN also could be caused by its action on presynaptic NAchRs on NE terminals within the PVN itself, in addition to activation of NAchRs on neurons within the NTS. Indeed, in vitro studies have shown that nicotine stimulated NE release from terminals in hypothalamic slices or synaptosomes (28, 29) and nicotine stimulated the release of CRH from hypothalamic explants (42, 43, 44). Taken together, the assumption would be that nicotine, acting presynaptically on NE terminals, caused the release of NE, which resulted in CRH release from the PVN. However, the current study demonstrated that presynaptic receptors were not involved in vivo, because microinjection of mecamylamine directly into the PVN was completely ineffective in blocking the release of NE in response to iv nicotine (Fig. 6Go). Furthermore, bilateral injections of mecamylamine into the PVN in vivo were ineffective in blocking nicotine-stimulated ACTH release (17). The basis for this discrepancy between in vitro and in vivo findings is not readily apparent. The doses of nicotine used in each model cannot be compared but may be a contributing factor. In addition, the response(s) of the deafferented terminals in in vitro studies may be altered.

A similar effect of systemic nicotine on NE release in the hippocampus also has been attributed to the action of nicotine on brainstem NE neurons, rather than on local presynaptic NE terminals (45). In that in vivo microdialysis study, blockade of NAchRs in hippocampus by a local microinjection of mecamylamine did not affect NE release via systemic nicotine, whereas blockade of NAchRs in locus coeruleus (the major input of NE to the hippocampus) effectively inhibited NE release in the ipsilateral hippocampus. A comparable relationship also has been shown between the action of systemic nicotine on NAchRs on ventral tegmental neurons and subsequent dopamine release in nucleus accumbens (46).

A direct action of nicotine on CRH neurons in the PVN, independent of NE modulation, is possible, because projections to PVN containing choline acetyltransferase have been demonstrated (47). In addition, both nicotinic ({alpha}-bungarotoxin binding) and muscarinic (propylbenzilcholine binding) cholinergic receptors have been identified in the hypothalamus by autoradiography (21, 48). Finally, in vitro studies have indicated that nicotine and acetylcholine directly stimulated CRH release from hypothalamic explants (42, 43, 44, 49, 50). However, only acetylcholine has been shown to activate CRH neurons in vivo (51). In this recent study, it was shown that acetylcholine-stimulated ACTH release was mediated via muscarinic cholinergic receptors, not NAchRs in the PVN, because it was sensitive to blockade by icv administration of the muscarinic antagonist, atropine, but was insensitive to the nicotinic antagonist, hexamethonium. Also in that study, the ACTH release, in response to an icv injection of acetylcholine, reached its peak around 30 min and returned to baseline within 120 min (51). In contrast, in another report, an icv injection of nicotine elevated plasma ACTH peak at 7 min and normalized in 30 min (17). These studies indicate a difference between nicotine- and acetycholine-induced ACTH secretion. Finally, bilateral injection of mecamylamine directly into the PVN did not block the ACTH response to systemic nicotine (17). Therefore, it is unlikely that nicotine, at the doses used in the present in vivo study, has a direct effect on CRH neurons of the PVN.

It is possible, however, that systemic nicotine may have a direct effect on CRH terminals in the median eminence. Immunoelectron microscopy has demonstrated the presence of the {alpha}4 subunit of NAchRs on CRH-containing terminals in the external layer of the median eminence (52). Although NE terminals also are present in the median eminence (53), NAchR subunits have not been colocalized to these. Therefore, an action of iv nicotine at CRH terminals is conceivable, although the effect of nicotine on NE terminals at this site is undetermined.

Although the rostral NE projections from the NTS to the PVN are involved in nicotine-stimulated ACTH release, an alternative, mecamylamine-sensitive pathway also could be affected by the action of nicotine at this site. Nicotinic activation of the descending sympathetic pathway from the NTS through the spinal cord to the sympathetic ganglia and subsequent nerve terminals in the peripheral vasculature would release NE into circulation. This pathway could potentially be an additional means whereby NE could contribute to ACTH secretion via direct noradrenergic action on the pituitary (54).

In summary, the results presented herein demonstrate that NAchRs in brainstem regions accessible from the fourth ventricle are involved in the PVN NE response to systemic nicotine. The data show a strong correlation between nicotine-stimulated NE release in the PVN and subsequent ACTH secretion. In addition, similar IC50 values for inhibition of both NE and ACTH secretion by mecamylamine were found (Figs. 5BGo and 5AGo). Taken together with previous studies showing that the administration of {alpha}-adrenergic antagonists into the hypothalamic region of the third ventricle significantly reduced iv nicotine-induced ACTH secretion (12), the present studies indicate that NE originating from the NTS is an important mediator of the ACTH response to iv nicotine.


   Acknowledgments
 
The authors would like to express their appreciation for the expert technical assistance of Kathleen McAllen and Tressa James.


   Footnotes
 
1 This work was supported by NIH Grant DA-03977 (to B.M.S.) and a postdoctoral fellowship in the Neuroscience Training in Drug Abuse Research program at the University of Minnesota (T32-DA-07234, to J.D.V.). Back

Received September 23, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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