This study examined hormone responses of the hypothalamic-pituitary-adrenal (HPA) axis to nicotine and mecamylamine in an in vitro model, using tissues from rats that underwent continuous nicotine administration and withdrawal. The results suggest that in vitro HPA responses are enhanced and sustained following continuous nicotine administration, but reduced following nicotine withdrawal. Additionally, tissue responses to mecamylamine were enhanced after continuous nicotine, resulting in decreased hormone release, but not during nicotine withdrawal. These findings contrast with the authors' previous in vivo studies, possibly due to removal of suprahypothalamic influences or dilution of nicotine concentrations in the in vitro system. Further studies are needed to better
Preclinical Screening for Neurodegenerative Disease (Parkinsonism)Drx Burade
This file includes the general introduction of Parkinson's, sign and symptoms of Parkinson's, treatment of Parkinson's and the main content that is the Preclinical Screening models for Neurodegenerative disease like Parkinson's
Preclinical Screening for Neurodegenerative Disease (Parkinsonism)Drx Burade
This file includes the general introduction of Parkinson's, sign and symptoms of Parkinson's, treatment of Parkinson's and the main content that is the Preclinical Screening models for Neurodegenerative disease like Parkinson's
Metabolic Adaptations: The Physiology of Cold Acclimatization and Exercise in...InsideScientific
Metabolic Adaptations to Exercise: Role of NAMPT and NAD+ in Skeletal Muscle
Jonas Treebak, PhD
NAD+ is a cofactor implicated in important metabolic reactions, such as oxidative phosphorylation. One of the highest rates of mitochondrial respiration is found in contracting skeletal muscle. Nicotinamide phosphoribosyltransferase (NAMPT) is essential for skeletal muscle NAD+ synthesis. Skeletal muscle-specific knockout of Nampt (SMNKO) reduces skeletal muscle NAD+ levels by 85-90%.This leads to mitochondrial dysfunction, energy deficiency, poor contractility, low exercise performance, and progressive muscle degeneration. The Treebak lab generated inducible SMNKO mice (iSMNKO) and achieved a comparable depletion of NAD+. iSMNKO mice did not show altered mitochondrial respiratory capacity at rest or after exercise in isolated intact fibers. Moreover, high resolution respirometry in mitochondria isolated from iSMNKO muscle exhibited unchanged respiration and ATP synthesis, as well as normal P/O and mitochondrial membrane potential. Interestingly, iSMNKO skeletal muscle has lower capacity for succinate-induced mitochondrial ROS production compared to the wildtype control. iSMNKO mice displayed normal VO2 at rest and during an acute bout of maximal exercise, they behaved similarly in response to prolonged voluntary wheel (VW) running, but failed to increase performance after VW training. Collectively, their data demonstrate that the role of NAD in mitochondrial function may be more nuanced than is often thought.
Cold Acclimatization – Fundamental Mechanisms and Popular Misconceptions
Alexander Bartelt, PhD
Homeotherm mammals like rodents or humans regulate their body temperature by means of thermogenesis. Below the thermoneutral zone, where basal metabolism is sufficient to maintain homeothermy, thermogenic mechanisms are activated to contribute to heat production. This adaptation to cold exposure is a phasic process with acute activation of existing stores, acquisition of extra nutrients as fuels, and finally extensive remodeling of cells, tissues, and physiology, altogether facilitating cold acclimatization. Most notably are shivering and non-shivering thermogenesis by myocytes and thermogenic adipocytes, respectively. In this seminar, Dr. Bartelt provides an overview of the phasic process of cold acclimatization, elaborates on novel, fundamental mechanisms, including his lab’s own work on the transcription factor Nfe2l1, and, finally, highlights misconceptions that are frequently found in the literature.
Thermal Physiology: The Effects of Environmental Temperatures on Energy Expen...InsideScientific
Mice are generally an excellent model of human biology with nearly identical metabolic pathways. In contrast, the 3000-fold difference in body mass causes huge differences in thermal physiology and energy homeostasis. Humans generally live in a thermoneutral environment, while mice live and are typically studied below thermoneutrality. A mouse housed singly at 22 °C devotes 42% of its energy expenditure to maintaining its body temperature; the corresponding value in humans is approximately 0%. Understanding this different physiology is important, allowing one to avoid incorrect application of mouse observations to humans. It also boosts elucidation of physiology that is subtle or difficult to study in humans.
The goal is to understand thermal physiology and to use it to develop conditions under which mice better model humans. This is important for studying the effectiveness of drug treatments for metabolic diseases, like obesity and diabetes. Marc and Oksana discuss what thermoneutrality means in the mouse and the concept of the thermoneutral point. They also explore the effects of cold, hot, and near-thermoneutral environments on mouse energy expenditure, body temperature, and behavior.
Based on Capillary Gate Theory and Tissue Repair Theory, this presentation will explain the recently identified “Stress Repair Mechanism” (SRM) that enables the long-anticipated Universal Theory of Medicine postulated by Hans Selye in 1954. The SRM maintains and repairs vertebrate tissues and accounts for most of the mysterious manifestations of allostasis that remain unexplained by Hypothalamic-Pituitary-Axis (HPA) hormones. SRM activity explains hemodynamic physiology, capillary hemostasis, infarction, Korotkoff sounds, blood pressure, hypertension, diabetes, allostasis, allostatic load, anesthesia, analgesia, atherosclerosis, apoptosis, malignancy, eclampsia, sepsis, Multi-System Organ Failure (MSOF), the surgical stress syndrome, the fight or flight response, and numerous other manifestations of physiology, pathology, and allostasis. SRM function comprises the autonomic nervous system, the vascular endothelium, and the dynamic enzymatic interaction of blood-borne hepatic Factors VII, VIIIC, IX and X that produces thrombin, soluble fibrin and insoluble fibrin, whose combined effects account for all SRM manifestations. The vascular endothelium is a diaphanous neuroendocrine organ that lines all blood vessels and is the sole constituent of capillary walls. It secretes tissue factor into extravascular tissues, and insulates those tissues from the hepatic enzymes, so that tissue disruption exposes tissue factor to the enzymatic interaction and activates tissue repair. The vascular endothelium also releases nitric oxide and von Willebrand Factor into blood in accord with autonomic balance to regulate the enzymatic interaction to govern tissue perfusion and organ function. Therefore, continuously fluctuating combinations of nervous stimuli that affect autonomic balance and forces that disrupt tissues determine SRM activity.
Metabolic Adaptations: The Physiology of Cold Acclimatization and Exercise in...InsideScientific
Metabolic Adaptations to Exercise: Role of NAMPT and NAD+ in Skeletal Muscle
Jonas Treebak, PhD
NAD+ is a cofactor implicated in important metabolic reactions, such as oxidative phosphorylation. One of the highest rates of mitochondrial respiration is found in contracting skeletal muscle. Nicotinamide phosphoribosyltransferase (NAMPT) is essential for skeletal muscle NAD+ synthesis. Skeletal muscle-specific knockout of Nampt (SMNKO) reduces skeletal muscle NAD+ levels by 85-90%.This leads to mitochondrial dysfunction, energy deficiency, poor contractility, low exercise performance, and progressive muscle degeneration. The Treebak lab generated inducible SMNKO mice (iSMNKO) and achieved a comparable depletion of NAD+. iSMNKO mice did not show altered mitochondrial respiratory capacity at rest or after exercise in isolated intact fibers. Moreover, high resolution respirometry in mitochondria isolated from iSMNKO muscle exhibited unchanged respiration and ATP synthesis, as well as normal P/O and mitochondrial membrane potential. Interestingly, iSMNKO skeletal muscle has lower capacity for succinate-induced mitochondrial ROS production compared to the wildtype control. iSMNKO mice displayed normal VO2 at rest and during an acute bout of maximal exercise, they behaved similarly in response to prolonged voluntary wheel (VW) running, but failed to increase performance after VW training. Collectively, their data demonstrate that the role of NAD in mitochondrial function may be more nuanced than is often thought.
Cold Acclimatization – Fundamental Mechanisms and Popular Misconceptions
Alexander Bartelt, PhD
Homeotherm mammals like rodents or humans regulate their body temperature by means of thermogenesis. Below the thermoneutral zone, where basal metabolism is sufficient to maintain homeothermy, thermogenic mechanisms are activated to contribute to heat production. This adaptation to cold exposure is a phasic process with acute activation of existing stores, acquisition of extra nutrients as fuels, and finally extensive remodeling of cells, tissues, and physiology, altogether facilitating cold acclimatization. Most notably are shivering and non-shivering thermogenesis by myocytes and thermogenic adipocytes, respectively. In this seminar, Dr. Bartelt provides an overview of the phasic process of cold acclimatization, elaborates on novel, fundamental mechanisms, including his lab’s own work on the transcription factor Nfe2l1, and, finally, highlights misconceptions that are frequently found in the literature.
Thermal Physiology: The Effects of Environmental Temperatures on Energy Expen...InsideScientific
Mice are generally an excellent model of human biology with nearly identical metabolic pathways. In contrast, the 3000-fold difference in body mass causes huge differences in thermal physiology and energy homeostasis. Humans generally live in a thermoneutral environment, while mice live and are typically studied below thermoneutrality. A mouse housed singly at 22 °C devotes 42% of its energy expenditure to maintaining its body temperature; the corresponding value in humans is approximately 0%. Understanding this different physiology is important, allowing one to avoid incorrect application of mouse observations to humans. It also boosts elucidation of physiology that is subtle or difficult to study in humans.
The goal is to understand thermal physiology and to use it to develop conditions under which mice better model humans. This is important for studying the effectiveness of drug treatments for metabolic diseases, like obesity and diabetes. Marc and Oksana discuss what thermoneutrality means in the mouse and the concept of the thermoneutral point. They also explore the effects of cold, hot, and near-thermoneutral environments on mouse energy expenditure, body temperature, and behavior.
Based on Capillary Gate Theory and Tissue Repair Theory, this presentation will explain the recently identified “Stress Repair Mechanism” (SRM) that enables the long-anticipated Universal Theory of Medicine postulated by Hans Selye in 1954. The SRM maintains and repairs vertebrate tissues and accounts for most of the mysterious manifestations of allostasis that remain unexplained by Hypothalamic-Pituitary-Axis (HPA) hormones. SRM activity explains hemodynamic physiology, capillary hemostasis, infarction, Korotkoff sounds, blood pressure, hypertension, diabetes, allostasis, allostatic load, anesthesia, analgesia, atherosclerosis, apoptosis, malignancy, eclampsia, sepsis, Multi-System Organ Failure (MSOF), the surgical stress syndrome, the fight or flight response, and numerous other manifestations of physiology, pathology, and allostasis. SRM function comprises the autonomic nervous system, the vascular endothelium, and the dynamic enzymatic interaction of blood-borne hepatic Factors VII, VIIIC, IX and X that produces thrombin, soluble fibrin and insoluble fibrin, whose combined effects account for all SRM manifestations. The vascular endothelium is a diaphanous neuroendocrine organ that lines all blood vessels and is the sole constituent of capillary walls. It secretes tissue factor into extravascular tissues, and insulates those tissues from the hepatic enzymes, so that tissue disruption exposes tissue factor to the enzymatic interaction and activates tissue repair. The vascular endothelium also releases nitric oxide and von Willebrand Factor into blood in accord with autonomic balance to regulate the enzymatic interaction to govern tissue perfusion and organ function. Therefore, continuously fluctuating combinations of nervous stimuli that affect autonomic balance and forces that disrupt tissues determine SRM activity.
Advanced nutrition for the brain series: stress, the HPA-axis and neuroinflammation. Targeted nutritional interventions for successful treatment of mental health conditions.
Inflammation is a major contributing factor to chronic modern illness and is driven, in part, by chronic stress and HPA-axis over stimulation. Mental health conditions, in particular clinical depression, are increasingly linked with neuroinflammation. As such, anti-inflammatory interventions are known to result in significant clinical benefits.
During this webinar Dr Bailey will discuss the biological mechanisms linking stress, chronic inflammation and mood disorders, together with a review of the current evidence for a targeted, anti-inflammatory nutrition approach to treatment. Nina will also clarify why some of the recent trials have failed to report benefits and how to optimise your anti-inflammatory interventions to treat clients with anxiety, depression, schizophrenia and PTSD.
Sugarcane Ash and Sugarcane Ash-Derived Silica Nanoparticles Alter Cellular M...Arthur Stem
Multiple epidemics of chronic kidney disease of an unknown etiology (CKDu), primarily in young healthy agricultural workers, have emerged in agricultural communities around the world. It is proposed that heat stress, dehydration and/or toxicant exposures may be a cause of this emerging disease. We have hypothesized that the harvest and burning of sugarcane leading to inhalation of sugarcane ash may contribute to development of CKDu. Sugarcane stalks consist of ~80% amorphous silica and we have demonstrated that following burning of sugarcane, nano-sized silica particles (~200 nm) are generated.
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.