Nicotine, the principal psychoactive component of tobacco, acutely stimulates secretion of the stress-responsive hypothalamo–pituitary–adrenal (HPA) axis hormones, adrenocorticotropic hormone (ACTH) and corticosterone (Matta et al. 1987). In addition, chronic nicotine self-administration (Chen et al. 2008) and other stressors (Aguilera 1994) augment ACTH and corticosterone responses to a novel stressor, such as mild footshock stress. However, the neuroplastic changes underlying this effect of chronic nicotine exposure on the stress response are only partially understood.
In the hypothalamic paraventricular nucleus (PVN), the critical output nucleus controlling the HPA axis, corticotropin-releasing factor (CRF) neurons in the parvocellular division (pcPVN), integrate the HPA responsiveness to diverse stressors and stimulate plasma ACTH secretion (Herman et al. 2005). We have shown that acutely injected nicotine and chronically self-administered nicotine are stressors that activate CRF neurons and elevate plasma ACTH levels (Valentine et al. 1996; Chen et al. 2008; Yu et al. 2008). Chronic nicotine self-administration also alters the phenotype of pcPVN CRF neurons by inducing the co-expression of arginine vasopressin (AVP). Stressor stimulation of these CRF+/AVP+ neurons would potentiate CRF-dependent ACTH secretion by co-releasing AVP, another modulator of pituitary corticotrophs (Rivier and Vale 1983; Yu et al. 2008). We have reported that PVN responsiveness to norepinephrine, a primary regulator of CRF neurons, is selectively enhanced in rats self-administering nicotine, but only during stress (Yu and Sharp 2010). Neuroplasticity in the phenotype of CRF neurons and their responsiveness to norepinephrine during stress would both contribute to the enhanced secretion of ACTH and corticosterone during mild footshock stress. However, the contribution of other neurotransmitters to this interaction between nicotine self-administration and a stressor is unknown.
We have proposed that nicotine self-administration is a chronic stressor, sensitizing the HPA to novel stressors (Chen et al. 2008; Yu et al. 2008). We have shown that increased c-Fos expression in CRF+/AVP+ neurons after stress is a distal component of the HPA sensitization induced by chronic nicotine self-administration. However, apart from norepinephrine (Yu and Sharp 2010), the effect of nicotine self-administration on neurotransmitters modulating these critical neurons is unknown. We hypothesized that nicotine self-administration would alter the balance of glutamatergic stimulation and GABAergic inhibition during mild footshock stress, thereby augmenting the response of CRF neurons to the stressor. We determined the effects of nicotine self-administration on footshock-induced release of (i) PVN glutamate and GABA, and (ii) plasma ACTH and corticosterone after blockade of glutamate or GABA receptors in the PVN. These experiments demonstrate that chronic nicotine self-administration both enhanced the PVN glutamate release and magnified the reduction in PVN GABA levels by footshock, providing new evidence of plasticity in neurotransmission that mediates enhanced HPA responsiveness.
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- Materials and methods
We have previously shown that chronic nicotine self-administration augments the HPA axis response to mild footshock stress (Chen et al. 2008). To our knowledge, the current studies are the first to characterize concomitant changes in glutamate and GABA within the PVN during stress in animals self-administering drug and control. These experiments demonstrate that chronic nicotine self-administration altered both excitatory and inhibitory neurotransmission within the PVN during mild stress by augmenting glutamate and diminishing GABA release, in each case approximately twofold more than in controls. The significance of these changes for HPA function was determined in pharmacological studies that showed blockade of PVN glutamate receptors prevented the augmented ACTH and corticosterone responses to footshock during nicotine self-administration. Additionally, blockade of inhibitory GABA-B receptors in the PVN enhanced the corticosterone response to footshock to a greater extent in rats self-administering saline compared to nicotine, indicating that CRF neurons are relatively disinhibited during mild footshock stress in rats self-administering nicotine. These findings indicate that both enhanced PVN glutamate and reduced GABA neurotransmission participate in augmenting HPA responsiveness to footshock during nicotine self-administration (Fig. 7).
Stimulation of the HPA by stressors depends, in part, on glutamatergic inputs to pcPVN (Herman et al. 2004). Forced swim stress, restraint, and immobilization have been reported to increase glutamate release in medial prefrontal cortex, hippocampus, and central amygdala (Moghaddam 2002; Ebner et al. 2005; Pung et al. 2006; Zhu et al. 2008). To our knowledge, the effect of stress on PVN glutamate levels has not been reported. Mild footshock stress significantly increased PVN glutamate release in rats self-administering saline and to a greater extent in those receiving nicotine. Intra-PVN kynurenic acid prevented the nicotine self-administration-enhanced release of corticosterone induced by footshock, but did not attenuate the overall corticosterone response to stress in rats self-administering saline. The peak corticosterone response was, however, reduced by 20% in the saline group. The failure of kynurenic acid to block the overall corticosterone response, which differs from a report showing that intra-PVN microinjection of kynurenic acid significantly reduced restraint stress (30 min)-induced corticosterone release by 24%, may reflect the magnitude of both the stressor stimulus and the corticosterone response to restraint stress versus mild footshock stress (Ziegler and Herman 2000). Corticosterone was elevated to a greater degree and for a much longer duration (150 min) after acute restraint compared with footshock (duration: 35 min) (Ziegler and Herman 2000). Although kynurenic acid induced a small but significant reduction in the peak corticosterone level of rats self-administering saline (20%), the rapidly declining corticosterone levels make it difficult to detect a difference at the later time points (i.e. 35 and 55 min). Nevertheless, a higher dose of kynurenic acid might be more efficacious. Therefore, it is likely that footshock-induced glutamate release contributes to the elevation of corticosterone in rats self-administering saline. In the nicotine self-administration group, the exaggerated release of PVN glutamate is necessary for the enhanced corticosterone response to footshock, which depends on the augmented release of ACTH (see Fig. 5). The efficacy of intra-PVN kynurenic acid in blocking the augmentation of ACTH implicates pcPVN hypophysiotropic CRF neurons in the action of this enhanced glutamate release.
The activity of GABAergic inputs to the PVN, which restrain HPA responses to stressors, diminishes during stress (Cullinan et al. 2008). Restraint stress suppressed inhibitory post-synaptic potentials in the PVN, and cold stress decreased PVN GABA levels (Ohtani et al. 1999; Verkuyl et al. 2005). Moreover, intra-PVN microinjection of muscimol, a GABA-A receptor agonist, blunted the rise in plasma corticosterone induced by restraint (Cullinan et al. 2008). Lastly, blockade of PVN GABA-A and especially GABA-B receptors increased ether-induced corticosterone secretion (Marques de Souza and Franci 2008). These findings accord with the current studies, in which footshock significantly reduced PVN GABA levels in the saline group; this reduction was magnified twofold in rats self-administering nicotine. Studies with saclofen, which blocks GABA-B receptors expressed on pcPVN neurons (Margeta-Mitrovic et al. 1999), clarified the functional significance of these differences in PVN GABA levels. The disinhibition of CRF neurons by intra-PVN saclofen caused significantly less augmentation of the corticosterone response to footshock in rats self-administering nicotine compared with saline. Lower PVN GABA levels were, therefore, associated with less effect of saclofen in the nicotine group. In summary, the additional decrement in PVN GABA levels reduced the inhibition of pcPVN CRF neurons, contributing to the augmented HPA response to footshock in rats self-administering nicotine.
The GABAergic input to the PVN is primarily from the peri-PVN region and the bed nucleus of stria terminals. Glutamatergic projections from the medial prefrontal cortex and hippocampus innervate the peri-PVN and the bed nucleus of stria terminals GABAergic neurons, increasing the inhibitory control of PVN CRF neurons (Herman et al. 2005). Similar to a previous report showing that peri-PVN kynurenic acid increased the corticosterone response to restraint stress (Ziegler and Herman 2000), we found that peri-PVN kynurenic acid augmented the corticosterone response to footshock in the saline group. Although the activity of GABAergic inputs to the PVN is reduced by stress, kynurenic acid blockade of glutamate receptors on peri-PVN GABA neurons would be expected to further reduce this activity, augmenting the stress-induced activation of CRF neurons and the HPA axis. In contrast, in rats self-administering nicotine, peri-PVN kynurenic acid had no affect on the corticosterone response to footshock, indicating that nicotine self-administration may further reduce the activity of glutamatergic inputs to peri-PVN GABAergic neurons during mild footshock stress.
Nicotine self-administration altered the HPA responsiveness to stress without affecting basal levels of PVN glutamate and GABA. This coheres with the fact that basal ACTH and corticosterone levels were unaffected by chronic nicotine self-administration in this study. Although both acutely injected and self-administered nicotine act as stressors, stimulating ACTH and corticosterone secretion, desensitization occurs during chronic nicotine self-administration (Chen et al. 2008; Yu et al. 2008). Similar to chronic stressors (Aguilera and Rabadan-Diehl 2000), chronic nicotine self-administration alters the phenotype of hypophysiotropic CRF neurons in pcPVN (Yu et al. 2008). Regarding potential plasticity within other functional units of the HPA, exposure to chronic stressors, including cocaine, has been shown to induce increased expression of proopiomelanocortin, CRF1 receptor and AVP V1b receptor in anterior pituitary corticotrophs (Aguilera and Rabadan-Diehl 2000; Zhou et al. 2003). However, the effects of nicotine on these structures have not been evaluated.
Homeostatic plasticity, one of multiple paradigms of brain plasticity, stabilizes the function of neurons and circuits despite alterations induced by other forms of plasticity (e.g. synaptic plasticity) (Nelson and Turrigiano 2008). Numerous molecular and cellular mechanisms promote homeostatic plasticity at the neuron level. For example, the firing rate of individual neurons is maintained within a preferred range by tuning synaptic strengths up or down (i.e. synaptic scaling), while holding constant the relative strength of each synapse (Turrigiano 2008). At the systems level, homeostatic plasticity, particularly synaptic scaling, is known to govern the ocular dominance plasticity that occurs in the visual cortex, and augments responses to the open eye after chronic closure of the opposite eye (Mrsic-Flogel et al. 2007). The present study suggests that circuits regulating the HPA stress response are dysregulated during chronic self-administration of nicotine. Although PVN glutamate release is enhanced, GABA release is diminished further rather than increased. This serves to facilitate the responses to increased glutamate neurotransmission. These findings, which are contrary to the precepts of homeostatic plasticity, may be characteristic of the dysregulation induced by drugs. Abused drugs exploit native brain circuitry through specific molecular interactions, altering the function of neurons and circuits, both short- and long-term (Kauer and Malenka 2007). This neuroplasticity can produce alterations in neuron, circuit and behavioral function that far outlast the presence of drug.
We have proposed that chronic nicotine self-administration functions as a chronic stressor by inducing the expression of CRF+/AVP+ neurons in pcPVN, which are activated by footshock, thereby contributing to the enhanced HPA responsiveness to footshock (Chen et al. 2008; Yu et al. 2008). Chronic nicotine self-administration also diminishes footshock-induced norepinephrine release in the PVN, yet enhances noradrenergic responsiveness concurrent with the application of a stressor (i.e. mild footshock stress) (Yu and Sharp 2010). The present study demonstrates that chronic nicotine self-administration differentially modulates glutamate and GABA transmission within the PVN. Both the augmented release of glutamate and the exaggerated reduction of GABA in the PVN are necessary for the enhanced HPA response to footshock. Disinhibition of the pcPVN, because of the reduction of GABA, is also the probable mechanism for the enhanced PVN responsiveness to norepinephrine during stress in rats self-administering nicotine (Yu and Sharp 2010). The net effect of these alterations in PVN neurotransmission is to increase the activation of pcPVN CRF+/AVP+ neurons, thereby sensitizing the HPA to a submaximal stressor such as mild footshock stress. These findings offer new mechanistic insights that run contrary to the concept of homeostatic plasticity and support the concept that smoking functions as a chronic stressor, sensitizing the HPA to stress.