Pavlovian Defensive Reactions Are Modulated By Norepinephrine In The Central Nucleus Of Amygdala

Previous researches suggest that,the amygdala plays a crucial role in elicits animal defensive reactions initiated by an environmental threat.Dysregulation of threat processing in the amygdala may contribute to the etiology of fear and anxiety disorders.In addition,given that pharmacological treatments for fear and anxiety disorders often target monaminergic systems,it is possible that changes in tonic levels of monamine neuromodulators in the amygdala may contribute.In particular,norepinephrine(NE)has long been implicated in fear and anxiety,yet relatively little is known about the contribution of NE and its adrenergic receptors(ARs)to amygdala-dependent reactions to threat.Studies have found that the modulatory role of NE in the amygdala,specifically the lateral nucleus of the amygdala(LA),has been recently found critical for initial acquisition,short-term memory(STM)and long-term memory(LTM),but did not affect expression,by using aversive Pavlovian paradigms.However,the central nucleus of the amygdala(CeA)also receives dense noradrenergic innervations,the function of which is not well understood.The CeAis the major output nucleus of the amygdala and is responsible for defensive responses to aversive stimulis.We therefore hypothesized that NE acting in CeA modules the expression of defensive reactions.To study this question,we are using a combination of pharmacology,behavioral readouts and immunohistochemistry to uncover the role of NE modulation of CeA functions in a Pavlovian threat conditioning paradigm.Expression of conditioned defensive responses involves much more complex circuits than we initially thought,including coordinated interactions between excitatory and inhibitory extra-amygdala and intra-amygdala microcircuits.We therefore tested the balance of excitatory and inhibitory drive within the CeA on conditioned responses using Designer Receptors Exclusively Activated by Designer Drugs(DREADDs).Next we used viral vectors expressing DREADDs and engineered to specifically target NE-expressing neurons to study the role of the locus coeruleus(LC),a maj or source of norepinephrine release in the brain,in CeA-mediated defensive responses.Before memory expression tests,NE-expressing neurons in LC were specifically activated or inhibited by systemic Clozapine-N-oxide(CNO)injections.Afterwards,we directly activated or inhibited axon terminals from the LC in CeA using intra-CeA CNO infusions followed by exposure to conditioned stimulis(tones).This created a perfect animal model to explore the pre-synaptic and post-synapyic functions of LC-NE circuit modulation in CeA-mediating defensive reactions,respectively.Results:1.Decreased expression of defensive responses(freezing)was observed following systemic injection of the ?-receptor antagonist Propranolol,and increased freezing was observed following injection of the ?-receptor agonist Isoproterenol,?2-receptor agonist Procaterol and ?1-receptor antagonist Prazosin;2.Increased and decreased expression of defensive responses was observed following direct CeA infusions of the Norepinephrine or ?-receptor antagonist Propranolol,while the ?-receptor agonist Isoproterenol or ?2-receptor agonist Procaterol has no effect on freezing during the expression test.Furthermore,increased and decreased expression of defensive responses was observed following direct CeA infusions of the ?1-receptor antagonist Terazosin or ?1-receptor agonist Cirazoline;3.Consistent with the pharmacology studies,we found that hM3Dq-mediated activation and hM4Di-mediated inhibition of LC terminals in CeA increased and decreased freezing behaviors respectively,through intraperitoneal or intracranial CNO injections during discrete phases of a cued Pavlovian threat conditioning paradigm.4.Activation of excitatory DREADD receptors(hM3Dq)in CeA caused decreased freezing compared to the control group in the long-term memory test;In contrast,activation of inhibitory DREADD receptors(hM4Di)in CeA significantly enhanced freezing compared to the control groupConclusion:Our findings support the hypothesis that tonic changes in norepinephrine in the central nucleus of the amygdala profoundly affect the expression of defensive responses elicited by conditioned threats,which suggests that this may contributed by norepinephrine from LC activates adrenergic receptors in CeA to bidirectional modulate defensive responses to threatening stimulus...