Opposing regulation of stress-related peptides on noradrenergic activity
Our laboratory described that the locus coeruleus (LC) is finely tuned by co-regulation between the endogenous opioids, enkephalin (ENK), dynorphin (DYN), and corticotropin releasing factor (CRF). During stress (physiological or psychological), CRF is released (from the central nucleus of the amygdala CNA) to shift the activity of LC neurons to a high tonic state that would promote scanning of the environment and behavioral flexibility. At the same time, endogenous opioids acting at ·u opioid receptor (OR) in the LC (via ENK) exert an opposing inhibitory effect that may serve to restrain the excitatory actions of CRF and help to bring neuronal activity back to baseline.
Our laboratory described that CRF and ENK that regulate the LC derive from distinct sources (CNA and medulla, respectively) but their axon terminals converge onto common LC neurons which can respond to both peptides because they co-express µ-OR and CRFr. Both ENK and CRF axon terminals co-localize glutamate, which mediates the short-lived LC activation by sensory stimuli. We also demonstrated that the dynorphin-µ-OR receptor system exerts another layer of regulation on the LC system by presynaptic inhibition of excitatory LC afferents. Our research has unveiled the complex circuitry by which CRF and endogenous opioids co-regulate activity of the LC-NE system. The circuitry that links these peptides to the LC-NE system and the conditions that engage this circuitry were identified and highlighted the amygdala as a key structure in its afferent regulation.
Finally, we discovered that coping strategy determines the magnitude of mu-OR/CRF balance on LC activity. The finding that coping style determines stress-response circuitry that is engaged during social stress has implications for using cognitive therapies in treating stress-related disorders.
Figure 1: The LC is finely tuned by co-regulation between the endogenous opioids, ENK and DYN, and CRF. Our previous studies have characterized the anatomical, physiological and behavioral basis for interactions between endogenous opioids and the LC-NE system. Specifically, we have shown that NE activity is regulated via distinct CRF and ENK afferents targeting postsynaptically distributed CRF and µ-opioid receptors (µ-OR). We identified the source of DYN afferents to the LC as originating from the CNA (B.A. Reyes et al., 2008) and demonstrated that DYN and CRF are co-transmitters in monosynaptic afferents to the LC where they are poised to coordinately impact LC functions (B.A. Reyes, Carvalho, et al.). We discovered that the DYN-κ-OR system regulates LC neurons at a presynaptic level, inhibiting excitatory afferent input (A.S. Kreibich et al., 2008). In addition to presynaptic modulation of glutamatergic and CRF afferents (A.S. Kreibich et al., 2008), we further demonstrated that κ-ORs modulate DYN afferents to the LC (B.A. Reyes et al., 2009) and that exposure to a κ-OR agonist induces internalization of κ-ORs and impacts cortical catecholaminergic expression levels (B.A. Reyes, Chavkin, & Van Bockstaele).
Figure 2: Potential mechanisms underlying sensitization to stress following exposure to chronic morphine include: (1) increased CRFr on the plasma membrane arising from increased synthesis (1a), or altered trafficking to the membrane (1b) or decreased internalization (1c); (2) more efficient CRFr signaling by enhanced Gs coupling or stronger affinities for RGS-binding to positive regulators; or (3) increased CRF release from LC afferents. Ongoing studies are directed at establishingrequired to establish which mechanism is involved.
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Regulation of the coeruleo-cortical pathway by the endocannabinoid system
Our laboratory has demonstrated that norepinephrine (NE), a biogenic amine integral to the stress-response and regulation of higher cognitive functions is modulated by the endocannabinoid system. We showed that cannabinoid receptor agonists increase indices of noradrenergic activity by increasing norepinephrine (NE) efflux in the rat frontal cortex (FC) and stimulating c-Fos expression in noradrenergic neurons of the locus coeruleus (LC). Likewise, repeated WIN 55,212-2 administration evoked an anxiogenic-like response with a concomitant increase in tyrosine hydroxylase (TH) expression in the LC as well as an increase in NE efflux in the FC. These effects were prevented by pre-treatment with a cannabinoid receptor (CB1R) antagonist indicating involvement of CB1 receptors.
Our group has also described functional interactions between CB1r and adrenergic receptor (AR) systems in the FC using in vitro intracellular electrophysiology techniques. Whole cell patch clamp recordings of layer V/VI cortical pyramidal neurons in rats revealed that clonidine-induced α2-AR-mediated elevations in cortical pyramidal cell excitability were significantly decreased following pre-treatment with the synthetic CB1r agonist, WIN 55,212-2, suggesting cannabinoid stimulation of NE release and desensitization of α2-AR. The receptor interaction was both action potential and GABAA receptor-independent as the desensitization occurred similarly in the presence or absence of tetrodotoxin or the GABAA receptor antagonist bicuculline indicating that CB1-α2-AR interactions are likely direct rather than mediated by synaptic afferents. We compared α2-AR-mediated responses in FC slices from WT and CB1r knockout (KO) mice. Similar to results that we reported for rats, results show that WT mice showed a clonidine-induced α2-AR-mediated increase in mPFC cell excitability coupled with an increase in input resistance. In contrast, CB1r KO mice showed an α2-AR-mediated decrease in mPFC cell excitability. The neurobehavioral consequences of exogenous cannabinoid exposure suggest that cannabinoid receptor ligands produce, in part, cellular and molecular changes in noradrenergic neurons.
Figure 3: Schematic diagram depicting cannabinoid-adrenergic interactions in stress-integrative circuitry. The basolateral complex of the amygdala (BLA) has been implicated in the consolidation of emotionally arousing experiences and involves glucocorticoid-mediated increases in eCB release and interactions with norepinephrine. (1) eCBs are posited to increase BLA activity by decreasing GABAergic neurotransmission. (2) Disinhibition of GABAergic interneurons results in an increase of glutamatergic signaling in the central nucleus of the amygdala (CNA), a source of excitatory afferents to the LC. (3) Activation of the LC causes an increase in noradrenergic signaling and norepinephrine (NE) release in postsynaptic targets, such as the prefrontal cortex (PFC). Given that the PFC represents a critical region in mediating the extinction of traumatic/aversive memories, treatments involving the eCB system that target this region may help alleviate symptoms of anxiety disorders by increasing extinction of such memories. For example, (4) CBs have been shown to inhibit monoamine oxidase (MAO), representing another mechanism in which CB signaling can regulate NE levels. (5) Targeting GABAergic projections to the LC with CB ligands can potentially modulate LC afferent activity to the PFC. Achieving the proper balance in frontal cortical activity by targeting cannabinoid-adrenergic interactions may result in enhancing extinction of aversive memories and diminish anxiety-like behaviors that are precipitated by stress.
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Sex differences in the mechanism of action of alcohol
Recent investigations in the laboratory have focused on alterations in neuronal activity in key stress-sensitive areas such as LC and the central nucleus of the amygdala (CeA). We demonstrated changes in activity patterns following repeated ethanol use that varied in a sex dependent manner. In the CeA, we examined a marker of recent neuronal activity, c-Fos, and a marker of long-term neuronal activity, ΔFosB, to identify differences in neuronal activation patterns across the sexes. Following exposure to chronic ethanol, there was an increased amount of ΔFosB immunoreactivity (IR) in male versus female subjects. When compared with c-Fos (marker of recent neuronal activation), male ethanol-treated subjects had similar c-Fos IR levels compared to controls, whereas female ethanol-treated subjects exhibited robust activation when compared to both control and male counterparts. In males, a significant increase in ΔFosB (marker of long-term activity) contrasted with c-Fos levels that were similar between ethanol and control groups, suggesting that males habituate to the neuronal activation induced by ethanol exposure. The pattern seen in females, with ethanol-treated subjects showing an increase in both c-Fos and ΔFosB seems to indicate that the female subjects do not habituate to chronic ethanol exposure in the same way, and this may be a mechanism underlying sex differences emerging in the alcohol addiction field. Similar findings were observed in the LC, where significant increases in c-Fos were observed in the female ethanol-treated subjects compared to males, again illustrating a lack of habituation to chronic ethanol exposure in females. These findings point to a dysregulation of the LC-NE system in females exposed to chronic ethanol that may pre-dispose them to increased vulnerability to continued stressors.
Figure 4. (Top Left) The activity patterns of the CeA and LC are dysregulated after chronic ethanol exposure, with males showing a neuronal habituation to chronic use, and females showing a higher rate of activation at baseline. (Bottom Left) The stress activation response of the CeA is potentiated after chronic ethanol exposure in both sexes. In females, neuronal activity levels after a stressor are similar to those after chronic ethanol exposure, while males show differential activation patterns in ethanol-treated compared to control. (Top Right) The LC shows a sexually dimporphic response to chronic ethanol exposure with CRFr distribution patterns mimicking a stress response. Habituation is observed in males, while females do not show such neuronal adaptations.
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Influence of Locus Coeruleus-Noradrenergic Stress Integrative Circuitry on Abeta Peptides
An ongoing study in the lab aims to investigate the underappreciated role of the locus coeruleus (LC)-norepinephrine (NE) stress system in Aβ production and secretion in the context of Alzheimer's Disease (AD). A culmination of evidence in the literature has demonstrated the importance of the LC-NE system in maintaining attention and arousal, sleep-wake patterns, and modulating inflammation; all of which are perturbed during the progression of AD. A number of clinical studies have identified the LC as one of the earliest regions to undergo degeneration, and further, that degeneration of the LC and corresponding reductions in cortical NE levels may contribute to both cognitive and behavioral disturbances observed in the clinic.
Stress is a risk factor for developing AD, and is supported by multiple clinical and preclinical studies demonstrating that amplification of the stress system disrupts cellular and molecular processes at the synapse, promoting the production and accumulation of the amyloid beta (Aβ) peptide. The aggregation of Aβ peptides results in the formation of senile plaques, a primary component of AD pathology, and marker of AD pathology that develops prior to neurodegeneration. Thus the focus of our ongoing project in the lab is to elucidate the role of stress-induced LC activation on A production and how this may play a role in the accumulation of Aβ at the synapse, which is predicted to result in LC neuronal death and a cascade of neurodegenerative events downstream of LC-NE dysregulation.
Figure 5. Model of proposed contribution of LC-NE Dysregulation in Alzheimer's disease. Our current working model proposes that aberrant activation of the LC at the synaptic level (a), leads to global aberrant LC activity in its widespread projection areas (A). With increased synaptic activity, increased transmission of NE may occur concomitantly with increased Aβ production in synaptic vesicles such as the LDCVs (B). Increased Aβ production and secretion may result from the described mechanisms of adrenergic receptor influence on APP processing (b). Further, the ability of NE to undergo volume transmission, engaging extrasynaptic mechanisms of transmission on neighboring neuronal and glial cells, has significant implications for the microenvironment (B, C). Finally, following a prolonged period of Aβ accumulation, cortical thinning, decreased noradrenergic transmission, and downstream loss of inflammatory modulation results in the detrimental activation of glial cells, and decreased dendritic spine density, and potentially network de-synchronization.
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