Absence of fear renewal and functional connections between prefrontal cortex and hippocampus in infant mice

Highlights

• Achieving context-independent extinction is critical to enhance exposure therapy.

• Little is known about the nature of fear memory extinction in the infants.

• Extinction of fear memory is context-independent in infant mice (P17).

• IL-BLA connections are functioning while CA1-PFC connections are not in P17 mice.

Abstract

Impairment in fear extinction is widely viewed as a major contributor to, or even an underlying mechanism of, the pathogenesis of anxiety disorders and PTSD. Children with traumatic experience have a higher risk for developing anxiety disorders and PTSD in the adult. Little is known about the nature of fear memory extinction and its underlying mechanism during this period. Here we showed that while renewal of fear memory is context-specific in adult mice, it is absent in infant mice (P17). Using local injection of GABAa receptor antagonist picrotoxin, we found that there is no functional connectivity between infralimbic prefrontal cortex and hippocampus in P17 mice, while prefrontal cortex projection to amygdala is functioning. Hence, the lack of fear renewal is likely caused by the lack of connections between hippocampus and prefrontal cortex which are known to be involved in the regulation of extinction memory.

Introduction

Context-dependent modulation of learned skills or information is vital to the appropriate function of an organism since this modulation allows modification or update of the stored information based on changes in its values or relationship to the external world (LaBar & Cabeza, 2006). Such modulation is also important for treating psychiatric diseases, such as PTSD and anxiety disorders. For example, exposure therapy has been used extensively and effectively in reducing the excessive expression of aversive responses in PTSD patients (Bowers and Ressler, 2015, Foa and McLean, 2016) The biological basis of exposure therapy is believed to involve modification of stored memory by repetitive retrieval of this memory in the absence of the reinforcer (Boschen et al., 2009, Maren, 2011, Maren et al., 2013).

With auditory fear conditioning, a neutral auditory cue (conditioned stimulus, CS) acquires the ability to predict the occurrence of an unconditioned stimulus (US, foot shock). During extinction learning in the adult, context information becomes associated with extinction which leads extinction recall to be highly context-dependent in that fear is reduced in the extinction context but returns in the non-extinction contexts (such as the conditioning context or a novel context) (termed fear renewal; Bouton and Moody, 2004, Quirk et al., 2006). Interestingly, renewal has been shown to be absent in the infant rats (Gogolla et al., 2009, Kim and Richardson, 2007a, Park et al., 2017). Conditioning involving different sensory modalities emerges at different time points during development (Stanton, 2000). For example, conditioning to an auditory cue emerges around postnatal day 15, while conditioning to a visual cue does not emerge until postnatal day 17 (Moye & Rudy, 1985). Therefore, the differences in fear response during extinction recall between infant and adult are unlikely accounted for by the immatureness of sensory afferents (such as auditory inputs) in the infants. It has been suggested that fear memory is erased in the infant (Gogolla et al., 2009, Kim and Richardson, 2010). Young children at critical developmental ages are more vulnerable to trauma than older children and adults (Hamiel et al., 2017, Simpson et al., 2012, Slone and Mann, 2016). Hence, a better understanding of the nature of fear extinction and whether/how context may modulate fear memory in the infants will likely provide new thinking and strategy in treating psychiatric diseases.

Fear extinction and retrieval requires a complex neural circuit involving hippocampus (HPC), medial prefrontal cortex (mPFC) and amygdala. mPFC is critical for extinction in the adult rats by regulating and integrating information related to fear memory (Quirk and Mueller, 2008, Sotres-Bayon et al., 2012). Numerous studies have suggested that prelimbic (PL) mPFC is associated with the expression of conditioned fear while infralimbic (IL) mPFC with suppression of conditioned fear during extinction learning and recall of extinction memory (Quirk and Mueller, 2008, Sharpe and Killcross, 2015). This IL-mediated fear memory suppression has been proposed to be carried out via its dense projections to clusters of GABAergic neurons (intercalated cell masses, ITCs) in the amygdala (Duvarci and Pare, 2014, Orsini and Maren, 2012, Pinto and Sesack, 2008). The amygdala receives direct cortical and thalamic sensory inputs and is reciprocally connected with many forebrain structures including mPFC and HPC (Pitkanen, Pikkarainen, Nurminen, & Ylinen, 2000). Basal and lateral amygdala is required for the acquisition, storage and expression of conditioned fear memory (Johansen et al., 2011, Schafe et al., 2001). Basal amygdala (BA) receives strong projections from the ventral hippocampus (vHPC), and sends a largely separate projection to the mPFC (Jin and Maren, 2015, Orsini and Maren, 2012).

Recent studies in the infant rodents have suggested that fear extinction is mediated by erasure of fear memory (Gogolla et al., 2009, Kim and Richardson, 2007a, Kim and Richardson, 2007b, Park et al., 2017). There is limited examination of the underlying mechanism in the infant rodents. For example, inactivation of mPFC during extinction training prevented the formation of long-term extinction memory in P24 rats but not in the P17 rats (Kim, Hamlin, & Richardson, 2009), suggesting that mPFC is not required for fear extinction, in clear contrast to the adult and adolescence. In addition, perineuronal net (PNN) level in the amygdala has been implicated in the “erasure” of fear memory since renewal was absent in P16 rats (when PNN level was low) or in adult rats with PNN level reduced pharmacologically (Gogolla et al., 2009).

In this study, we first confirmed the absence of renewal of cued fear memory after extinction training in the P17 mice. Then, by using immunocytochemistry analysis we showed that inputs to HPC appear to be functional while connections between HPC and mPFC did not appear to be functioning in P17 mice. Thus, our results suggest that the lack of mature connections between critical brain regions may mediate the absence of fear renewal in the infant rodents.