Behavioral and immunohistochemical characterization of rapid reconditioning following extinction of contextual fear

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In many cases of trauma, the same environmental stimuli that become associated with aversive events are experienced on other occasions without adverse consequence. We examined neural circuits underlying partially reinforced fear (PRF), whereby mice received tone-shock pairings on half of conditioning trials. Tone-elicited freezing was lower after PRF conditioning than fully reinforced fear (FRF) conditioning, despite an equivalent number of tone-shock pairings. PRF preferentially activated medial prefrontal cortex (mPFC) and bed nucleus of the stria terminalis (BNST). Chemogenetic inhibition of BNST-projecting mPFC neurons increased PRF, not FRF, freezing. Multiplexing chemogenetics with in vivo neuronal recordings showed elevated infralimbic cortex (IL) neuronal activity during CS onset and freezing cessation; these neural correlates were abolished by chemogenetic mPFC→BNST inhibition. These data suggest that mPFC→BNST neurons limit fear to threats with a history of partial association with an aversive stimulus, with potential implications for understanding the neural basis of trauma-related disorders. eLife digest While walking home alone late one night, you hear footsteps behind you. Your heart starts to beat faster as you wonder whether someone might be following you. Being able to identify and evade threats is essential for survival. A key part of this process is learning to recognize signals that predict potential danger: the sound of footsteps behind you, for example. But many such cues are unreliable. The person behind you might simply be heading in the same general direction as you. And if you spend too much time and energy responding to such false alarms, you may struggle to complete other essential tasks. To be useful, responses to cues that signal potential threats must thus be proportionate to the likelihood that danger is actually present. By studying threat detection in mice, Glover et al. have identified a brain circuit that helps ensure that this is the case. Two groups of mice learned to fear a tone that predicted the delivery of a mild footshock. In one group of animals, the tone was followed by a shock on every trial (it was said to be ‘fully reinforced’). But in the other group, the tone was followed by a shock on only 50% of trials (‘partially reinforced’). After training, both groups of mice froze whenever they heard the tone – freezing being a typical fear response in rodents. But the animals trained with the partially reinforced tone showed less freezing than their counterparts in the fully reinforced group. Moreover, freezing in response to the partially reinforced tone was accompanied by activity in a specific neural pathway connecting the frontal part of the brain to an area called the bed nucleus of the stria terminalis. Inhibiting this pathway made mice respond to the partially reinforced tone as though it had been reinforced on every trial. This suggests that activity in this pathway helps dampen responses to unpredictable threat cues. In people with anxiety disorders, cues that become associated with unpleasant events can trigger anxiety symptoms, even if the association is unreliable. The findings of Glover et al. suggest that reduced activity of circuits that constrain excessive responses to threats might contribute to anxiety disorders. Introduction In many cases of psychological trauma, encounters with contexts and stimuli during aversive experience(s) are interleaved with occasions when the same stimuli are experienced without consequence. Most standard rodent assays of fear (i.e., threat) memory, however, present the subject with a conditioning stimulus (CS) that on each occasion is paired with an aversive unconditioned stimulus (US) (Fanselow and Poulos, 2005). This discrepancy is pertinent to modeling traumatic memories in rodents, via back translation from human to rodent. Theoretical accounts of associative learning predict that conditioned responses to CSs with a mixed or partial reinforcement history, which render the CS uncertain or ambiguous with regard to its expected outcome, may differ in certain respects from those that are consistently reinforced. For example, as compared to fully reinforced CSs, partially reinforced CSs can be more difficult to extinguish and produce lesser conditioned responses, due to associative strength accruing to the conditioning context or through the endowment of the CS with inhibitory (CS = no US) properties (Humphreys, 1939; Fitzgerald, 1963; Rawlins et al., 1985; Rescorla, 2007; Tsetsenis et al., 2007; Miguez et al., 2012; Harris et al., 2019). Fear behavior that arises from partial reinforcement could involve neural circuits distinct from the well-described circuits implicated in standard (i.e., fully reinforced) fear conditioning (Pape and Pare, 2010; Bukalo et al., 2014; Tovote et al., 2015). Two brain regions that could be important for the acquisition and expression of partially reinforced fear (PRF) are the medial prefrontal cortex (mPFC, comprising, in the rodent, the prelimbic [PL], infralimbic [IL], and anterior cingulate [ACC] cortices) and the bed nucleus of the stria terminalis (BNST) (Lebow and Chen, 2016; Goode et al., 2019). The mPFC is engaged in experimental situations requiring integration of higher-order cues or disambiguation between conflicting cues to gate a level of response appropriate to the value of outcome (Sharpe and Killcross, 2018; Marek et al., 2019), while the BNST has been shown to support learning when a stimulus poorly predicts threat (Lebow and Chen, 2016; Goode et al., 2019). These structures are also anatomically connected, with a particularly dense connection between the IL and the anterior regions of the BNST (Hurley et al., 1991; McDonald et al., 1999; Dong et al., 2001; Vertes, 2004; Radley and Sawchenko, 2011; Radley et al., 2013; Johnson et al., 2016; Glangetas et al., 2017; Tillman et al., 2018; Johnson et al., 2019). Additionally, BNST-projecting IL cells are activated by ‘unpredictable’ threat in a backward conditioning paradigm (Goode et al., 2019). Moreover, stimulation of glutamatergic mPFC inputs produces synaptic depression in the BNST (Glangetas et al., 2013). Together, these findings suggest the mPFC and BNST might form a functional circuit regulating fear to ambiguous and uncertain threats. Here, we sought to elucidate the potential role of the mPFC and BNST and other neural circuits in PRF, using a paradigm in which a CS was paired with a footshock US on only half of the trials (McHugh et al., 2015; Glover et al., 2017). By combining immediate-early gene mapping, neuronal pathway tracing, in vivo chemogenetics, and a multiplexed approach combining in vivo chemogenetics and in vivo neuronal recordings, we demonstrate that the mPFC→BNST circuit negatively gates PRF. Results Lower freezing to a partially reinforced CS The PRF conditioning procedure entailed presenting male C57BL/6J (B6) mice with three pairings of a tone CS and a footshock US, along with three interspersed presentations of the same CS without concomitant footshock (McHugh et al., 2015; Glover et al., 2017). For comparison, a fully reinforced fear (FRF) group received 3x CS+US pairings, and a CS-only control group received 6x CS presentations without the US (Figure 1A,B). Figure 1 with 2 supplements see all Download asset Open asset Lower freezing during retrieval of partially reinforced fear; effects of genetic strain. (A) Schematic depiction of experimental procedure for assessing, in B6 mice, PRF and FRF, along with CS-only controls. (B) Schematic depiction of experimental procedure for assessing, in B6 mice, PRF and FRF retrieval in a novel context (context B) and the conditioning context (context A) (C) Lower CS-related freezing during retrieval in PRF mice than in FRF mice. Higher baseline and CS-related freezing in PRF and FRF mice relative to CS-only controls (n = 4–8 mice per group). (D) Schematic depiction of experimental procedure for assessing PRF and FRF retrieval in the B6 and S1 genetic strains. (E) Lower CS-related freezing during retrieval in PRF than in FRF in B6, not S1, mice (n = 7–8 mice per group/strain). Data are means ± SEM. *p<0.05. Figure 1—source data 1 PRF versus FRF (Figure 1C). https://cdn.elifesciences.org/articles/60812/elife-60812-fig1-data1-v2.xls Download elife-60812-fig1-data1-v2.xls Figure 1—source data 2 Strain comparison (Figure 1E). https://cdn.elifesciences.org/articles/60812/elife-60812-fig1-data2-v2.xls Download elife-60812-fig1-data2-v2.xls Freezing increased to a similar extent over the six conditioning trials in the PRF group and over the three conditioning trials, plus the corresponding three no-trial periods, in FRF group, but did not significantly increase in the CS-only group (analysis of variance [ANOVA] group-effect: F(2,17)=5.74, p=0.0125; trial-effect: F(5,85)=13.49, p<0.0001; interaction: F(5,85)=1.64, p=0.1099). On a retrieval test conducted in a novel context (context B) the following day, the PRF and FRF groups froze more than CS-only controls during pre-CS baseline and CS presentation. Notably, however, CS-evoked freezing was lower in the PRF, relative to the FRF, group (ANOVA group-effect: F(2,17)=53.02, p<0.0001; CS-effect: F(1,17)=216.90, p=0.0001; interaction: F(2,17)=25.51, p=0.0001, followed by post-hoc tests: CS-only vs PRF p<0.0001, CS-only vs FRF p<0.0001, PRF vs FRF p=0.0008) (Figure 1C, Figure 1—figure supplement 1). These data show that B6 mice express less freezing in the PRF, as compared to FRF, procedure despite the number of CS–US pairings being equivalent in both conditions. These differences are in line with lower freezing in the PRF procedure in a mixed C57BL/6J;CBA/J;129S6/SvEvTac genetic background (Tsetsenis et al., 2007) but, indicating a degree of strain dependency of PRF, differ from data in outbred CD-1 mice, in which freezing is equivalent between PRF and FRF groups (Glover et al., 2017). Mice with an abnormal fear phenotype do not exhibit lower PRF We next reasoned that an inbred strain (S1), which exhibits impaired contextual (and cued) fear discrimination, deficits in limiting fear following extinction and conditioned inhibition, and high fear expression in a different assay for PRF (Camp et al., 2012), might exhibit deficits in the current PRF assay (Camp et al., 2009; Camp et al., 2012; Figure 1D). Across conditioning trials, there was increased freezing in the PRF and FRF groups, irrespective of strain (ANOVA group-effect: F(1,25)=0.15. p=0.7023, trial-effect: F(5,125)=38.02, p<0.0001; strain-effect: F(1,25)=7.68, p=0.0103; three-way interaction: F(5,125)=0.66, p=0.6539). On retrieval in context B, PRF B6 mice showed less CS-related freezing than their FRF counterparts, whereas freezing was equivalent in PRF and FRF S1 mice (ANOVA strain-effect: F(1,27)=8.72, p=0.0065; conditioning-type effect: F(1,27)=6.15, p=0.0197; CS-effect: F(1,27) = 524.00, p<0.0001; three-way interaction: F(1,27)=7.03, p=0.0132, followed by post-hoc tests: PRF vs FRF in B6 p=0.0038, PRF vs FRF in S1 p=0.2322) (Figure 1E, Figure 1—figure supplement 1). The finding that S1 mice exhibit similar freezing to the PRF and FRF procedures aligns with the excessive fear shown by this strain to innocuous stimuli and following extinction (Camp et al., 2009; Camp et al., 2012) and further illustrates the strain dependency of PRF. Increased latency to feed in the novelty-suppressed feeding test after PRF An earlier study by Glover et al., 2017 found that following PRF conditioning, CD-1 mice had a higher latency to feed, as compared to a FRF group, in the novelty-suppressed feeding (NSF) test, an assay sensitive to anxiolytics and antidepressants (Ramaker and Dulawa, 2017). To test whether PRF had a similar effect in B6 mice, NSF was assessed under either high or low illumination levels the day after B6 mice underwent either PRF or FRF. Under high, but not low, illumination, latencies to feed were higher in the PRF and FRF groups than unconditioned controls (ANOVA group-effect: F(1,44)=10.93, p=0.0019; illumination-effect: F(1,44)=4.11, p=0.0230; interaction: F(1,44)=2.83, p=0.0699, followed by post hoc tests: PRF vs Con p=0.0004, FRF vs Con p=0.0222) (Figure 1—figure supplement 2). These data show that PRF conditioning increases anxiodepressive-like anxiety-like behavior under relatively aversive (high illumination) conditions of approach–avoidance conflict. Ex vivo neuronal regional activity correlates of PRF The complex behavioral sequelae of PRF suggest that this form of fear may have different neural substrates than FRF. We, therefore, sought to identify neural correlates of PRF by quantifying the number of c-Fos+ cells, as a proxy for neuronal activity, in forebrain regions following retrieval (for corresponding behavioral data, see Figure 1B). There were a higher number of c-Fos+ cells in the basolateral amygdala (BLA) of FRF mice (ANOVA group-effect: F(2,17)=6.79, p=0.0068, followed by post hoc tests: FRF vs CS-only p=0.0038, FRF vs PRF p=0.0132), as compared to either PRF mice or a set of controls that had received CS-only trials during conditioning. In the paraventricular nucleus of the thalamus (PVT), another region implicated in fear (Penzo et al., 2015), c-Fos+ counts were higher in the PRF and FRF groups than controls (ANOVA group-effect: F(2,17)=4.01, p=0.0374, followed by post-hoc tests: CS-only vs PRF p=0.0281, CS-only vs FRF p=0.0145). No group differences were evident in the lateral or medial habenula, or ventral or dorsal hippocampus (Figure 2A–I, Figure 2—figure supplement 1). Figure 2 with 2 supplements see all Download asset Open asset PRF preferentially activates subregions of mPFC and BNST. (A) Schematic depiction of experimental procedure for assessing ex vivo neuronal regional activity (via c-Fos immunohistochemistry) after PRF or FRF retrieval, along with CS-only controls. Representative images and c-Fos+ cell count differences for basal amygdala (B), paraventricular nucleus of the thalamus (C), infralimbic cortex (D), prelimbic cortex (E), posterior portion of the anterior cingulate cortex (F), anteroventral BNST (G), anterodorsal BNST (H), and lateral habenula (I). For corresponding behavioral data, see Figure 1B. Scale bars = 30 µm (B,D–F), 100 µm (C,I), 300 µm (G,H). n = 4–8 mice per group. Data are means ± SEM. *p<0.05. Figure 2—source data 1 c-Fos. https://cdn.elifesciences.org/articles/60812/elife-60812-fig2-data1-v2.xls Download elife-60812-fig2-data1-v2.xls In subregions of the mPFC, however, there were more c-Fos+ cells in the IL (F(2,17)=8.21, p=0.0032, followed by post-hoc tests: CS-only vs PRF p=0.0009, CS-only vs FRF p=0.0411, FRF vs PRF p=0.0420), but not the posterior ACC (F(2,17)=1.01, p=0.3862) of PRF and FRF mice, relative to CS-only controls. Counts in the PL were higher in PRF mice relative to controls and trended higher in the FRF group (F(2,17)=3.60, p=0.0499, followed by post hoc tests: CS-only vs PRF p=0.0196). The same pattern of elevated activity in the PRF group, relative to the other groups, was also evident in the BNST, though specifically in the anteroventral BNST (avBNST) (F(2,17)=19.43, p=0.0001, followed by post hoc tests: CS-only vs PRF p=0.0001, CS-only vs FRF p=0.0294, PRF vs FRF p=0.0005), not the anterodorsal BNST (adBNST) (F(2,14)=1.38, p=0.2831) (Figure 2A–I). These findings show that retrieval of a PRF CS, despite being characterized by lower freezing than FRF, associates with a unique pattern of regional brain activation, with preferentially high activation in the IL and PL subregions of the mPFC and the avBNST. Connectivity between mPFC, BNST, and downstream targets Previous studies in the rat have demonstrated a direct (GABAergic) input from the mPFC to the BNST that is particularly dense between the IL and avBNST (Dong et al., 2001), but also present between the PL and avBNST (Johnson et al., 2016; Johnson et al., 2019). As our c-Fos data indicated activation of the IL, PL, and avBNST by PRF, we sought to verify an mPFC-to-BNST projection in mice. In a combinatorial viral tracing approach to label postsynaptic targets of mPFC neurons in the BNST (Zingg et al., 2017; Sengupta and Holmes, 2019), a construct containing a Cre-containing anterograde trans-synaptic virus was infused into the mPFC and a Cre-dependent, synaptophysin-containing, mCherry-fused construct infused into the BNST (Figure 2—figure supplement 2). Indicative of monosynaptic input from the mPFC, mCherry labeling was apparent in BNST neurons, mainly in the ventral areas below the anterior commissure. In the rat, PL neurons form close appositions with GABAergic cells in the avBNST that in turn send efferents to the paraventricular nucleus of the hypothalamus (PVN), a key mediator of responses to stress and defensive behaviors (Johnson et al., 2016; Johnson et al., 2019). Indicating that a similar connection is likely present in mice, inspection of our tissue revealed mCherry/synaptophysin expression originating from mPFC-innervated BNST neurons in the PVN, as well as lateral hypothalamus. A corollary to the existence of a disynaptic mPFC–BNST–PVN circuit in mice is whether the PVN in turn targets other fear-mediating regions in this species. To gain initial insight into this question, we infused a Cre-dependent, YFP-fused construct containing either channelrohodpsin2 (ChR2) or synaptophysin into the PVN of oxytocin-Cre mice, to label a major population of (oxytocin-positive) PVN cells. This indicated labeling in the ventrolateral periaqueductal gray (vl/PAG) (Figure 2—figure supplement 2), a region known to regulate defensive behaviors including freezing (Tovote et al., 2015). Together these data provide evidence of input from the mPFC to the BNST in the mouse, as well as onward connections from the BNST to the PVN and in turn possibly on to the vl/PAG. Thus, PRF engagement of the mPFC and BNST can be viewed in the context of a direct connection between these regions and their downstream access to a broader fear-regulating neural circuitry. Inhibition of mPFC→BNST neurons increases freezing to a PRF CS To causally interrogate the contribution of the mPFC→BNST pathway to PRF, a retrogradely transported Cre-containing construct viral construct was infused into the BNST and a construct containing a Cre-dependent form of hM4Di (or mCherry control) infused into the mPFC, enabling the expression of the inhibitory DREADD in mPFC→BNST neurons to inhibit their activity, via systemic injection of clozapine N-oxide (CNO), during retrieval (Figure 3A,B). Figure 3 with 4 supplements see all Download asset Open asset Inhibition of mPFC→BNST neurons increases PRF. (A) Schematic depiction of experimental procedure for assessing effects of chemogenetic inhibition of mPFC→BNST neurons during retrieval. (B) Cartoon of viral strategy and representative images of hM4Di–mCherry labeling in BNST neurons receiving mPFC input (scale bars = 200 µm). (C) Lower CS-related freezing during retrieval in PRF mice than in FRF mice transfected with mCherry, not hHM4Di. Data are means ± SEM. *p<0.05. During conditioning, freezing increased over trials to a similar extent in all groups (ANOVA trial-effect: F(5,145)=23.54, p<0.0001; group effect: F(3,145)=0.91, p=0.4467; interaction: F(15,145)=0.61, p=0.08647) (Figure 3—figure supplement 1). Following CNO administration, CS-related freezing during retrieval was lower in PRF mice than in FRF mice expressing the control virus, replicating our earlier data. By contrast, there was no difference in freezing in mice expressing hM4Di (ANOVA conditioning-type effect: effect: p<0.0001; three-way interaction: followed by post-hoc tests: mCherry PRF vs mCherry FRF p<0.0001, hM4Di PRF vs hM4Di FRF mCherry PRF vs hM4Di PRF mCherry PRF vs hM4Di PRF (Figure of the freezing during retrieval indicated no differences in despite a for freezing trials in the mCherry PRF group (ANOVA trial-effect: group-effect: p<0.0001; trial group interaction: (Figure 3—figure supplement 1). These data show that inhibition of mPFC→BNST neurons increases freezing to a PRF This finding suggests that engagement of these mPFC→BNST neurons the expression the PRF, though it that inhibition of these neurons also produces an increase in PRF which may have been due to high levels of freezing. IL cells signal CS onset and freezing The finding that mPFC to the BNST pathway increases freezing to a PRF CS that mPFC neurons likely of To this we an approach chemogenetic inhibition of mPFC→BNST neurons with in vivo recordings of mPFC activity via which we the IL (Figure 3—figure supplement 2). The of did not differ between groups ± FRF ± FRF ± FRF ± the data to the of the CSs during retrieval revealed of IL with activity to the onset of the CS (Figure 3—figure supplement activity from baseline in 100 time the of CS onset were as CS showed a in neuronal activity in response to the CS ± ± paired (Figure 3—figure supplement and for see Figure 3—figure supplement responses of CS onset and were in the mCherry FRF group (Figure 3—figure supplement when the of was and compared the conditioning and virus groups, this revealed a higher of in the mCherry groups than in hM4Di groups for PRF mice but no differences between virus groups in the FRF mice and no difference between PRF and FRF groups, irrespective of virus group test in in (Figure 3—figure supplement To whether IL cells were also associated with the behavior of mice during their activity was to of freezing and those cells a relative to either the onset or of freezing (i.e., of from baseline in 100 time the of the These as and showed a in activity ± ± paired ± ± paired (Figure 3—figure supplement and for see Figure 3—figure supplement a freezing which was evident in both of the mCherry groups, while increased the of freezing in both groups (Figure 3—figure supplement the of these cell were compared groups, there was a higher of in the mCherry PRF group than in the hM4Di PRF group whereas there was no group difference in FRF mice and no difference between PRF and FRF groups in either the mCherry or hM4Di virus conditions (Figure 3—figure supplement there was no difference between the mCherry and hM4Di groups in the of irrespective of whether mice had PRF or FRF. Discussion Here, we sought to provide insight into the neural substrates regulating the fear response to an an assay of partial reinforcement in B6 mice, we found that PRF conditioning a lower fear response than FRF, which was associated with neuronal activation in the mPFC and BNST. We also show that the mPFC and BNST a monosynaptic circuit when a increase in the expression of PRF and an of in vivo correlates of both CS onset and freezing in IL The current findings with and the mPFC and BNST in situations in which there is and a For example, the mPFC is engaged in that integration of higher-order cues to gate learned responses and 2015; and 2017; and Killcross, 2018; Marek et al., 2019), or there is between and inhibitory CS for in fear extinction and 2012; et al., 2018; et al., fear et al., conditioning et al., 2014; et al., 2019), and et al., 2017; et al., The BNST, learning in the of the et al., 2010; and and when a stimulus poorly predicts threat (Lebow and Chen, 2016; Goode et al., et al., either it is et al., et al., 2012; et al., 2015; et al., 2018; Goode et al., et al., 2004; et al., et al., 2009; et al., 2010; et al., 2012; et al., 2013; et al., or ambiguous or et al., et al., 2009; et al., 2015; et al., 2016; Goode and 2017; et al., with to the These known of the mPFC and BNST these structures well to fear under conditions of partial reinforcement the CS is experienced both with and without the As we show the mPFC and BNST form a neural through a direct that to limit the expression of partially reinforced This is of a study that IL neurons to the avBNST are activated by by a of unpredictable threat in which US the CS (Goode and 2017). In with the current data, there is evidence a key role for the mPFC→BNST circuit in fear of threat and The of this role to be fully is that when cues are relatively of other environmental such as the expression of fear in a that the mPFC to control over the BNST. In support of this the mPFC is to higher-order of conditioned responding (Sharpe and Killcross, a study in found that of either the PL or IL impaired one of such known as occasion and Killcross, 2019). for the increase in PRF by mPFC→BNST inhibition is that presentations during conditioning the CS with inhibitory properties that are by mPFC→BNST neurons during retrieval. inhibition is a to the mPFC and in the For example, and chemogenetic inhibition of the IL the retrieval of fear extinction memories and 2009; Bukalo et al., 2015; et al., 2015; et al., 2016; et al., Bukalo et al., and the expression of learned through CS–US et al., of a signal during stress activity in a lateral area of BNST avBNST et al., this area fear to a CS in with anxiety et al., a number of and functional studies in and have implicated the BNST in the of uncertain threat Goode et al., and 2019). Together, these findings suggest that inhibitory properties of the partially reinforced CS could be by the IL downstream to the BNST, limiting the expression of fear to a level appropriate to its partial reinforcement is important to in this regard that while we our virus to the IL and avBNST – on evidence of a dense connection between these subregions – the and ventral of these areas

Although a great deal is known about neurobiological mechanisms of initial conditioning and extinction, relatively little is known about mechanisms involved in the return of behavior following extinction. In this article, we examine the effects of temporarily inactivating the bed nucleus of the stria terminalis (BNST) on initial conditioning and postextinction reconditioning. We investigate effects in unsignaled contextual fear conditioning, in which animals initially receive strong contextual conditioning, followed by three sessions of nonreinforced context exposure (extinction), and then receive a single context-shock reconditioning trial. In 2 experiments with male Long Evans rats, we evaluated the effects of delivery of a muscimol/baclofen cocktail to the BNST prior to initial conditioning or reconditioning. In Experiment 1, we found that a single context-shock pairing results in more freezing following extinction than when it is the initial conditioning trial. This rapid reconditioning effect was impaired by BNST inactivation. In Experiment 2, we found that BNST inactivation also causes a deficit in freezing after strong initial conditioning. These findings suggest that the BNST is involved in both initial conditioning and postextinction reconditioning. We discuss implications of these findings for current thinking about BNST function in learning and memory processes. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Involvement of the prelimbic prefrontal cortex on cannabidiol-induced attenuation of contextual conditioned fear in rats

Numerous studies have attributed the psychopathology of post-traumatic stress disorder (PTSD) to maladaptive behavioral responses such as an inability to extinguish fear. While exposure therapies are mostly effective in treating these disorders by enhancing extinction learning, relapse of PTSD symptoms is common. Although several studies indicated a role for cholinergic transmission and nicotinic acetylcholine receptors (nAChRs) in anxiety and stress disorder symptomatology, very little is known about the specific contribution of nAChRs to fear extinction OBJECTIVES: In the present study, we examined the effects of inhibition and desensitization of α4β2 nAChRs via a full antagonist (Dihydro-beta-erythroidine (DhβE)) and two α4β2 nAChR partial-agonists (varenicline and sazetidine-A) on contextual fear extinction, locomotor activity, and spontaneous recovery of contextual fear in mice. We trained and tested the subjects in a contextual fear extinction as well as an open field paradigm and spontaneous recovery following injections of DhβE, varenicline, and sazetidine-A. Our results demonstrated that lower doses of DhβE (1mg/kg) and sazetidine-A (0.01mg/kg) enhanced contextual fear extinction whereas higher doses of varenicline (0.1mg/kg) and sazetidine-A (0.1mg/kg) resulted in impaired contextual fear extinction. However, the higher dose of sazetidine-A (0.1mg/kg) decreased locomotor activity, which may contribute to increased freezing response observed during fear extinction. Finally, we found that the low dose of DhβE, but not sazetidine-A, also decreased spontaneous recovery of contextual fear following fear extinction. Overall, these results suggest that inhibition and desensitization of α4β2 nAChRs enhance extinction of contextual fear memories. This suggests that modulation of α4β2 nAChRs may be employed as an alternative pharmacological strategy to aid exposure therapies associated with PTSD by augmenting contextual fear extinction processes.

BackgroundIn the fear memory network, the hippocampus modulates contextual aspects of fear learning while mutual connections between the amygdala and the medial prefrontal cortex are widely involved in fear extinction. G-protein-coupled receptors (GPCRs) are involved in the regulation of fear and anxiety, so the regulation of GPCRs in fear signaling pathways can modulate the mechanisms of fear memory acquisition, consolidation and extinction. Various studies suggested a role of M-type K+ channels in modulating fear expression and extinction, although conflicting data prevented drawing of clear conclusions. In the present work, we examined the impact of M-type K+ channel blockade or activation on contextual fear acquisition and extinction. In addition, regarding the pivotal role of the hippocampus in contextual fear conditioning (CFC) and the involvement of the axon initial segment (AIS) in neuronal plasticity, we investigated whether structural alterations of the AIS in hippocampal neurons occurred during contextual fear memory acquisition and short-time extinction in mice in a behaviorally relevant context.ResultsWhen a single systemic injection of the M-channel blocker XE991 (2 mg/kg, IP) was carried out 15 minutes before the foot shock session, fear expression was significantly reduced. Expression of c-Fos was increased following CFC, mostly in GABAergic neurons at day 1 and day 2 post-fear training in CA1 and dentate gyrus hippocampal regions. A significantly longer AIS segment was observed in GABAergic neurons of the CA1 hippocampal region at day 2.ConclusionsOur results underscore the role of M-type K + channels in CFC and the importance of hippocampal GABAergic neurons in fear expression.

Females and males are different in brain and behavior. These sex differences occur early during development due to a combination of genetic and hormonal factors and continue throughout the lifespan. Previous studies revealed that male rats exhibited significantly higher levels of contextual fear memory than female rats. However, it remains unknown whether a sex difference exists in the contextual fear extinction. To address this issue, male, normally cycling female, and ovariectomized (OVX) female Sprague-Dawley rats were subjected to contextual fear conditioning and extinction trials. Here we report that although male rats exhibited higher levels of freezing than cycling female rats after contextual fear conditioning, female rats subjected to conditioning in the proestrus and estrus stage exhibited an enhancement of fear extinction than male rats. An estrogen receptor (ER) beta agonist diarylpropionitrile but not an ERalpha agonist propyl-pyrazole-triol administration also enhanced extinction of contextual fear in OVX female rats, suggesting that estrogen-mediated facilitation of extinction involves the activation of ERbeta. Intrahippocampal injection of estradiol or diarylpropionitrile before extinction training in OVX female rats remarkably reduced the levels of freezing response during extinction trials. In addition, the locomotion or anxiety state of female rats does not vary across the ovarian cycle. These results reveal a crucial role for estrogen in mediating sexually dimorphic contextual fear extinction, and that estrogen-mediated enhancement of fear extinction involves the activation of ERbeta.

Baclofen administration alters fear extinction and GABAergic protein levels

Anxiety, depression, and social impairment exhibit high clinical comorbidity, yet their underlying shared neural circuitry remains poorly defined. Using a mouse model of chronic social isolation combined with circuit tracing and chemogenetic tools, we identified a key role for the basolateral amygdala (BLA) in relaying prefrontal cortex (PFC) signals to the bed nucleus of the stria terminalis (BNST) to drive behavioral changes. Further circuit dissection identified two distinct BNST microcircuits segregated by their input sources: one receives indirect PFC input relayed through the BLA (PFC → BLA → BNST), while the other is innervated by direct PFC projections (PFC → BNST). Chemogenetic inhibition of BLA neurons in the indirect pathway ameliorated anxiety-like behavior, depression-like behavior, and social deficits. Within the BNST, however, inhibition of neurons in PFC → BLA → BNST pathway selectively alleviated affective phenotypes without altering social behavior. In contrast, inhibition of neurons in PFC → BNST pathway specifically restored social recognition while leaving emotional behaviors intact. Thus, the BLA integrates PFC-derived signals to broadly modulate behavior, while downstream BNST microcircuits dissociate these influences. The indirect, BLA-relayed pathway within the BNST specifically drives affective symptoms, whereas the direct PFC → BNST pathway selectively governs social recognition. This dissociable circuit model offers a new framework for understanding clinical comorbidity and may inform targeted interventions for distinct symptom dimensions.

Sex differences in fear extinction and involvements of extracellular signal-regulated kinase (ERK)

Maternal separation exaggerates spontaneous recovery of extinguished contextual fear in adult female rats

Surviving threats in the environment requires brain circuits for detecting (or anticipating) danger and for coordinating appropriate defensive responses (e.g., increased cardiac output, stress hormone release, and freezing behavior). The bed nucleus of the stria terminalis (BNST) is a critical interface between the “affective forebrain”—including the amygdala, ventral hippocampus, and medial prefrontal cortex—and the hypothalamic and brainstem areas that have been implicated in neuroendocrine, autonomic, and behavioral responses to actual or anticipated threats. However, the precise contribution of the BNST to defensive behavior is unclear, both in terms of the antecedent stimuli that mobilize BNST activity and the consequent defensive reactions. For example, it is well known that the BNST is essential for contextual fear conditioning, but dispensable for fear conditioning to discrete conditioned stimuli (CSs), at least as indexed by freezing behavior. However, recent evidence suggests that there are circumstances in which contextual freezing may persist independent of the BNST. Furthermore, the BNST is involved in the reinstatement (or relapse) of conditioned freezing to extinguished discrete CSs. As such, there are critical gaps in understanding how the BNST contributes to fundamental processes involved in Pavlovian fear conditioning. Here, we attempt to provide an integrative account of BNST function in fear conditioning. We discuss distinctions between unconditioned stress and conditioned fear and the role of BNST circuits in organizing behaviors associated with these states. We propose that the BNST mediates conditioned defensive responses—not based on the modality or duration of the antecedent threat or the duration of the behavioral response to the threat—but rather as consequence the ability of an antecedent stimulus to predict when an aversive outcome will occur (i.e., its temporal predictability). We argue that the BNST is not uniquely mobilized by sustained threats or uniquely involved in organizing sustained fear responses. In contrast, we argue that the BNST is involved in organizing fear responses to stimuli that poorly predict when danger will occur, no matter the duration, modality, or complexity of those stimuli. The concepts discussed in this review are critical to understanding the contribution of the human BNST to fear and anxiety disorders.

Pre-adolescent and adolescent mice are less sensitive to the effects of acute nicotine on extinction and spontaneous recovery

Panic disorder (PD) is associated with anticipatory anxiety, a sustained threat response that appears to be related to the bed nucleus of the stria terminalis (BNST). Individuals with panic disorder may demonstrate significant differences in causal connectivity of the BNST in comparison to healthy controls. To test this hypothesis, resting-state functional magnetic resonance imaging (fMRI) was used to identify aberrant causal connectivity of the BNST in PD patients. 19 PD patients and 18 healthy controls (HC) matched for gender, age and education were included. Granger causality analysis (GCA) utilizing the BNST as a seed region was used to investigate changes in directional connectivity. Relative to healthy controls, PD patients displayed abnormal directional connectivity of the BNST including enhanced causal connectivity between the left parahippocampal gyrus and left BNST, the right insula and the right BNST, the left BNST and the right dorsolateral prefrontal cortex (dlPFC) and right BNST to the left and right dlPFC. Furthermore, PD patients displayed weakened causal connectivity between the right dlPFC and the left BNST, the left dlPFC and the right BNST, the left BNST and the left dorsomedial prefrontal cortex (dmPFC), right insula, right fusiform, and right BNST to the right insula. The results suggest that PD strongly correlates with increased causal connectivity between emotional processing regions and the BNST and enhanced causal connectivity between the BNST and cognitive control regions.

Because of the use of radiation in cancer therapy, the risk of nuclear contamination from power plants, military conflicts, and terrorism, there is a compelling scientific and public health interest in the effects of environmental radiation exposure on brain function, in particular hippocampal function and learning and memory. Previous studies have emphasized changes in learning and memory following radiation exposure. These approaches have ignored the question of how radiation exposure might impact recently acquired memories, which might be acquired under traumatic circumstances (cancer treatment, nuclear disaster, etc.). To address the question of how radiation exposure might affect the processing and recall of recently acquired memories, we employed a fear conditioning paradigm wherein animals were trained, and subsequently irradiated (whole-body X-ray irradiation) 24 h later. Animals were given 2 weeks to recover, and were tested for retention and extinction of hippocampus-dependent contextual fear conditioning or hippocampus-independent cued fear conditioning. Exposure to irradiation following training was associated with reduced daily increases in body weights over the 22-days of the study and resulted in greater freezing levels and aberrant extinction 2 weeks later. This was also observed when the intensity of the training protocol was increased. Cued freezing levels and measures of anxiety 2 weeks after training were also higher in irradiated than sham-irradiated mice. In contrast to contextual freezing levels, cued freezing levels were even higher in irradiated mice receiving 5 shocks during training than sham-irradiated mice receiving 10 shocks during training. In addition, the effects of radiation on extinction of contextual fear were more profound than those on the extinction of cued fear. Thus, whole-body irradiation elevates contextual and cued fear memory recall.

Both the basal amygdala (BA) and the bed nucleus of the stria terminalis (BNST) can participate in contextual fear, but it is unclear whether contextual fear engrams involve a direct interaction between these two brain regions. To determine whether dorsal BNST (dBNST)-projecting neurons in the BA participate in contextual fear engrams, we combined the TetTag mouse with a retrograde tracer to label dBNST-projecting cells in the BA. We identified a population of neurons located in the anterior subdivision of the BA (aBA) that was activated during fear conditioning and reactivated during retrieval but that did not project to the dBNST. In contrast, dBNST-projecting neurons located in the posterior BA (pBA) were activated during contextual fear conditioning but were not reactivated during retrieval. Similarly, we found neurons in the oval BNST subdivision (ovBNST) that were activated during contextual fear conditioning without being reactivated during retrieval. However, the anterodorsal BNST (adBNST) subdivision was not activated during either contextual fear conditioning or retrieval, underscoring the divergent functionality of these two dBNST subdivisions. Finally, we found that the ovBNST receives a monosynaptic projection from neurons located in the BA. Our results indicate that aBA neurons that do not project to the dBNST participate in contextual fear engrams. In contrast, dBNST-projecting neurons in the BA do not appear to participate in contextual fear engrams, but might instead contain a BA → ovBNST pathway that is active during the initial encoding of contextual fear memories.