Pharmacological depletion of serotonin in the basolateral amygdala complex reduces anxiety and disrupts fear conditioning

https://doi.org/10.1016/j.pbb.2015.09.021Get rights and content

Highlights

  • 5,7-DHT injections into BLC/CeA + i.p. desipramine increase social interaction behaviors.

  • 5,7-DHT injections into BLC/CeA + i.p. desipramine disrupt acquisition of conditioned fear.

  • 5,7-DHT injections into BLC/CeA + i.p. desipramine selectively deplete local 5-HT.

Abstract

The basolateral and lateral amygdala nuclei complex (BLC) is implicated in a number of emotional responses including conditioned fear and social anxiety. Based on previous studies demonstrating that enhanced serotonin release in the BLC leads to increased anxiety and fear responses, we hypothesized that pharmacologically depleting serotonin in the BLC using 5,7-dihydroxytryptamine (5,7-DHT) injections would lead to diminished anxiety and disrupted fear conditioning. To test this hypothesis, 5,7-DHT(a serotonin-depleting agent) was bilaterally injected into the BLC. Desipramine (a norepinephrine reuptake inhibitor) was systemically administered to prevent non-selective effects on norepinephrine. After 5 days, 5-7-DHT-treated rats showed increases in the duration of social interaction (SI) time, suggestive of reduced anxiety-like behavior. We then used a cue-induced fear conditioning protocol with shock as the unconditioned stimulus and tone as the conditioned stimulus for rats pretreated with bilateral 5,7-DHT, or vehicle, injections into the BLC. Compared to vehicle-treated rats, 5,7-DHT rats had reduced acquisition of fear during conditioning (measured by freezing time during tone), also had reduced fear retrieval/recall on subsequent testing days. Ex vivo analyses revealed that 5,7-DHT reduced local 5-HT concentrations in the BLC by ~ 40% without altering local norepinephrine or dopamine concentrations. These data provide additional support for 5-HT playing a critical role in modulating anxiety-like behavior and fear-associated memories through its actions within the BLC.

Introduction

Serotonin (5-HT) plays a critical role in regulating adaptive stress responses to aversive stimuli and is strongly implicated in stress-related anxiety disorders including post-traumatic stress disorder and panic disorder. Serotoninergic neurotransmission is a major therapeutic target for treating these disorders [see review (Hale et al., 2012)]. Yet, serotonin regulation of anxiety and fear-associated behaviors and associated autonomic and endocrine responses to stressful stimuli is complex in due to functional heterogeneity among subpopulations of serotonergic neurons and the large number of serotonin receptors; in addition, serotonin's effects on physiological and behavioral responses to aversive stimuli appear to depend on the brain region where it is released (Hale et al., 2013).

One area where serotonin plays an important role in modulating anxiety and fear responses is the basolateral amygdala complex (BLC; which includes the basolateral and lateral nuclei). The BLC is highly responsive to stressful stimuli (Brydges et al., 2013, Butler et al., 2011, Henderson et al., 2012, Johnson et al., 2008, Singewald et al., 2003) and plays a critical role in fear conditioning, which is critical for survival [see reviews (Johansen et al., 2011, Johansen et al., 2012)]. Serotonergic neurons located in the brainstem dorsal and median raphe nuclei project to the amygdala, hippocampus, and ventromedial prefrontal cortex (PFC). Within the BLC, extracellular levels of 5-HT increase rapidly during conditioned fear (Zanoveli et al., 2009) and following exposure to inescapable stress (Amat et al., 1998). Following inescapable stress the increase in extracellular 5-HT is prolonged relative to either escapable stress or restraint stress, and remains elevated 100% above escapable stress or restraint stress controls for 24 h. The persistent increases in extracellular 5-HT concentrations within the amygdala following stress may contribute to a net loss of local GABA inhibition and subsequent increase in excitation of glutamatergic projection neurons. In support of this, serotonin acutely increases GABAergic tone in the BLC by exciting local GABAergic interneurons via the postsynaptic 5-HT2A receptors (Jiang et al., 2009, McDonald and Mascagni, 2007, Rainnie, 1999), but stress downregulates the 5-HT2A receptor and reduces serotonin's effects on local GABAergic tone (Jiang et al., 2009). In general, increases in the excitability of amygdala glutamatergic projection neurons lead to enhanced fear conditioned behavior, so, stress-induced downregulation of 5-HT2A receptors, loss of GABAergic tone, and disinhibition of glutamatergic projection neurons should also enhance fear conditioning. This hypothesis is supported by work done by Bosker and Ravinder where a single systemic injection of serotonin reuptake inhibitor in rats increases extracellular 5-HT in the amygdala by ~ 150% (Bosker et al., 2001) and also enhances acquisition of fear associated freezing responses, and increased fear conditioned freezing responses (Ravinder et al., 2013). In contrast, reduction of 5-HT tone in the amygdala using the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) reduces conflict anxiety (Sommer et al., 2001), but little is known about how the depletion of 5-HT affects acquisition of fear conditioning.

In the present article, we hypothesized that chronic reduction in serotonergic tone within the BLC region would severely disrupt both the acquisition of conditioned fear, as well as extinction and extinction recall responses. In order to reduce serotonin tone within the BLC, we used 5,7-DHT, which, although the mechanism is not entirely clear, reproducibly depletes local 5-HT by up to 90% in forebrain structures such as the amygdala (Bjorklund et al., 1975, File et al., 1979, Sommer et al., 2001, Tran et al., 2013). To test this hypothesis, we bilaterally injected 5,7-DHT into the BLC to chronically reduce local serotonergic neurotransmission, then assessed anxiety-like behavior and conditioned fear responses, and validated depletion of local 5-HT ex vivo. Since 5,7-DHT has been shown to also reduce local norepinephrine levels at higher doses, a norepinephrine reuptake inhibitor (desipramine) was administered systemically since it has been shown to block this effect (Bjorklund et al., 1975), and norepinephrine, and dopamine were also assessed to confirm that the depletion was specific to 5-HT.

Section snippets

Animals

All experiments were conducted on adult male Wistar rats (300–325 g), which were purchased from Harlan Laboratories and were housed individually in plastic cages under standard environmental conditions (22 °C; 12/12 light/dark cycle; lights on at 7:00 A.M.) for 7–10 days prior to the surgical manipulations. Food and water were provided ad libitum. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth Edition (Institute for Laboratory Animal

Open-field behavior and social interaction test

Rats receiving intra-BLC injections of vehicle (n = 8) or 5,7-DHT (n = 11) did not show differences in general locomotor associated behaviors (i.e., distance traveled, t(16) =  0.7, p = 0.477) in the open-field (one less n for vehicle group due to malfunctioning video). Although the 5,7-DHT-treated rats did not show a preference for spending more time in the center regions of the open-field (center time, t(7) = 0.5, p = 0.630; data not shown), they did show an increase in social interaction, compared to

Discussion

Here we show that depletion of serotonin within the BLC using 5,7-DHT decreased anxiety-associated behaviors in a social interaction test, but also reduced acquisition of cue-induced fear conditioned freezing (as well as an expected proportionate reduction in recall during extinction sessions). In Experiment 1, ex vivo analyses of microdissected tissue revealed that 5,7-DHT reduced local 5-HT concentrations in the BLC/CeA by ~ 40% without altering local norepinephrine or dopamine concentrations,

Conclusions

The present data, in combination with data showing that pharmacologically increasing 5-HT with SSRIs enhances fear conditioning in rodent and in humans, further support an important role for 5-HT in the modulation of anxiety-like behavior and fear-associated memories through its actions within the BLC. Furthermore, our data are consistent with previous experiments where increasing or depleting 5-HT levels in the BLC region respectively enhances or diminishes fear conditioned behaviors. These

Declaration of conflicting interests

No author has a conflict of interest for the data presented here.

Acknowledgements

This work was supported with K01 AG044466 to PLJ and R01 MH52619 and MH52619 to AS.

References (37)

Cited by (43)

View all citing articles on Scopus
View full text