1. ABSTRACTThe hypothalamic-pituitary-adrenal (HPA) axis is a three-component endocrine system that modulates physiological responses to stress. To better
understand the biology of nicotine (NIC) addiction, we developed an in vivo model of continuous NIC administration and withdrawal in laboratory rats, in
order to study HPA axis stress responses to NIC. Our earlier studies demonstrated that HPA responses to NIC were reduced and transient following
continuous NIC administration, but were enhanced and sustained following NIC “withdrawal” by mecamylamine (MEC; a NIC receptor antagonist).
In the present study, following in vivo continuous NIC administration and withdrawal, we determined HPA axis hormone responses to NIC and MEC in
a three-flask, in vitro model of the HPA axis. Hypothalami, pituitaries, and adrenal glands were collected from male rats under two dosing conditions: 1)
immediately following 2-week continuous NIC via thrice daily injections plus voluntary consumption of NIC in drinking water (to model NIC habituation),
and 2) 24 h after cessation of 2-week continuous NIC (to model NIC withdrawal). For each axis studied, one-half hypothalamus, one-half pituitary, and
one adrenal gland were placed individually into three temperature-controlled flasks connected by tubing and perfused in series with modified Bradbury
buffer. Sampling ports between flasks were used to collect buffer at intervals before and after addition of NIC and MEC, for measurement of
corticotropin-releasing hormone from the hypothalamus flask, adrenocorticotropic hormone from the pituitary flask, and corticosterone from the adrenal
flask. Hormones were measured by highly specific immunoassays.
The in vitro system maintained stable temperatures, flow rates, pH and hormone baselines. In vitro HPA responses were significantly higher in the
continuous NIC group than in the NIC withdrawal group. MEC addition to the hypothalamus flask decreased HPA axis activity in the continuous NIC
group but had little effect in the NIC withdrawal group.
These results suggest that in vitro HPA responses are enhanced and sustained following continuous NIC, and reduced following NIC withdrawal. The
findings stand in contrast to our previous in vivo results with continuous NIC and its withdrawal, as described above. Possible mechanisms include
absence of suprahypothalamic influences in the in vitro system and dilution of NIC concentrations in the second and third flasks. Further in vitro as well
as in vivo studies addressing the complex relationships among NIC, stress, and the HPA axis may help elucidate new approaches to the understanding
and treatment of nicotine addiction.
Animals. Six, eight-week old, male Sprague-Dawley rats weighing 200-225 grams (Hilltop Lab Animals, Inc.) were used in this study. Rats were singly housed on a 12 h light/dark cycle with food and water available ad libitum.
Continuous NIC Administration. Rats received thrice-daily injections of NIC (0.3 mg/kg) by intraperitoneal (IP) administration for 14 days (Matta et al 2007). Injections occurred at approximately 0900, 1300, and 1700 h. In addition to ad libitum tap water, rats also were
provided a second drinking source containing 0.006% NIC. The volume of NIC solution consumed daily was recorded to determine oral NIC intake. When combined with daily injections, total NIC intake ranged from 1.1–2.3 mg/kg/day (Figure 2), the approximate exposure of a
smoker who uses a total of one-third to one-half pack per day.
Experimental Design Summary. At the conclusion of the two week NIC administration, the tissues from 3 rats were studied immediately following the 2-week NIC administration (therefore, these animals should have been experiencing NIC habituation;
i.e., these animals modeled NIC addiction), and the tissues from the other 3 rats were studied 24 hours following 2-week NIC administration (therefore, these animals should have been experiencing NIC withdrawal).
In Vivo Groups (N = 6) In Vitro Axes (N = 12) In Vitro Drug Additions
Habituation Group: Continuous NIC for two weeks (N = 3 male rats) Habituation following continuous NIC for two weeks (N = 6) NIC (N = 6), MEC (N = 6)
Withdrawal Group: 24 h Following Continuous NIC for two weeks (N = 3 male rats) Withdrawal following continuous NIC for two weeks (N = 6) NIC (N = 6), MEC (N = 6)
Tissue Isolation. The hypothalamus was isolated by the “block method” (Hatton et al 1980). The hypothalamus and pituitary were bisected, and the adrenal glands were removed by ventral approach and cleared of adipose tissue. Next, the tissues were incubated individually
in modified Bradbury tissue culture medium (pH 7.4) at 37°C (Bradbury et al 1974; Garrido et al 1999), weighed, contained within stainless steel screens, and immediately placed in jacketed tissue-baths comprising the in vitro perfusion system (Figure 1).
In Vitro Perfusion System. Flow rates of the culture medium ranged between 1.6–3.7 ml/min. Mean temperatures were 36.2 ± 0.2°C in the hypothalamus tissue baths, 36.8 ± 0.1°C in the pituitary tissue baths, and 36.7 ± 0.1°C in the adrenal tissue baths.
At the end of each experiment, the tissues were exposed to 60 mM potassium chloride (KCl) to test tissue responsiveness and viability to membrane depolarization (Gao et al 2000) (Figure 3). The duration of each in vitro experiment was 80 min.
In Vitro NIC and MEC Addition and Sampling. Three 0.2 mL NIC injections (each 0.2 nmol free base NIC), were added into the hypothalamic bath yielding a final flask concentration approximating the plasma NIC concentration after smoking one cigarette
(50 ng/mL) (Cam et al., 1979). Following a post-NIC injection equilibration, three 0.2mL MEC injections (each 2 nmol MEC) were added into the hypothalamic bath yielding a final bath concentration of approximately 1000 nM. Culture medium samples were
collected 10 min prior to, and 5 and 15 min after, drug additions and analyzed for CRH, ACTH, and CORT.
Hormone Assays. CRH, ACTH, and CORT were analyzed with enzyme immunoassays. Inter- and intra-assay coefficients of variation for all assays were 5-9% and 6-8% respectively.
Statistical Analysis. Statistically significant differences were determined by three-way ANOVA ( condition: habituation vs. withdrawal,  drug: NIC vs. MEC vs. KCl, and  time: -10, 5, and 15). Post hoc comparisons were made with Tukey-Kramer and
Fisher’s LSD tests to determine the location(s) of significance. Statistical significance was considered as p < 0.05.
 Our results suggest that in vitro HPA responses are enhanced and sustained following continuous NIC administration for 2
weeks, and reduced following NIC withdrawal. As well, our results suggest that tissue responses to MEC are enhanced
following continuous NIC (resulting in decreased hormone release), but not during NIC withdrawal.
 Our earlier in vivo studies demonstrated that HPA responses to NIC were reduced and transient following continuous NIC
administration, but were enhanced and sustained following NIC withdrawal by MEC. Our in vitro findings stand in contrast to
our previous in vivo results. Several possibilities may explain these reciprocal findings:
 Supra-hypothalamic brain areas that may have been sensitized to the effects of NIC were removed in the in vitro system, and their removal
may have influenced the results of the present study. Previous studies have demonstrated that NIC stimulation of HPA axis activity results from
activation of brain stem areas, particularly adrenergic pathways of the solitary tract nucleus that extend to the paraventricular nucleus of the
hypothalamus (Matta et al, 1998). As well, NIC addiction and withdrawal have been shown to influence other brain areas that directly or
indirectly influence HPA axis activity, including the amygdala, bed nucleus of the stria terminalis, and hippocampus (Zorrilla et al, 2014).
 Dilution of NIC concentrations as buffer perfused through the in vitro system may have contributed to the results of the present study. Future
studies will include measurement of NIC concentrations at each tissue bath for correlations with HPA hormone measurements.
 Extension of these studies to include female groups to parallel our previous in vivo studies on sexually diergic effects of
continuous NIC and its withdrawal will be a prudent next step.
 These studies may help elucidate new approaches to the understanding and treatment of nicotine addiction.
Bradbury M.W.B., Burden J., Hillhouse E.W. (1974) Stimulation electrically and by acetylcholine of the rat hypothalamus in vitro. J. Physiol. 239: 269-283.
Cam G.R., Bassett J.R., Cairncross K.D. (1979) The action of nicotine on the pituitary-adrenal cortical axis. Arch. Int. Pharmacodyn. 237:49-66.
Gao L.Z., Zhang W.H., Ju G. (2000) Suppression of adrenocorticotropic hormone release by stimulation of the nerve fibers in the anterior pituitary. J. Endocrinol. 12: 753-757.
Garrido M.M., Manzanares J., Fuentes J.A. (1999) Hypothalamus, anterior pituitary and adrenal gland involvement in the activation of adrenocorticotropin and corticosterone secretion by gastrin-releasing peptide. Brain. Res. 828: 20-26.Cam, G.R.; Bassett, J.R.; Cairncross, K.D. (1979) The action of nicotine on the pituitary-
adrenal cortical axis. Arch. Int. Pharmacodyn. 237:49-66.
Gentile N.E., Andrekanic J.D., Karwoski T.E., Czambel R.K., Rubin R.T., Rhodes M.E. (2011) Sexually diergic hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine, continuous nicotine infusion, and nicotine withdrawal by mecamylamine in rats. Brain Res Bull. 85:145-152.
Hatton G.I., Doran A.D., Salm A.K., Tweedle C.D. (1980) Brain slice preparation: hypothalamus. Brain. Res. Bull. 5: 405-414.
Matta S.G., Yitong F., Valentine J.D., Sharp B.M. (1998) Response to the hypothalamic-pituitary-adrenal axis to nicotine. Psychoneuroendocrinology. 23:103-113.
Matta SG, Balfour DJ, Benowitz NL, Boyd RT, Buccafusco JJ, Caggiula AR, et al. Guidelines on nicotine dose selection for in vivo research. Psychopharmacology (Berl). 2007;190:269-319.
McKlveen J.M., Wilson J.M., Rubin R.T., Rhodes M.E. (2010) Sexually diergic, dose-dependent hypothalamic-pituitary-adrenal axis responses to nicotine in a dynamic in vitro perfusion system. J. Pharmacol. Toxicol. Methods. 61:311-318.
Moidel M.A., Belz E.E., Czambel R.K., Rubin R.T., Rhodes M.E. (2006) Novel in vitro perfusion system for the determination of hypothalamic-pituitary-adrenal axis responses. J. Pharmacol. Toxicol. Methods. 53:264-271.
Rhodes M. E., O'Toole S. M., Wright S. L., Czambel R. K., Rubin R. T. (2001a) Sexual diergism in rat hypothalamic-pituitary-adrenal axis responses to cholinergic stimulation and antagonism. Brain Res Bull. 54:101-113.
Rhodes M.E., O’Toole S.M., Czambel R.K., Rubin R.T. (2001b) Male-female differences in rat hypothalamic-pituitary-adrenal axis responses to nicotine stimulation. Brain Res. Bull. 54:681-688.
Rhodes, M. E., Balestreire, E. M., Kenneth Czambel, R., Rubin, R. T. (2002) Estrous cycle influences on sexual diergism of HPA axis responses to cholinergic stimulation in rats. Brain Res Bull. 59:217-225.
Rhodes, M. E., Kennell, J. S., Belz, E. E., Czambel, R. K., Rubin, R. T. (2004) Rat estrous cycle influences the sexual diergism of HPA axis stimulation by nicotine. Brain Res Bull. 64:205-213.
Skwara A.J., Karwoski T.E., Czambel R.K., Rubin R.T., Rhodes M.E. (2012) Influence of environmental enrichment on hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine, continuous nicotine by osmotic mini-pumps, and nicotine withdrawal by mecamylamine in male and female rats. Behav. Brain Res.
234:1-10.
Zorrilla E.P., Logrip M.L., Koob G.F. (2014) Corticotropin releasing factor: A key role in the neurobiology of addiction. Front. Neuroendocrinol. 35:234-244.
Our in vitro system maintained stable physiological parameters and paralleled hormone responses reported in our
previous in vitro studies with NIC (Moidel et al 2006, McKlveen et al 2010).
 Figure 4 (left column): Effects of NIC addition in continuous NIC (pink) and withdrawal (purple) groups: In vitro
CRH, ACTH, and CORT responses to NIC were higher in continuous NIC groups compared to the NIC
withdrawal groups. CRH and CORT hormones were significantly higher before and after NIC addition (p’s <
0.05).
 Figure 4 (right column): Effects of MEC addition in continuous NIC (pink) and withdrawal (purple) groups: The
in vitro CRH baseline was higher in the continuous NIC group compared to the NIC withdrawal group.
Following MEC addition, CRH responses at 5 and 15 min were significantly reduced compared to baseline CRH
concentrations (p’s < 0.05). ACTH responses to MEC also were lower at 15 min.
METHODS
INTRODUCTION
RESULTS
Tissue hypothalamic-pituitary-adrenal axis responses to nicotine and mecamylamine
following in vivo continuous nicotine administration and withdrawal
Michael E. Rhodes1
, Lauren E. Harbaugh1
, Julie A. Rutkauskas1
, Robert T. Rubin2
1
Department of Biology, Saint Vincent College, Latrobe, PA and 2
Department of Psychiatry, VA Greater Los Angeles Healthcare System, Los Angeles, CA
DISCUSSION & CONCLUSIONS
REFERENCES
Figure 2: Total NIC intake during the 14 day NIC administration period. Rats that were presented with
voluntary 0.006% NIC solution consumed 0.2–1.4 mg/kg/day. Combining the thrice-daily injections with
drinking water consumption, rats were delivered a semi-continuous administration of NIC at
approximately 1.1–2.3 mg/kg/day for 14 days (the approximate exposure of a smoker who uses
approximately one-third to one-half pack of cigarettes per day).
Each bar represents the mean ± SEM of 6 rats.
Figure 3: CRH and ACTH concentrations pre- and post-KCl addition. Hormone responses to KCl
provide validation of tissue viability and responsiveness to stimulation post-experimentally. *
represents a hormone concentration difference compared to baseline values at indicated time points
(p < 0.05).
Each bar represents the mean ± SEM of 6 rats.
Figure 1: In vitro perfusion system that
models the HPA axis.
NIC at 0 min MEC at 0 min
a
a
a a
b
a a
a
a
b b
 Studies addressing the complex relationships among sex, NIC intake and withdrawal,
and stress may help to develop new approaches to the understanding and treatment
of NIC addiction.
 The hypothalamic-pituitary-adrenal (HPA) axis is a three-gland component of the
endocrine system and an important modulator of the stress response. Our
laboratory’s focus has been to study sexual diergism of HPA axis responses to
cholinergic stimulation and antagonism (Rhodes et al, 2001a, 2001b, 2002, 2004).
 In recent years, our laboratory has developed an in vivo model to better understand
the biology of NIC addiction and withdrawal by studying HPA axis hormones
following continuous NIC administration and its withdrawal in male and female
laboratory rats. Results from these studies demonstrated that HPA responses were
reduced and transient following continuous NIC but were enhanced and sustained
following NIC withdrawal (Gentile et al, 2011; Skwara et al, 2012).
 In the present study, following in vivo continuous NIC administration and withdrawal,
we determined HPA axis tissue responses to NIC and the NIC antagonist,
mecamylamine (MEC), in a dynamic, three-flask, in vitro model (Figure 1) of the
HPA axis.
*
* *
Figure 4: CRH, ACTH, and CORT concentrations before and after drug additions at 0 min. Left
column = nicotine (NIC) addition at 0 min; right column = mecamylamine (MEC) addition at 0 min. a
= difference between continuous NIC and NIC withdrawal at indicated time points (p < 0.05), b =
difference from baseline at indicated time points (p < 0.05). Each point represents the mean ± SEM
of 6 axes.
Continuous NIC = pink circles.
NIC Withdrawal = purple squares.
Supported by 2014-2015 Faculty Research Grant to Michael E. Rhodes, Ph.D.