Enkephalin knockdown in the basolateral amygdala reproduces vulnerable anxiety-like responses to chronic unpredictable stress

Abstract

The endogenous enkephalins (ENKs) are potential candidates participating in the naturally occurring variations in coping styles and determining the individual capacities for adaptation during chronic stress exposure. Here we demonstrate that there is a large variance in individual behavioral, as well as in physiological outcomes, in a population of Sprague-Dawley rats subjected to 3 weeks of chronic unpredictable stress (CUS). Separation of resilient and vulnerable subpopulations reveals specific long-term neuroadaptation in the ENKergic brain circuits. ENK mRNA expression was greatly reduced in the posterior basolateral nucleus of amygdala (BLAp) in vulnerable individuals. In contrast, ENK mRNA levels were similar in resilient and control (unstressed) individuals. Another group of rats were used for lentiviral-mediated knockdown of ENK to assess whether a decrease of ENK expression in the BLAp reproduces the behavioral disturbances found in vulnerable individuals. ENK knockdown specifically located in the BLAp was sufficient to increase anxiety in the behavioral tests, such as social interaction and elevated plus maze when compared with control individuals. These results show that specific neuroadaptation mediated by the ENKergic neurotransmission in the BLAp is a key regulator of resilience, whereas a decrease of the ENK in the BLAp is a maladaptation mechanism, which mediates the behavioral dichotomy observed between vulnerable and resilient following 3 weeks of CUS.

Figure 1

Representative autoradiographs for enkephalin (ENK) mRNA within the medial prefrontal cortex, nucleus accumbens (NAc), dorsal striatum and amygdaloid complex with corresponding bregma coordonates (a–c) (Swanson, 2003). (d) ACAd, anterior cingulate area, dorsal part; ILA, infralimbic area; PL, prelimbic area; Ald, agranular insular area, dorsal part; PIR, piriform area. (e) 1- striatum, dorsolateral part; 2- striatum, central part; 3- striatum, medial part; 4- NAc, ventral core; 5- NAc, dorsal core; 6- NAc, dorsal shell; 7- NAc, medial shell; NAc, lateral shell. (f) CeAl, central amygdalar nucleus, lateral part; LA, lateral amygdalar nucleus; MEApd, medial amygdalar nucleus, posterodorsal part; MEApv, medial amygdalar nucleus, posteroventral part; COApl, cortical amygdalar nucleus, posterior part, lateral zone; BLAp, basolateral amygdalar nucleus, posterior part.

Figure 2

Matrix scatter diagram displaying three groups that have been differentiated by k-means analysis. This was done by a partition of four behavioral parameters into three clusters (duration of social interaction, open-arms time ratio in the elevated plus maze (EPM), total arm distance traveled in the EPM and sucrose intake). A line connects each point to its cluster centroid. The black cluster represents control (n=25), the blue cluster represents vulnerable (n=29) and the green cluster represents resilient (n=19) individuals.

Figure 3

Resilience and vulnerability to 21 days of chronic unpredictable stress (CUS) are marked by specific long-term behavioral, physiological and neuroadaptation in the enkephalinergic (ENKergic) brain circuit. (a) Vulnerable displayed lower duration of social interaction compared with control and resilient individuals. (b) Vulnerable showed lower weight gain compared with resilient individuals. (c) Vulnerable individuals showed higher basal corticosterone compared with control individuals. (d) Vulnerable displayed decreased locomotion as measured by total arm distance in the elevated plus maze (EPM) compared with resilient individuals. (e) Resilient displayed higher exploration as measured by the open-arm time ratio in the EPM compared with control. (f) Enkephalin (ENK) mRNA is reduced only in posterior part of the amygdalar nucleus (BLAp) in vulnerable compared with control and resilient individuals 1 week after the last stress session. In contrast there is no difference between control and resilient individuals. (g) Vulnerable displayed enhanced sucrose consumption compared with control and resilient individuals. (h) No difference in the sucrose preference test between groups. Social interaction was assessed 3 days after the end the CUS. EPM was evaluated 24 h after the social interaction. Sucrose intake and preference tests were performed before (days −3 to −1), during (days 9–10 and 19–20), and after (days 4–6) the CUS. Bars represent mean±SEM; *p<0.05.

Figure 4

Enkephalin (ENK) knockdown increased anxiety-like behaviors. (a) Representative immunoreactive for copepod green fluorescent protein (copGFP) and autoradiographs for ENK mRNA within the amygdaloid complex. (b) We observed a 59% reduction of ENK mRNA level in ENK shRNA animals when compared with scrambled shRNA animals. ENK knockdown decrease the duration of social interaction (c); the number of social interaction (f); and the open-arm time ratio in the elevated plus maze (EPM) (g). ENK knockdown did not affect the total arm distance in the EPM (d); the sucrose intake (e); and the sucrose preference (h). Social interaction, sucrose intake, and sucrose preference tests were evaluated in the same animals before the administration of the viral vector (initial) and after the 14 days of the recovery period (final). Bars represent mean±SEM (n=13 for ENK and scrambled n=8); *p<0.05.