Changes in GRK3 and Norepinephrine Responsiveness in Locus Coeruleus Neurons are Associated with Learned Helplessness After Repeated Forced Swim Stress

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In brain, locus coeruleus plays an important role in mediating the stress responses. Two important neurotransmitter/hormones released in locus coeruleus (LC) during exposure to stress include corticotrophin releasing factor (CRF) and norepinephrine (NE). CRF and NE predominantly act on G protein-coupled receptors (GPCR), the corticotrophin-releasing factor type 1 receptor (CRF1-R) and the alpha2A–adrenoceptors (α2A-AR), respectively. Activation of CRF1-R increases LC neuronal firing and activation of postsynaptic α2A-AR inhibits firing of LC neurons. Additionally, presynaptic α2A-ARs act as autoreceptors inhibiting release of NE in LC. Both the agonist-occupied CRF1-R and α2A-AR are preferentially desensitized by G protein-coupled receptor kinase 3 (GRK3). This desensitization contributes to termination of the signaling of CRF1-R and α2A-AR, observed during the persistent presence of neurotransmitters during stress. At present, there is a gap in knowledge as to what changes in GPCR signaling occur during single and repeated stress. The present study provides a contribution towards filling this gap. This study observed the effects of single and repeated forced swim stress on the escape task performance of rats in a shuttle-box. The study also observed the effects of GRK3 and NE responsiveness in LC neurons in slices of rat brainstem. Both single and repeated forced swim stress segregated the stressed rats into two clusters based on their performance in the escape task. One cluster showed impaired escape behavior compared to controls and was designated Learned Helpless (LH), showing susceptibility to the adverse effects of stress. The other cluster of stressed rats showed escape behavior similar to the controls and was termed Non-Helpless (NH), showing resilience against the adverse consequences of stress. Thus this study demonstrated that a milder stress than inescapable electric shock, the stress paradigm of forced swim, could induce deficits in escape behavior. These deficits are a well-established index of depression-associated behavior. Biochemical analysis showed that single forced swim stress did not cause any change in the levels of GRK3, CRF1-R and α2A-AR in the LC of the LH rats compared to the control and NH rats. However, repeated forced swim stress caused a decrease in the levels of GRK3 and an increase in the levels of CRF1-R and α2A-AR in the LC of LH rats compared to the control and NH rats. Also, repeated forced swim stress was accompanied by an increase in the responsiveness of α2A-AR upon application of lower concentrations of NE as observed by measuring the changes in membrane current in response to different concentrations of NE in single LC neuron. Moreover, the increases in immobility, LH behavior, decreases in GRK3 and increase in CRF1-R and α2A-AR in LC after repeated forced swim stress alone were not observed in rats pretreated with desipramine (DMI). In conclusion, a much milder and more physiological stressor than electric shock, repeated forced swim stress, enables the identification of a sub-population of stress-susceptible rats that display LH. This LH behavior was associated with a decrease in GRK3 and an increase in α2A-AR levels and responsiveness in LC, accompanied by an increase in CRF1-R levels. The repeated forced swim-induced changes in responsiveness of the postsynaptic α2A-AR may indicate that when the stressful stimuli are removed there is a rebound compensatory mechanism by which the α2A-AR may reduce LC hyperactivity. Although, the method used in this study measured only postsynaptic α2A-AR function, if similar changes in the presynaptic α2A-AR occur, this would decrease NE release in LC during stress. Thus the pre- and postsynaptic α2A-ARs would cancel each other functionally. This would lead to predominance of excitatory effects of CRF1-R, potentially contributing to the hyperresponsiveness of LC to CRF and to hyperactivity of the LC that is characteristically observed on exposure to stress. DMI pretreatment, by increasing the availability of norepinephrine in the LC, will maintain an inhibitory tone on the neurons and prevent the hyperactivity of LC neurons associated with exposure to repeated stress. This will lead to prevention of repeated forced swim stress-induced decrease in GRK3 and increase in the levels and responsiveness α2A-AR and CRF1-Rs in the LC, resulting in the prevention of subsequent impairment of escape behavior in rats.