Vagus Nerve Stimulation Enhances Extinction of Conditioned Fear in Rats and Modulates Arc Protein, CaMKII, and GluN2B-Containing NMDA Receptors in the Basolateral Amygdala

Abstract

Vagus nerve stimulation (VNS) enhances the consolidation of extinction of conditioned fear. High frequency stimulation of the infralimbic cortex (IL) produces long-term potentiation in the basolateral amygdala (BLA) in rats given VNS-paired extinction training, whereas the same stimulation produces long-term depression in sham-treated rats. The present study investigated the state of synaptic plasticity-associated proteins in the BLA that could be responsible for this shift. Male Sprague-Dawley rats were separated into 4 groups: auditory fear conditioning only (fear-conditioned); fear conditioning + 20 extinction trials (extended-extinction); fear conditioning + 4 extinction trials paired with sham stimulation (sham-extinction); fear conditioning + 4 extinction trials paired with VNS (VNS-extinction). Freezing was significantly reduced in extended-extinction and VNS-extinction rats. Western blots were used to quantify expression and phosphorylation state of synaptic plasticity-associated proteins such as Arc, CaMKII, ERK, PKA, and AMPA and NMDA receptors. Results show significant increases in GluN2B expression and phosphorylated CaMKII in BLA samples from VNS- and extended-extinction rats. Arc expression was significantly reduced in VNS-extinction rats compared to all groups. Administration of the GluN2B antagonist ifenprodil immediately after fear extinction training blocked consolidation of extinction learning. Results indicate a role for BLA CaMKII-induced GluN2B expression and reduced Arc protein in VNS-enhanced extinction.

Figure 1

Increased expression of p-CaMKII and decreased expression of Arc protein in the BLA of animals given VNS during extinction training. (a) Timeline of behavioral protocol. On days 1 and 2 rats were subjected to auditory fear conditioning (AFC) followed by a conditioned fear response test (CFRT) on day 3. On day 4 rats underwent extinction training (EXT) and were sacrificed 45 min later. (b) Rats given VNS during extinction training show higher levels of p-CaMKII compared to rats given sham stimulation (^∗ p < 0.05). (c, d) No significance difference in expression of CaMKIIα or CaMKIIβ across groups. (e) VNS-treated rats show lower levels of Arc protein expression compared to sham-treated rats (^∗ p < 0.05). (f) No difference in p-PKA expression across groups.

Figure 2

Increased expression of GluN2B in the BLA of rats given VNS during fear extinction training. (a) Rats given VNS during extinction training show higher levels of GluN2B compared to rats given sham stimulation (^∗ p < 0.05). (b–e) No significant difference in expression of GluN2A, p-GluA1 at S845 or S831, or p-ERK across groups.

Figure 3

VNS accelerates molecular mechanisms of extinction learning. (a) Timeline of behavioral protocol. On days 1 and 2 rats were subjected to auditory fear conditioning (AFC). One cohort was sacrificed 45 min after AFC. Another cohort underwent a conditioned fear response test (CFRT; day 3), followed by extinction training (EXT) or extended-extinction (EE) training (day 4), and was sacrificed 45 min later. (b-c) Rats given VNS during extinction training show higher levels of p-CaMKII and GluN2B compared to rats given sham during extinction training and rats that underwent auditory fear conditioning alone (FC; ^∗ p < 0.05) and do not differ from rats that underwent extended extinction. (d) Rats given VNS during extinction training show lower levels of Arc protein compared to all other groups (^∗ p < 0.05).

Figure 4

Ifenprodil blocks extinction learning in VNS-extinction and EE rats. (a) Timeline of behavioral protocol. On days 1 and 2 rats were subjected to auditory fear conditioning (AFC) followed by a conditioned fear response test (CFRT) on day 3. On day 4 rats underwent extinction training (EXT) or extended-extinction (EE) training that was immediately followed by an i.p. injection of ifenprodil or vehicle. On day 5 rats underwent a second CFRT (CFRT 2). (b) VNS-extinction and EE rats given a vehicle injection immediately after extinction training show a significant decrease in freezing levels during the second conditioned fear response test (CFRT 2) compared to sham-vehicle rats (∗). VNS-vehicle rats show significantly lower freezing levels during CFRT 2 compared to VNS-ifenprodil rats (#).