Extinction Learning from a Mechanistic and Systems Perspective - Oliver T. Wolf

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Sangha Extinction-Induced Plasticity by signals that inform the organism if the environment is safe alone and the compound fear+safety cue, were examined in or not. The inability to discriminate among danger, safety, and response to the fear cue during extinction training and recall as reward cues can lead to generalized fear responses that are fear behavior decreased. The hypothesis tested is that there is a enhanced in Post-traumatic Stress Disorder (PTSD) patients bias for the neurons that are safety cue responsive during DC to (Jovanovic et al., 2012). become responsive to the fear cue as fear extinction progresses. Behavioral therapy for maladaptive fear often involves repeated exposures to the danger cue in the absence of an aversive outcome, a procedure known as extinction. Through repeated MATERIALS AND METHODS exposures, the subject feels an increasing sense of control over the situation and fear diminishes. Safety conditioning is another Subjects method of reducing fear. During safety conditioning, a safety cue Fourteen Long Evans male rats (Harlan) weighing 350–400 g in conjunction with a danger cue signifies no aversive outcome at the beginning of experiments were single housed under a whereas the danger cue on its own does result in an aversive 12 h light/dark cycle (lights on 07:00) and handled for 1 week outcome. Thus, extinction and safety conditioning are related before commencing experiments. All procedures were performed but distinct phenomena. Safety cues can even act as positive during the light cycle and approved by the Gallo Center reinforcers, suggesting the mechanisms of safety learning may Institutional Animal Care and Use Committee in accordance overlap with reward learning (Christianson et al., 2012; Sangha with the National Institute of Health guidelines. Rats had ad et al., 2013). libitum access to food and water up until the third reward The amygdala has been consistently implicated in processing learning session, at which point they were restricted to 22 g of and regulating a myriad of emotional responses (for review see food per day for the remainder of the experiment. Janak and Tye, 2015). The basal amygdala (BA) in particular is important for discriminating among sensory stimuli that Behavioral Apparatus signal multiple outcomes of a similar valence (Málková et al., The experimental chambers, used in all experiments and 1997; Corbit and Balleine, 2005; Balleine and Killcross, 2006), obtained from MedAssociates, were Plexiglas boxes (32 cm and it possesses neuronal populations selective for valence length × 31 cm width × 35 cm height) encased in sound- (Schoenbaum et al., 1999; Paton et al., 2006; Belova et al., 2007; attenuating shells. A recessed port 3 cm above the floor and Shabel and Janak, 2009; Sangha et al., 2013). located in the center of one wall was used to deliver sucrose. Two Evidence suggests that fear extinction learning induces lights (28 V, 100 mA) located 12 cm from the floor on the wall plasticity and remodeling of inhibitory circuits and synapses opposite the port provided constant illumination. A light (28 V, within the amygdala (Heldt and Ressler, 2007; Lin et al., 2009; 100 mA) located 33 cm above the floor on the wall opposite the Sangha et al., 2012), as well as decreased synaptic efficacy in the port served as the 20 s continuous light cue. A high-frequency medial prefrontal cortex-BA pathway (Cho et al., 2013). Within “tweeter” speaker (ENV-224BM) located 25 cm from the floor the BA, “extinction” neurons have been reported (Herry et al., on the wall opposite the port was used to deliver the auditory 2008). These are neurons that are unresponsive to a fear cue cues. Footshock was delivered through a grid floor via a constant before extinction but become responsive to the fear cue after current aversive stimulator (ENV-414S). A video camera located extinction, when fear behavior is diminished. Diminished fear at the top of the sound-attenuating shell recorded the rat’s behavior is also seen during safety conditioning in response behavior for offline video analysis. to a safety cue. Using a discriminative conditioning task that allows assessment of fear, safety and reward cue learning together, Discriminative Conditioning Sangha et al. (2013) demonstrated significant suppression of The three cues signifying reward, fear or safety were a 20 s freezing behavior in response to a compound fear+safety cue continuous 3 kHz tone (70 dB), a 20 s pulsing 11 kHz tone compared to the high freezing seen in response to a fear cue. (200 ms on, 200 ms off; 70 dB) or a 20 s continuous light (28 V, In addition, this study also reported several neural microcircuits 100 mA), counterbalanced across subjects, with the caveat that within the BA that showed a discriminative response to these the light cue was reserved for the safety cue in most subjects, cues. In particular, 24% of recorded neurons were responsive 12 out of 14 rats. Training first consisted of five reward sessions to the compound fear+safety cue but unresponsive to the fear (Figure 1A; R1–5), in which a 20 s reward cue was paired with 3 s cue when presented alone suggesting these neurons are encoding delivery of a 10% sucrose solution (100 µL) into a port accessible safety. Similar to these “safety” neurons, the “extinction” neurons to the rat (3 s sucrose delivery commenced pseudorandomly reported by Herry et al. (2008) were also unresponsive to the between 10 and 20 s after reward cue onset for 25 trials, ITI 90– fear cue before extinction training. Since safety conditioning and 130 s). This was followed by a single session of habituation (H) extinction are related phenomena, neurons classified as “safety” to the future fear cue and safety cue during a session in which neurons in Sangha et al. (2013) were here examined through reward cue training continued (25 reward trials, ITI 90–130 s). extinction to see if they became “extinction” neurons, similar to The future fear cue and safety cue were presented separately the neurons reported by Herry et al. (2008). five times each for 20 s without reinforcement to allow subjects To do this, firing rates of neurons classified as discriminative, to habituate to their presentation thereby reducing any baseline nondiscriminative or unresponsive during discriminative freezing to these novel cues. Four sessions of discriminative conditioning (DC), based on their responses to the fear cue conditioning followed (DC1–4): reward cue training continued Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 9 Sangha Extinction-Induced Plasticity FIGURE 1 | (A) Summary of experimental design. S, surgical implantation of electrodes into the BA bilaterally followed by 10 d surgical recovery. R1–5, reward sessions in which the reward cue was paired with sucrose delivery. H, habituation in which, in addition to the reward cue-sucrose pairings, rats also received unreinforced presentations of the future fear and safety cues. DC1–4, discriminative conditioning in which reward cue-sucrose pairings continued as well as the addition of trials where the fear cue was paired with footshock, the fear cue was paired with the safety cue without footshock, or the safety cue was presented alone without footshock. E1–2, extinction in which the fear and reward cues were presented unreinforced. (B) Locations of each electrode tip from 14 rats. All 111 recorded neurons were in the BA. (C) Mean (±SEM) percentage of time spent freezing during each cue comparing early vs. late DC sessions (DC1 vs. DC3+4). During late DC, (Continued) Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 10 Sangha Extinction-Induced Plasticity FIGURE 1 | Continued rats froze significantly more to the fear cue compared to the fear+safety cue, reward cue or safety-alone cue, demonstrating discriminatory fear behavior (*p < 0.05). (D) Mean (±SEM) percentage of time spent freezing for each fear cue trial during E1 and E2. Freezing was significantly suppressed compared to the first trial beginning at trial 7 and remained significantly suppressed for the remainder of trials during E1 and E2 (*p < 0.05). (E) Summary of fear cue unresponsive and responsive neurons before extinction and during late extinction. Above, neurons were assigned to one of four groups based on their response to the fear cue and fear+safety cue during late DC (DC3+4); i.e., before extinction. A neuron was considered responsive if there was a significant change in firing frequency during the first 200 ms of the cue compared to pre-cue baseline. Below, a summary of the subset of neurons from each of the four groups to switch their response to the fear cue during late extinction (trials 10–20 of E1 and trials 1–5 of E2 in which freezing behavior was significantly lowered). From left to right, before extinction, one group (n = 27) showed no response to the fear cue but did show a significant change in firing frequency in response to the fear+safety cue. During late extinction, 11 of these neurons switched to being fear cue responsive. The next group (n = 48) showed no response to either the fear or fear+safety cue before extinction. But during late extinction, 21 of these neurons became fear cue responsive. In contrast, the next group (n = 19) showed a significant change in firing frequency in response to both the fear and fear+safety cue before extinction and nine of these neurons became fear cue unresponsive during late extinction. The last group (n = 17) showed a significant change in firing frequency in response to the fear cue but not the fear+safety cue before extinction. Of these neurons, eight became fear cue unresponsive. (F) Comparison of the number of neurons that were fear cue responsive, irrespective to its responding to the other cues, before extinction (DC3+4) to late extinction. The number of neurons being fear cue responsive increased from 36 before extinction to 50 during late extinction, a 39% increase. (3 s sucrose delivery commenced 18 s after reward cue onset; 15 amplifiers, filters (0.4 and 5 KHz), and multichannel spike- trials), along with the additional presentation of the 20 s fear sorting software (Plexon). Implanted rats were connected to the cue followed by a mild 0.5 s footshock at the offset of the fear recording apparatus via a swivel commutator. Discrimination cue (0.4 mA; four trials). On separate trials this same 20 s fear of individual units was performed offline by using principal cue was simultaneously paired with a 20 s safety cue resulting component analysis of waveform shape. Single cells were in no footshock (fear+safety cue; 15 trials). Trials in which identified by constancy of waveform shape, cross-correlograms, the 20 s safety cue was presented alone without any footshock and interspike intervals (Janak, 2002). In addition, quantitative J3 were also included (safety-alone cue; 10 trials) to assess if any and Davies Bouldin validity index (DB) statistics were calculated. freezing developed to the safety cue as a result of being paired High J3 values and low DB values are indicative of good to the fear cue as well as providing the animal with additional single unit isolation (Davies and Bouldin, 1979; Nicolelis et al., trials that contained a safety cue-no shock contingency. Trials 2003; Herry et al., 2008; Sangha et al., 2013). Stability of units were presented pseudorandomly (ITI 100–140 s). Two sessions across sessions was assessed by calculating principal component of extinction followed (E1–2), in which the fear and reward cues space cylinders using WaveTracker (Plexon). In addition, linear were presented unreinforced (E1: 20 trials each of the fear and correlation values between time-shifted average waveforms were reward cues, E2: five trials each of the fear and reward cues; trials calculated (Jackson and Fetz, 2007; Herry et al., 2008; Sangha were presented pseudorandomly, ITI 90–130 s). et al., 2013). As a control, the r-values from average waveforms of randomly paired neurons and sessions were computed. Only Behavioral Analyses units deemed stable across sessions using these procedures were Fear behavior was assessed, offline from videos, by measuring included in the analysis. freezing, defined as complete immobility with the exception of respiratory movements, which is an innate defensive behavior (Blanchard and Blanchard, 1969; Fendt and Fanselow, 1999). The Classification of Neurons total time spent freezing was quantified during the entire 20 s For each neuron, significance of cue-evoked firing rates was of each cue presentation and expressed as percent time spent determined as previously published (Sangha et al., 2013), using freezing. Calculating the percent time spent in the port assessed a 10,000-round paired permutation test (Hesterberg et al., 2005) reward-seeking behavior. Behavioral data were analyzed using comparing the averaged 20 s pre-cue baseline period to the one- or two-way repeated measures ANOVA, followed by Tukey’s first 200 ms after cue onset during the last two DC sessions post-hoc test when indicated by significant (p < 0.05) main effects and during late extinction (trials 10–20 of E1 and trials 1– or interactions. 5 of E2). That is, the 20 s pre-cue baseline firing rates and the 200 ms post-cue firing rates for a given cue were shuffled Surgery and redistributed independently 10,000 times. The differences Rats were anesthetized with isoflurane and stereotaxically between the baseline and post-cue firing for the single real case implanted bilaterally with fixed eight-electrode arrays and the 10,000 reshuffled cases were used to create a distribution. (NeuroBiological Laboratories) directed at the BA (relative In accordance with the permutation test, if the actual mean to bregma: AP: −2.04 to −2.92 mm posterior, ML: 4.1–4.9 mm, difference was within <2.5% of either tail, it was considered DV: 6.6–7.5 mm ventral from brain surface (Paxinos and Watson, significant. P-values were then adjusted for multiple corrections 2007) (Figure 1B). Rats were allowed 7–10 d to recover in which using the Benjamini-Hochberg correction with a corrected cutoff they had ad libitum access to food and water. of 0.05 (Benjamini and Hochberg, 1995). To avoid false positives, neurons that showed a significant cue-evoked inhibition using In Vivo Single Unit Recordings this permutation test were only included in the final analyses Neuronal activity was recorded with commercial hardware and if the baseline firing frequency was >0.05 Hz. Neurons were software, including headstage amplifiers and programmable classified as “fear cue responsive” if there was a significant Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 11 Sangha Extinction-Induced Plasticity increase or decrease in firing rate to the fear cue during late DC. repeated-measures ANOVA on percent time spent freezing These neurons were then segregated based on whether there was revealed a main effect of trial [F(24, 336) = 6.35, p < 0.0001] also a significant change in firing rate to the fear+safety cue. and Dunnett’s multiple comparisons test showed freezing was Neurons that did not show a significant change in firing rate to significantly lower (p < 0.05) during trial 7 and each subsequent the fear cue during late DC (i.e., before extinction) were classified trial compared to the first trial. Thus, freezing was significantly as “fear cue unresponsive.” A subset of these neurons did however suppressed compared to the first trial beginning at trial 7 and show a significant increase or decrease in firing rate compared remained significantly suppressed for the remainder of trials to baseline to the fear+safety cue and were analyzed separately. during E1 and E2. Similarly, neurons were classified as “reward cue responsive” if there was a significant increase or decrease in firing rate to the Neural Recordings of Fear and Safety reward cue during late DC. Neurons In order to compare neuronal responding during discriminatory Histology fear behavior to significant fear suppression during extinction, Rats were deeply anesthetized with sodium pentobarbital. A neuronal responding was analyzed during late DC (DC3+4) 10 s 19 µA DC current was passed through each wire to mark and compared to late extinction (Figure 1E). Late extinction each electrode tip. Rats were then perfused with formalin consisted of the last 10 trials of E1 and the 5 trials of E2; freezing containing 3% potassium ferrocyanide. Sections (50 µM) were behavior during each of these trials was significantly lower than stained against acetylcholinesterase and only units recorded from the beginning of extinction (trial 1 of E1, Figure 1D). Z-scores electrode wires verified to be in the BA were included in the were calculated for each neuron’s response to the first 200 ms analyses. of each cue (see Materials and Methods) and used to make comparisons among different neuronal populations. RESULTS Neurons Unresponsive to the Fear Cue Before In a previous study neurons of the BA were tracked over the Extinction course of a discriminative conditioning task (Sangha et al., Neurons classified as “fear cue unresponsive” before extinction 2013). In this task rats learn to discriminate among fear, safety, had no significant change in firing rates to the fear cue compared and reward cues. In the present study, the same BA neurons to baseline during DC3+4 (permutation tests, p > 0.05). These were followed into fear and reward cue extinction to assess the neurons were then segregated based on whether or not they plasticity of neurons that were fear cue responsive and fear cue showed significant changes in firing rates to the fear+safety cue unresponsive before extinction. compared to baseline during DC3+4 (Figure 1E). This was done Recordings were made during each behavioral training session in an effort to assess if the “safety” neurons become “extinction” (Figure 1A, see Materials and Methods). A total of 111 single neurons. In other words, does one subpopulation preferentially neurons located in the BA from 14 rats (Figure 1B) were isolated switch to being fear cue responsive? from recordings made during discriminative conditioning and Before extinction, 27 neurons were fear cue unresponsive extinction. Most neurons had low mean firing rates (Median = but fear+safety cue responsive (Figures 1E, 2A), showing a 0.83 Hz, Max = 20.35 Hz, Min = 0.06 Hz), suggesting the sample discriminative response to the fear+safety cue vs. fear cue. This was predominantly putative projection neurons (Likhtik et al., subpopulation showed either an excitatory (n = 15, Figure 2A 2006). upper) or inhibitory (n = 12, Figure 2A lower) response to the fear+safety cue. Five of these fear cue unresponsive neurons Fear Behavior developed an excitatory response to the fear cue in late extinction Discriminative Conditioning and six developed an inhibitory response (permutation tests, p < The percent time spent freezing during each cue was averaged 0.05). The remaining 16 neurons remained unresponsive to the across early (first DC session, DC1) and late (final two DC fear cue (permutation tests, p > 0.05). sessions, DC3+4) discriminative conditioning (Figure 1C). A Before extinction, 48 neurons were both fear cue and Two-way repeated-measures ANOVA on percent time spent fear+safety cue unresponsive (Figures 1E, 2B). Of these 48 freezing revealed a significant interaction between phase of unresponsive neurons, five developed an excitatory response to training and cue type [F(3, 39) = 8.575, p < 0.001] and a main the fear cue in late extinction and 16 developed an inhibitory effect of phase of training [F(1, 13) = 5.118, p < 0.05] and cue response (permutation tests, p < 0.05). The remaining 27 type [F(3, 39) = 29.331, p < 0.001]. Freezing to the fear cue was neurons remained unresponsive to the fear cue (permutation significantly greater than the fear+safety cues, safety-alone cue, tests, p > 0.05). and reward cue during late DC (post-hoc Tukey’s, p < 0.001 Together, of all the neurons that were unresponsive to the fear each comparison), demonstrating discriminatory fear behavior cue before extinction (n = 76), 43% (32 of 76 neurons) switched by these animals. to being responsive during late extinction (Figure 1E). Contrary to the hypothesis, neurons that responded to the fear+safety Fear Extinction cue, but not the fear cue, before extinction did not appear to The percent time spent freezing during each fear cue trial of E1 preferentially switch to being fear cue responsive during late and E2 was averaged across animals (Figure 1D). A One-way extinction compared to neurons unresponsive to both cues. Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 12 Sangha Extinction-Induced Plasticity FIGURE 2 | Continued before extinction. Five of these fear cue unresponsive neurons developed an excitatory response to the fear cue in late extinction and six developed an inhibitory response. (B) Neurons that were both fear cue and fear+safety cue unresponsive before extinction. Of these 48 unresponsive neurons, five developed an excitatory response in late extinction and 16 developed an inhibitory response to the fear cue. The remaining 27 neurons remained unresponsive to the fear cue. Neurons Responsive to the Fear Cue Before Extinction Neurons classified as “fear cue responsive” before extinction had significant increases or decreases in firing rates to the fear cue compared to baseline during DC3+4 (permutation tests, p < 0.05). These neurons were then segregated based on whether or not they also showed significant changes in firing rates to the fear+safety cues compared to baseline during DC3+4 (Figure 1E). This was done to assess if one subpopulation preferentially switched to being fear cue unresponsive. Before extinction, 19 neurons were both fear cue and fear+safety cue responsive (nondiscriminative; Figures 1E, 3A). This subpopulation showed either an excitatory (n = 6, Figure 3A upper) or inhibitory (n = 13, Figure 3A lower) response to the fear+safety cue. All neurons showing an excitatory response to both types of cues before extinction maintained their response through late extinction (n = 6; permutation tests, p < 0.05). Nine neurons showing an inhibitory response to both cues before extinction lost their inhibitory response in late extinction (permutation tests, p > 0.05). The remaining four neurons maintained their inhibitory response through late extinction (permutation tests, p < 0.05). That is, within this subpopulation of neurons, all excitation responses were maintained through extinction but the majority of inhibition responses were lost through extinction. Before extinction, 17 neurons were fear cue responsive but fear+safety cue unresponsive (Figures 1E, 3B), showing a discriminative response to the fear cue vs. fear+safety cue. Only 1 neuron showed an excitatory response to the fear cue (Figure 3B) while the remaining 16 neurons showed an inhibitory response to the fear cue. Eight neurons that showed significant inhibition to the fear cue before extinction lost the inhibitory response in late extinction (permutation tests, p > 0.05) and one neuron switched its inhibitory response to the fear cue before extinction to an excitatory response to the fear cue in late extinction. The remaining one excitatory response and seven inhibitory responses were maintained through late extinction (permutation tests, p < 0.05). Together, of all the neurons that were responsive to the fear FIGURE 2 | Neurons unresponsive to the fear cue before extinction. cue before extinction (n = 36), 47% (17 of 36 neurons) switched Z-scores were calculated for each neuron’s response to the first 200 ms of each cue. Mean (±SEM) Z-scores are shown to the fear and fear+safety (f+s) to being unresponsive during late extinction (Figure 1E). cues before extinction and to the fear cue during late extinction. *p < 0.05, In summary, extinction induced a gain in response to the firing frequency during first 200 ms of cue of a given neuron compared to its fear cue in 43% of fear cue unresponsive neurons and a loss in pre-cue baseline firing frequency. Significant positive Z-score values indicate response to the fear cue in 47% of fear cue responsive neurons. an excitatory response and significant negative Z-score values indicate an The number of fear cue responsive neurons before extinction was inhibitory response. Non-significant values indicate unresponsive to the cue. (A) Neurons that were fear cue unresponsive but fear+safety cues responsive also compared to late extinction (Figure 1F) to determine if there (Continued) was an overall increase or decrease in the absolute number of neurons being fear cue responsive as a result of extinction. Before Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 13 Sangha Extinction-Induced Plasticity FIGURE 3 | Continued extinction maintained their response through late extinction (n = 6). Nine neurons showing an inhibitory response to both cues before extinction lost their inhibitory response in late extinction. The remaining 4 neurons maintained their inhibitory response through late extinction. (B) Neurons that were fear cue responsive but fear+safety cue unresponsive before extinction. Eight neurons that showed significant inhibition to the fear cue before extinction lost the inhibitory response in late extinction; one neuron switched its inhibitory response to the fear cue before extinction to an excitatory response to the fear cue in late extinction. The remaining one excitatory response and seven inhibitory response neurons maintained their responses through late extinction. extinction, 75 neurons were fear cue unresponsive and 36 were fear cue responsive (Figure 1F, upper). During late extinction, 61 neurons were fear cue unresponsive and 50 were fear cue responsive (Figure 1F, lower). Thus, there was a 39% increase in the number of fear cue responsive neurons as a result of extinction. However, a Fisher’s exact test revealed this increase was not significant (p = 0.073). Reward Behavior and Neural Recordings of Reward Responsive Neurons Since reward cue extinction occurred concurrently to fear cue extinction, neuronal responding to the reward cue before extinction (DC3+4) and during late extinction was also assessed. Discriminative Conditioning The percent time spent in the reward port during each cue was averaged across early (first DC session, DC1) and late (final two DC sessions, DC3+4) discriminative conditioning (Figure 4A). A Two-way repeated-measures ANOVA on percent time spent in port revealed a significant main effect of cue type [F(3, 39) = 71.56, p < 0.0001]. Reward seeking during the reward cue was significantly greater than the fear+safety cues, safety-alone cue, and fear cue during both early and late DC (post-hoc Tukey’s, p < 0.001 each comparison), demonstrating discriminatory reward seeking behavior by these animals. Reward Extinction The percent time spent in the reward port during each reward cue trial of E1 and E2 was averaged across animals (Figure 4B). A One-way repeated-measures ANOVA on percent time spent in port revealed a main effect of trial [F(24, 336) = 2.858, p < 0.05] and Dunnett’s multiple comparisons test showed reward seeking was significantly lower (p < 0.05) during trial 2 and each subsequent trial compared to the first trial with the exception of trials #8, 9, and 10 of E1 and trials #3 and 5 of E2. Thus, FIGURE 3 | Neurons responsive to the fear cue before extinction. compared to the first trial of E1, reward seeking was successfully Z-scores were calculated for each neuron’s response to each cue. Mean (±SEM) Z-scores are shown to the fear and fear+safety (f+s) cues before extinguished by the end of E1 and maintained into E2. extinction and to the fear cue during late extinction. *p < 0.05, firing frequency during first 200 ms of cue of a given neuron compared to its pre-cue baseline Neural Recording firing frequency. Significant positive Z-score values indicate an excitatory Similar to the analyses completed for the fear responsive neurons, response and significant negative Z-score values indicate an inhibitory the number of reward cue responsive neurons before extinction response. Non-significant values indicate unresponsive to the cue. (A) Neurons was compared to late extinction (Figure 4C) to determine that were both fear cue and fear+safety cues responsive before extinction. All neurons showing an excitatory response to both types of cues before if there was an overall increase or decrease in the absolute (Continued) number of neurons being reward cue responsive as a result of extinction. Before extinction, 62 neurons were reward cue Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 14 Sangha Extinction-Induced Plasticity FIGURE 4 | Changes in response to the reward cue. (A) Mean (±SEM) percentage of time spent in reward port during each cue comparing early vs. late DC sessions (DC1 vs. DC3+4). During both early and late DC, rats spent significantly more time in the port during the reward cue compared to the fear+safety cue, fear cue or safety-alone cue, demonstrating discriminatory reward-seeking behavior (*p < 0.05). (B) Mean (±SEM) percentage of time spent in reward port for each reward cue trial during E1 and E2. Reward seeking was significantly suppressed (*p < 0.05) compared to the first trial beginning at trial 2 and remained significantly suppressed for the remainder of trials during E1 and E2 with the exception of trials #8–10 of E1 and trials #3 and 5 of E2. (C) Comparison of the number of neurons that were reward cue responsive before extinction (DC3+4) to late extinction. The number of neurons being reward cue responsive decreased from 62 before extinction to 49 during late extinction. responsive and 49 were reward cue unresponsive. During late support extinction as an active process involving both gains and extinction, 49 neurons were fear cue responsive and 62 were fear losses of responses to the fear cue. cue unresponsive. This decrease in the number of reward cue The prevalent view in the extinction field is that extinction responsive neurons as a result of extinction was not significant is an active process, not a passive one (reviewed in Myers and (Fisher’s exact test, p > 0.05). Davis, 2002, 2007). There is ample evidence that extinction does not erase fear memories. In particular, it has been demonstrated DISCUSSION by others (Repa et al., 2001; Herry et al., 2008; An et al., 2012), and here in this study (Figure 3A, upper), that amygdala This study examined how neurons classified as discriminative, neurons maintain increased responsiveness to the CS, even nondiscriminative or unresponsive during discriminative after extinction. However, there is also evidence that extinction conditioning (DC), based on their responses to the fear and reverses the changes induced by fear learning. For example, fear fear+safety cues, responded to the fear cue during extinction conditioning-induced potentiation is reversed with extinction in training and recall as fear behavior decreased. The hypothesis both the thalamo-lateral amygdala and cortico-lateral amygdala tested was that there is a bias for the neurons that were safety cue pathways (Kim et al., 2007; Hong et al., 2009). The data in the responsive during DC to become responsive to the fear cue as current study are in agreement with both views. There was both extinction progresses. a gain of response to the fear cue (Figures 2A,B), which supports Although 41% of “safety” neurons became fear cue responsive extinction as new learning, and a loss of response to the fear as a result of extinction, the data revealed that there was no cue (Figure 3A, lower and Figure 3B), which may be due to bias for these neurons to become preferentially responsive during unlearning. fear extinction compared to the other identified subgroups. In Fear conditioning also induces synchronization at theta addition to the plasticity seen in the “safety” neurons, 44% frequencies within the amygdala-hippocampal-prefrontal cortex of neurons unresponsive to either the fear cue or fear+safety (PFC) network (Sangha et al., 2009; Lesting et al., 2011). cue during DC became fear cue responsive during extinction. After extinction the synchronization between the amygdala and Together these emergent responses to the fear cue as a result hippocampus is lost but theta synchronization is maintained of extinction support the hypothesis that new learning underlies between the amygdala and PFC, and between the hippocampus extinction. The overall increase in fear cue responsive neurons in and PFC. A similar effect has been reported in the PFC-BA circuit response to extinction also implies that these changes in neuronal in which fear extinction decreases excitatory transmission from responding during extinction are not a result of simple exposure PFC to BA while maintaining inhibitory transmission (Cho et al., to the sensory stimuli. If the shift were a result of repeated sensory 2013). These data demonstrate both reversal and maintenance exposures, one would expect the neurons across all groups to of learning-induced network activity occurring in parallel during show decreased responding to sensory stimuli after multiple extinction. exposures as a result of sensory habituation. In contrast, 47% This suggests that both new learning and unlearning of neurons responsive to the fear cue during DC, regardless mechanisms may occur in parallel during extinction. Both of its response to the fear+safety cue, became unresponsive to processes are active processes. During extinction, a new the fear cue during extinction. These findings are consistent association regarding the CS is learned; i.e., a CS-no US with a suppression of neural responding mediated by inhibitory association. And, similarly to learning the original association, learning, or, potentially, by direct unlearning. Together, the data long-term retention of extinction training requires both RNA and Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 15 Sangha Extinction-Induced Plasticity protein synthesis across several learning paradigms and species successfully recall fear extinction (Quirk et al., 2000; Laurent and (reviewed in Lattal et al., 2006). But, since extinction also involves Westbrook, 2009; Chang and Maren, 2010; Fontanez-Nuin et al., reactivation of the original memory, the integrity of the original 2011; Sierra-Mercado et al., 2011; Santini et al., 2012; Sangha memory is vulnerable to disruption through reconsolidation et al., 2014, but see Do Monte et al., 2015). mechanisms. When the original CS-US association is reactivated In summary, the data implicate multiple levels of plasticity in during extinction, it can be updated via reconsolidation response to fear extinction that most likely interact with multiple mechanisms resulting in a weakening/reversal of the memory. microcircuits within the BA. It also indicates that there may Extinction and reconsolidation have been demonstrated to be a general remapping of these neuronal microcircuits within occur in parallel in the basolateral amygdala complex during the BA in response to extinction. Together it suggests the final reactivation of a fear memory that is no longer reinforced output of the integrated BA circuit to influence fear behavior is with shock (Duvarci et al., 2006), supporting a view that both a balance of excitation and inhibition, and perhaps reversal of new learning and unlearning mechanisms are at play during learning-induced changes. Further exploration of the intricacies extinction. This view is also consistent with reports that briefly of upregulating or downregulating these BA microcircuits on reactivating a fear memory before employing fear extinction downstream targets and their effects on fear behavior will lead training results in persistent attenuation of fear in both rats to greater understanding of the mechanisms contributing to (Monfils et al., 2009) and humans (Schiller et al., 2010). In successful fear inhibition which is compromised in individuals this case, the brief reactivation of the fear memory may induce suffering from PTSD and similar disorders. unlearning via reconsolidation mechanisms and the extinction training results in the learning of a new CS-no US association. AUTHOR CONTRIBUTIONS The unlearning phenomena may be caused by reversal of learning-induced changes at the synapse and within the network, SS: experimental design, performed research, analyzed data, or it may be caused by suppression of neural responding wrote manuscript. mediated by increased inhibition. Several neurons reported here had decreased firing rates in response to the fear cue ACKNOWLEDGMENTS during extinction. It is not clear what the source of cue-evoked inhibition, nor its downstream effects, might be. However, it has This work was supported by funding from the State of California been shown that the balance between excitation and inhibition in for Medical Research on Alcohol and Substance Abuse through the PFC-BA pathway is shifted toward inhibition after extinction the University of California at San Francisco and NIH grant (Cho et al., 2013), suggesting that the upstream source for the AA014925 to Dr. P. H. Janak. The author greatly appreciates Dr. inhibitions seen in the data presented here may be the PFC. P. H. Janak for discussion and comments on the manuscript. The This would be consistent with the requirement of the infralimbic author also thanks R. Reese and I. Grossrubatscher for technical region of the prefrontal cortex to discriminate between the fear assistance, Dr. T. M. Gill for programming and recording and fear+safety cues in this task (Sangha et al., 2014), and to guidance and Dr. J. Z. Chadick for programming assistance. 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Neurosi. 17, 6011–6020. The reviewer Stefan Herlitze and handling Editor Denise Manahan-Vaughan Monfils, M. H., Cowansage, K. K., Klann, E., and LeDoux, J. E. (2009). Extinction- declared their shared affiliation, and the handling Editor states that the process reconsolidation boundaries: key to persistent attenuation of fear memories. nevertheless met the standards of a fair and objective review. Science 324, 951–955. doi: 10.1126/science.1167975 Myers, K. M., and Davis, M. (2002). Behavioral and neural analysis of extinction. Copyright © 2015 Sangha. This is an open-access article distributed under the terms Neuron 36, 567–584. doi: 10.1016/S0896-6273(02)01064-4 of the Creative Commons Attribution License (CC BY). The use, distribution or Myers, K. M., and Davis, M. (2007). Mechanisms of fear extinction. Mol. Psychiatry reproduction in other forums is permitted, provided the original author(s) or licensor 12, 120–150. doi: 10.1038/sj.mp.4001939 are credited and that the original publication in this journal is cited, in accordance Nicolelis, M. A., Dimitrov, D., Carmena, J. M., Crist, R., Lehew, G., Kralik, with accepted academic practice. No use, distribution or reproduction is permitted J. D., et al. (2003). Chronic, multisite, multielectrode recordings in which does not comply with these terms. Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2015 | Volume 9 | Article 354 | 17 ORIGINAL RESEARCH published: 24 November 2015 doi: 10.3389/fnbeh.2015.00314 The Memory System Engaged During Acquisition Determines the Effectiveness of Different Extinction Protocols Jarid Goodman and Mark G. Packard* Department of Psychology, Texas A&M Institute for Neuroscience, Texas A&M University, College Station, TX, USA Previous research indicates that extinction of rodent maze behavior may occur without explicit performance of the previously acquired response. In latent extinction, confining an animal to a previously rewarded goal location without reinforcement is typically sufficient to produce extinction of maze learning. However, previous studies have not determined whether latent extinction may be successfully employed to extinguish all types of memory acquired in the maze, or whether only specific types of memory may be vulnerable to latent extinction. The present study examined whether latent extinction may be effective across two plus-maze tasks that depend on anatomically distinct neural systems. Adult male Long-Evans rats were trained in a hippocampus-dependent place learning task (Experiment 1), in which animals were trained to approach a consistent spatial location for food reward. A separate group of rats were trained Edited by: in a dorsolateral striatum-dependent response learning task (Experiment 2), in which Oliver T. Wolf, Ruhr University Bochum, Germany animals were trained to make a consistent egocentric body-turn response for food Reviewed by: reward. Following training, animals received response extinction or latent extinction. Armin Zlomuzica, For response extinction, animals were given the opportunity to execute the original Ruhr University Bochum, Germany Michael E. Ragozzino, running approach response toward the empty food cup. For latent extinction, animals University of Illinois at Chicago, USA were confined to the original goal locations with the empty food cup, thus preventing *Correspondence: them from making the original running approach response. Results indicate that, relative Mark G. Packard to no extinction, latent extinction was effective at extinguishing memory in the place markpackard@tamu.edu learning task, but remained ineffective in the response learning task. In contrast, typical Received: 18 September 2015 response extinction remained very effective at extinguishing memory in both place and Accepted: 04 November 2015 response learning tasks. The present findings confirm that extinction of maze learning Published: 24 November 2015 may occur with or without overt performance of the previously acquired response, but Citation: that the effectiveness of latent extinction may depend on the type of memory being Goodman J and Packard MG (2015) The Memory System Engaged During extinguished. The findings suggest that behavioral treatments modeled after response Acquisition Determines the extinction protocols may be especially useful in alleviating human psychopathologies Effectiveness of Different Extinction Protocols. involving striatum-dependent memory processes (e.g., drug addiction and relapse). Front. Behav. Neurosci. 9:314. doi: 10.3389/fnbeh.2015.00314 Keywords: extinction, memory, hippocampus, striatum, memory systems Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 18 Goodman and Packard Multiple Memory Systems and Extinction INTRODUCTION DLS, impairs latent extinction (Gabriele and Packard, 2006; Gabriele, 2008). In contrast, inactivation of the DLS, but not Mammalian memory is not a unitary phenomenon, but the hippocampus, impairs response extinction (Gabriele and rather it transpires through distinct systems. These ‘‘memory Packard, 2006; Gabriele, 2008). A corollary to the contention systems’’ differ in terms of not only the type(s) of memory that these extinction protocols depend on operatively and they mediate, but also the brain regions that subserve anatomically distinct learning systems is that response and them. Although a variety of memory systems have been latent extinction may not be equally effective across all learning dissociated in the mammalian brain (Squire, 2004; White situations. For instance, if a critical feature needed for latent et al., 2013), significant attention has been devoted to extinction mechanisms to occur is absent from the learning anatomical dissociations between a spatial/cognitive memory situation, then it is reasonable to hypothesize that latent system mediated by the hippocampus and a stimulus-response extinction would not be effective, whereas response extinction (S-R)/habit system mediated by the dorsolateral striatum could still work. (DLS; Packard et al., 1989; Packard and McGaugh, 1992, One potential limitation to examining learning and memory 1996; McDonald and White, 1993; Packard and Teather, mechanisms using the straight-alley maze is that we do not 1997; Chang and Gold, 2003; Iaria et al., 2003; Compton, know what type of memory is being encoded during initial 2004). task acquisition. Initial learning in the straight alley maze Research from our laboratory indicates that multiple memory may involve acquisition of at least two distinct types of systems may not only be implicated in the initial acquisition memory: (1) a habit-like running approach response to the of a task, but also in its extinction (Gabriele and Packard, opposite end of the maze and/or (2) the spatial location of the 2006). Extinction constitutes a new, dissociable type of learning food reward, which in turn triggers a goal-directed running that occurs when a subject is placed in the original learning approach to the rewarded location at the opposite end of the situation but with the reinforcer—or the stimulus event that maze. Consequently, when using the straight alley maze to motivated initial learning—removed. Extinction is deemed to examine extinction mechanisms, the type of memory being have occurred when the behavioral response or responses that extinguished remains unknown. Moreover, studies using the indicated initial learning decrease. Learned behavior in the straight alley maze cannot determine whether latent extinction straight alley maze, a maze in which rats learn to traverse a is effective at extinguishing all types of memory or whether runway for food reward located at the opposite end of the latent extinction may only be effective for certain types of maze, may be extinguished using two distinct protocols. In a memory. Considering that latent extinction may partially operate typical ‘‘response extinction’’ protocol, rats are placed in the same by producing a new inhibitory spatial memory (see Gabriele starting position as during training, but with the food reward at and Packard, 2006), it is possible that latent extinction may the opposite end of the maze removed. Thus, during response only be effective in tasks whereby the spatial location of the extinction trials, animals can execute the running approach goal is an integral part of the to-be-extinguished memory, response, only now this response leads to an empty food well. In such as in spatial memory tasks. In contrast, latent extinction ‘‘latent extinction,’’ rats are confined to the original goal location may not be effective in tasks whereby the spatial location with the empty food well. Thus, during latent extinction, animals of the goal is irrelevant, such as in S-R/habit memory cannot execute the running approach response. Historically tasks. the effectiveness of latent extinction figured prominently in To examine whether only certain types of memory may learning theory, because it demonstrated that—in contrast to be vulnerable to latent extinction, the present study utilized the Hullian S-R view of extinction (Hull, 1943, 1952)—a subject two distinct versions of the plus-maze. In a ‘‘place learning’’ does not need to make the previously acquired response for version of the plus-maze dependent on the hippocampus extinction to occur (Seward and Levy, 1949; Deese, 1951; (Schroeder et al., 2002; Compton, 2004), rats were reinforced Moltz, 1955; Denny and Ratner, 1959; Dyal, 1962; Clifford, to approach a consistent spatial location. In a ‘‘response 1964). learning’’ version of plus-maze dependent on the DLS (Chang Although the behavior of the rat is ostensibly similar following and Gold, 2004; Palencia and Ragozzino, 2005; Asem and both extinction protocols, investigators have suggested that Holland, 2015), rats were reinforced to make a consistent response and latent extinction might be achieved through egocentric body-turn at the maze choice point. Thus, these distinct learning mechanisms. The effectiveness of typical place and response tasks tap into dissociable neural systems, response extinction is easily explained through classical S-R a hippocampus-dependent spatial/cognitive memory system models of extinction learning, whereas latent extinction has and a DLS-dependent S-R/habit memory system, respectively. summoned heated debates between proponents of expectancy Following initial learning in these tasks, animals were given theory and proponents of a neo-Hullian view involving the response extinction, latent extinction, or no extinction. It fractional anticipatory approach response (Moltz, 1957; Deese was hypothesized that latent extinction would be selectively and Hulse, 1967). Although the precise mechanisms underlying effective at extinguishing memory in the place learning latent extinction have yet to be completely elucidated, evidence task, but not the response learning task. Moreover, we from our laboratory indicates that latent extinction indeed hypothesized that typical response extinction would be effective depends on a dissociable neural system. In the straight- at extinguishing memory in both place and response learning alley maze inactivation of the hippocampus, but not the tasks. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 19 Goodman and Packard Multiple Memory Systems and Extinction MATERIALS AND METHODS were reinforced to approach a consistent spatial location. At the start of each training trial, the animal was placed on the north or Subjects south arm facing the outside of the maze (the start arm sequence The subjects were 46 male Long-Evans rats approximately 90 was counterbalanced across training), and the food reward (1/2 days old and weighing 375–425 g upon arrival. Animals were Froot Loop) was always located in the recessed food well of the subsequently food-restricted and maintained at 85% of the east arm. This place learning protocol presumably compelled their ad lib weight throughout all behavioral procedures. Water rats to acquire a cognitive map of the learning environment that was provided ad libitum. Animals were housed individually enabled them to guide behavior from different starting positions in a temperature-controlled vivarium with a 12 h light-dark to the correct spatial location. Extensive evidence indicates that cycle (lights on at 7 AM), and all behavioral procedures were spatial learning in the plus-maze critically involves hippocampal conducted during the light phase of this cycle. Age, weight, function (Packard and McGaugh, 1996; Packard, 1999; Schroeder and housing conditions did not differ between animals in et al., 2002; Colombo et al., 2003; Compton, 2004; Jacobson et al., Experiments 1 and 2. Animal use in this study was carried out in 2012). accordance with the ethical guidelines of the Institutional Animal In Experiment 2, animals (N = 25) received training in Care and Use Committee (IACUC) at Texas A&M University. a response learning version of the plus-maze task whereby The protocol was approved by IACUC. animals were reinforced to make a consistent egocentric body- turn response at the maze choice point (Leong et al., 2012, Apparatus 2015; Goodman and Packard, 2014; Wingard et al., 2015). An eight arm radial maze was modified by removing four of Animals were released from north and south starting positions the original arms to create a plus-maze configuration consisting (counterbalanced) throughout training. When animals began in of north, south, east, and west arms. The arms of the cross the north arm, the food reward (1/2 Froot Loop) was located maze measured 60 × 9 cm, and the center platform of the maze in the recessed food well of the east arm. When animals connecting the four arms measured 40 cm in diameter. At the began in the south arm, the food reward was located in the end of each arm was a recessed food well. A clear Plexiglas cross- west arm. Thus, regardless of the starting position, animals shaped structure was placed in the center of the cross maze, were reinforced to make a left body-turn response at the serving as the intersection of the four arms. A separate Plexiglas choice point to receive food reward. Learning in this task divider was used to block off the arm opposite to the start arm constitutes an exemplar of egocentric/S-R learning mediated for each trial, creating a T-maze configuration that could be by the DLS (Packard and McGaugh, 1996; Chang and Gold, adjusted between trials. The maze was situated in a room with 2004; Palencia and Ragozzino, 2005; Asem and Holland, 2015; multiple extra maze cues, including posters, a door, a cabinet, and for reviews, see Packard, 2009; Goodman and Packard, in a table. press). For each training trial in Experiments 1 and 2, if the animal made an initial full-body entry into the correct arm (i.e., the arm Behavioral Procedures containing the food), the trial was scored as correct. If the animal Maze Habituation made an initial full body entry into the incorrect arm, the trial was Before maze training, animals in Experiments 1 and 2 were given scored as incorrect. A trial ended once the animal found the food 2 days of habituation to the maze. For each day of habituation, or after 120 s had elapsed. When finding the food, the animal was a rat was placed on the maze apparatus (from the north arm on allowed to finish eating before being removed from the maze and day 1 and from the south arm on day 2) and was given 5 min to placed in an opaque holding container for a 30 s intertrial interval explore the maze. No food was located on the maze at this time. (ITI). The percentage of correct trials and the latency to reach the Immediately after the 5 min, each rat was removed from the maze correct food well were used as measures of acquisition. and placed in a holding container with three Froot Loops cereal pieces (Kellog’s). Rats were monitored to confirm consumption Extinction of the Froot Loops. Extinction was conducted 24 h after the last day of maze training and lasted 3 days. No food was located in the maze throughout Maze Training extinction training. The maze was rotated 90º after every two Maze training began 24 h following the last day of habituation trials to prevent the use of intramaze cues. and lasted 8 days. For the first 2 days of training, animals were In Experiment 1, rats that were previously given place learning given six trials per day, and for the remainder of training animals were subsequently assigned to response extinction (n = 7), were given 15 trials per day. The maze was rotated 90º after latent extinction (n = 7), or ‘‘no extinction’’ control (n = 7) every two trials to discourage the use of intramaze cues. A wide- groups. Groups were matched on average latency and percent angle digital camera was fixed over the maze and attached to a correct responses during the last 3 days of acquisition. Response computer monitor (only visible to the experimenter) allowing for extinction was conducted over 3 days (10 trials per day). For a clear aerial view of arm entries, and a stopwatch was used to each trial of response extinction, animals were started from the record latencies during task performance. north or south arm and were given the opportunity to run to In Experiment 1, animals (N = 21) received training for 8 days the previously correct food well. An animal was removed from in a place learning version of the plus-maze task whereby animals the maze after reaching the previously correct food well or after Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 20 Goodman and Packard Multiple Memory Systems and Extinction 120 s had elapsed. For each trial, if the animal made an initial of perseverative trials (see above) were recorded and used as full-body entry into the previously correct arm and ran directly measures of extinction. The experimenter conducting the probe to the food well, the trial was identified as ‘‘perseverative.’’ A trials and scoring the animals was blind to the experimental trial was not considered perseverative if the animal at any point conditions. made an entry into the incorrect arm or failed to enter either the correct or incorrect arm within 120 s. After each trial the RESULTS animal was removed from the maze and placed in an opaque holding container for a 30 s ITI. The behavioral procedure Experiment 1 for latent extinction was adapted from previous work from our laboratory indicating the effectiveness of latent extinction Initial Acquisition in the straight alley maze (Gabriele and Packard, 2006, 2007; Initial acquisition of the place learning task is depicted in Gabriele et al., 2009). For each trial of latent extinction, an Figure 1. A two-way repeated measures 3 × 8 ANOVA animal was confined to the previously correct goal arm (i.e., (Group × Day) computed on percentage of correct turning the east arm for the place learning task) for 60 s using a responses over the course of training (Figure 1A) indicated a Plexiglas shield secured 20 cm from the end of the maze arm. significant main effect of Day (F (7,126) = 22.22, p < 0.001), After each trial, the animal was placed in an opaque holding but no effect of Group (F (2,18) = 0.15, p = 0.860) and no container for a 30 s ITI. For the ‘‘no extinction’’ control group, Group × Day interaction (F (14,126) = 1.51, p = 0.118). Likewise, animals were not placed in the maze for the 3 extinction a 3 × 8 ANOVA (Group × Day) computed on latency days, but rather remained in their holding containers for the (Figure 1B) indicated a significant effect of Day (F (7,126) = duration of an extinction session, i.e., while animals in the 52.41, p < 0.001), but no effect of Group (F (2,18) = 0.00, latent and response extinction groups were receiving extinction p = 1.00) and no Group × Day interaction (F (14,126) = 1.47, training. p = 0.131). Together, these results indicate that all groups In Experiment 2, animals that previously received response acquired the task about equally over the course of training, and learning were subsequently assigned to response extinction any subsequent differences between groups during extinction (n = 6), limited latent extinction (n = 6), extended latent may not be readily attributed to differing rates of initial task extinction (n = 6), or ‘‘no extinction’’ control (n = 7) groups. acquisition. Groups were matched on average latency and percent correct responses during the last 3 days of acquisition. The behavioral Response Extinction procedures for response extinction and no extinction control Figure 2 depicts learning rates over the course of extinction groups were identical to that described for Experiment 1. training for animals in the ‘‘response extinction’’ group. Tests of For limited and extended latent extinction (conducted over within-subjects contrasts computed on number of perseverative 3 days), animals were confined to the east or west goal trials (Figure 2A) revealed a significant linear effect of Day arm for 60 s for each trial with the sequence of goal arm (F (1,6) = 39.06, p = 0.001), indicating a decrease in number confinements mimicking the counterbalanced sequence of food of perseverative trials during response extinction training. In locations throughout initial response learning. For each day of addition, within-subjects contrasts computed on latency for limited latent extinction, animals received 10 trials (five trials extinction training days 1–3 (Figure 2B) also revealed a linear on each arm). The parameters for limited latent extinction effect of Day (F (1,6) = 113.56, p < 0.001), indicating that latency were chosen based on previous evidence indicating that 10 increased over the course of response extinction training. latent extinction trials per day produced extinction in the straight alley (Gabriele and Packard, 2006). However, given Extinction Probes that latent extinction trials had to be divided between east The results from the extinction probe trials are depicted in and west goal arms, this only permitted five trials on each Figure 3. To assess the effectiveness of the different types of arm per day. In order to allow for 10 trials on each arm, extinction training for each group, comparisons were made an additional group was given extended latent extinction, between the probe day and the last day of initial acquisition. in which animals received 20 trials (10 trials on each arm) The first four trials (vs. the last four trials) of the last per day. acquisition day were selected for this comparison based on the observation that during initial acquisition, animals were Extinction Probes typically slower and more likely to make errors for the first Twenty four hours following the last day of extinction, all animals few trials of each training day vs. the final training trials of the in Experiments 1 and 2 were given four probe trials. No food was previous day (see Figures 1C,D). Therefore, it was reasonable located in the maze for the extinction probe trials. For each probe to expect that the extinction probe trials would also have trial, an animal was released from the north or south arm (start higher latencies and more errors than the terminal trials of arm sequence: SNNS), and after reaching the previously correct the last acquisition day, regardless of whether an extinction food well or after 120 s had elapsed, animals were removed protocol was effective. Thus, for a more accurate measurement from the maze and placed in an opaque holding container for of the effectiveness of each extinction protocol, we compared a 30 s ITI. The maze was rotated 90º after every two trials. the extinction probe trials with the first four trials of the final Latency to reach the previously correct food well and the number acquisition day. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 21 Goodman and Packard Multiple Memory Systems and Extinction FIGURE 1 | Acquisition of hippocampus-dependent place learning in the plus-maze. (A,B) The percentage of correct turns increased (A) and the latency to reach the correct food well decreased (B) over the course of training, with no differences between groups. (C,D) Subsequently, all groups were combined, and the trials of each day were averaged into trial bins (1 trial bin = 3 trials). Animals were more likely to make incorrect turns (C) and were slower (D) on the first few trials of a given training day vs. the last few trials of the previous day. A two-way repeated measures 3 × 2 ANOVA (Group × acquisition. For animals in the latent extinction group, Fisher’s Day) was computed for number of perseverative trials on the LSD test indicated that there was a significant decrease in the last acquisition day (i.e., training day 8; first four trials) and number of perseverative trials from the last acquisition day (M = the extinction probe day (Figure 3A). Results indicated no 3.57) to the probe day (M = 2.43), p = 0.007. In addition, significant main effect of Group (F (2,18) = 1.79, p = 0.195), the response extinction group showed a significant decrease but there was a significant effect of Day (F (1,18) = 10.89, p = in number of perseverative trials between the last acquisition 0.004) and a significant Group × Day interaction (F (2,18) = day (M = 3.29) and the probe day (M = 2.00), p = 0.003. 5.37, p = 0.015). Multiple pairwise comparisons using Fisher’s Animals given no extinction did not show a significant change LSD test indicated that there were no significant differences in number of perseverative trials from the last acquisition day in number of perseverative trials between groups on the last (M = 3.14) to the probe day (M = 3.43), p = 0.456. On acquisition day. This is consistent with data presented above the extinction probe day, Fisher’s LSD test indicated that the indicating that the groups did not differ during initial task latent extinction group (M = 2.42) displayed a significantly Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 22 Goodman and Packard Multiple Memory Systems and Extinction FIGURE 2 | Response extinction of hippocampus-dependent place learning. (A,B) For animals in the response extinction group, the number of perseverative trials decreased (A) and latency increased (B) over the course of extinction training, indicating the effectiveness of response extinction. lower number of perseverative trials than animals in the no extinction group (M = 2.00) was also significantly lower than extinction control group (M = 3.42), p = 0.026. Similarly, perseverative trials for the no extinction group, p = 0.002. In number of perseverative trials during probe day for the response contrast, perseverative trials for the latent extinction group and FIGURE 3 | Extinction probe trials in the hippocampus-dependent place learning task. (A) There were no between-group differences in perseveration during the first few trials of the last training day (i.e., training day 8). Response and latent extinction groups, but not the “no extinction” group, displayed a decrease in number of perseverative trials from the last acquisition day to the probe day. On the probe day, the latent and response extinction groups displayed lower perseveration than the no extinction group, but the latent and response extinction groups did not differ from each other in perseveration. (B) There were no differences in latency between groups on the last training day. Response and latent extinction groups, but not the “no extinction” group, increased latency from the last acquisition day to the probe day. On the probe day, the latent and response extinction groups had higher latency than the no extinction group. Latency was also higher in the latent extinction group vs. the response extinction group on the probe day. Results indicate the effectiveness of latent and response extinction protocols in extinction of hippocampus-dependent place learning. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 23 Goodman and Packard Multiple Memory Systems and Extinction response extinction group did not differ on the probe day, Response Extinction p = 0.327. Figure 5 depicts learning over the course of extinction training A two-way repeated measures 3 × 2 ANOVA (Group × for animals in the ‘‘response extinction’’ group. Tests of within- Day) was computed for latency on the last acquisition day subjects contrasts computed on number of perseverative trials (i.e., training day 8; first four trials) and the extinction probe (Figure 5A) for extinction days 1–3 revealed a significant day (Figure 3B). Results indicated a significant main effect linear effect of Day (F (1,5) = 24.98, p = 0.004), indicating that of Group (F (2,18) = 5.48, p = 0.014), a significant effect of the number of perseverative trials decreased over the course Day (F (1,18) = 36.84, p < 0.001), and a significant Group × of response extinction training. In addition, within-subjects Day interaction (F (2,18) = 17.92, p < 0.001). Multiple pairwise contrasts computed on latency (Figure 5B) also revealed a comparisons using Fisher’s LSD test indicated that there significant effect of Day (F (1,5) = 23.90, p = 0.005), indicating that were no significant differences in latency between groups on latency increased over the course of response extinction training. the last acquisition day. For animals given latent extinction, there was a significant increase in latency from the last Extinction Probes acquisition day (M = 14.77) to the probe day (M = 40.71), The results from the extinction probe trials are depicted p = 0.002. There was also a significant increase in latency in Figure 6. The rationale for comparing extinction probe between the last acquisition day (M = 11.61) and the probe performance with the first four trials of the final training day day (M = 65.00) for animals given response extinction, p < was described in the results for Experiment 1 (see above). A 0.001. Animals given no extinction did not show a significant two-way repeated measures 4 × 2 ANOVA (Group × Day) change in latency from the last acquisition day (M = 17.39) was computed for number of perseverative trials on the last to the probe day (M = 11.61), p = 0.419. On the probe acquisition day (i.e., training day 8; first four trials) and the day, Fisher’s LSD test indicated that latency for the latent extinction probe day (Figure 6A). Results indicated a significant extinction group (M = 40.71) was significantly higher than main effect of Group (F (3,21) = 3.73, p = 0.027), a significant latency in the no extinction control group (M = 11.61), effect of Day (F (1,21) = 7.66, p = 0.012), and a significant p = 0.002. In addition, probe day latency for animals in Group × Day interaction (F (3,21) = 4.48, p = 0.014). Multiple the response extinction group (M = 65.00) was significantly pairwise comparisons using Fisher’s LSD test indicated that there higher than latency in the no extinction control group, p < were no significant differences in number of perseverative trials 0.001. Latency in the response extinction group was also between groups on the last acquisition day. This is consistent significantly higher than latency in the latent extinction group, with data presented above indicating that the groups did not p = 0.009. differ during initial task acquisition. For animals in the ‘‘response Taken together, the results of Experiment 1 indicate that extinction’’ group, Fisher’s LSD test indicated that there was a following acquisition in a place learning task animals given significant decrease in the number of perseverative trials from latent or response extinction displayed higher latency and lower the last acquisition day (M = 3.50) to the probe day (M = perseveration during the extinction probe trials, relative to 1.33), p < 0.001. No other groups showed a significant change animals given no extinction. These results suggest that either in number of perseverative trials between the last acquisition a latent or response extinction protocol may be effective at day and the probe day. On the extinction probe day, Fisher’s extinguishing hippocampus-dependent place learning in the LSD test indicated that the response extinction group (M = plus-maze. 1.33) displayed a significantly lower number of perseverative trials than animals in the no extinction control group (M = 3.23), p < 0.001. Number of perseverative trials for the limited Experiment 2 latent extinction group (M = 3.00) did not differ from the no Initial Acquisition extinction group, p = 0.642. In addition, perseverative trials Initial acquisition of the response learning task is depicted for the extended latent extinction group (M = 3.17) did not in Figure 4. A two-way repeated measures 4 × 8 ANOVA differ from the no extinction group, p = 0.790. There was (Group × Day) computed on percentage of correct turning a significantly lower number of perseverative trials in the responses over the course of training (Figure 4A) indicated a response extinction group vs. the limited latent extinction group, significant main effect of Day (F (7,147) = 23.74, p < 0.001), p < 0.001, and the extended latent extinction group, p < but no effect of Group (F (3,21) = 0.224, p = 0.878) and no 0.001. Group × Day interaction (F (21,147) = 0.753, p = 0.771). Similarly, A two-way repeated measures 4 × 2 ANOVA (Group × Day) a two-way repeated measures 4 × 8 ANOVA (Group × Day) was computed for latency on the last acquisition day (i.e., training computed on latency (Figure 4B) also indicated a significant day 8; first four trials) and the extinction probe day (Figure 6B). effect of Day (F (7,147) = 95.52, p < 0.001), no effect of Results indicated a significant main effect of Group (F (3,21) = Group (F (3,21) = 0.330, p = 0.800), and no Group × Day 22.00, p < 0.001), a significant effect of Day (F (1,21) = 183.9, interaction (F (21,147) = 0.88, p = 0.620). These results indicate p < 0.001), and a significant Group × Day interaction (F (3,21) = that all groups acquired the task about equally. Therefore, 81.57, p < 0.001). Multiple pairwise comparisons using Fisher’s any subsequent differences between groups during extinction LSD test indicated that there were no significant differences in may not be readily attributed to differing rates of initial task latency between groups on the last acquisition day. Comparing acquisition. the mean latencies between the last acquisition day and the probe Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 24 Goodman and Packard Multiple Memory Systems and Extinction FIGURE 4 | Acquisition of DLS-dependent response learning in the plus-maze. (A,B) The percentage of correct turns increased (A) and the latency to reach the correct food well decreased (B) over the course of training in the response learning task. There were no differences between groups, suggesting all groups acquired the task about equally. (C,D) All groups were combined, and the trials of each day were averaged into trial bins (1 trial bin = 3 trials). Animals were more likely to make incorrect turns (C) and were slower (D) on the first few trials of a given training day vs. the last few trials of the previous day. day for each group indicated a significant increase in latency given response extinction displayed higher latency and lower between the 2 days for all groups: no extinction (last acquisition perseveration during the extinction probe trials, relative to day M = 8.46, probe day M = 16.32, p = 0.049), limited latent animals given no extinction. In contrast, animals given limited or extinction (last acquisition day M = 7.58, probe day M = 16.67, extended latent extinction protocols did not differ significantly p = 0.037), extended latent extinction (last acquisition day M = in latency or perseveration from animals given no extinction. 10.29, probe day M = 19.96, p = 0.027), and response extinction The results suggest that in contrast to typical response extinction, (last acquisition day M = 11.00, probe day M = 92.92, p < 0.001). latent extinction protocols may not be effective at extinguishing On the probe day, Fisher’s LSD test indicated that latency for the memory in a DLS-dependent response learning task. response extinction group (M = 92.92) was significantly higher than latency in the no extinction control group (M = 16.32), p < 0.001. Latency did not differ significantly between limited latent DISCUSSION extinction (M = 16.67) and the no extinction control group, p = 0.957, and latency also did not differ between extended latent The present findings indicate a dissociation regarding the extinction (M = 19.96) and the no extinction control group, p = effectiveness of latent extinction across two learning and memory 0.567. Response extinction latency was significantly higher than tasks. Latent extinction was effective at extinguishing memory in latency in limited latent extinction, p < 0.001, and extended a hippocampus-dependent place learning task, but not in a DLS- latent extinction groups, p < 0.001. dependent response learning task. In contrast, typical ‘‘response Taken together, the results of Experiment 2 indicate that extinction’’ was effective in both place and response learning following acquisition in the response learning task, animals tasks. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 25 Goodman and Packard Multiple Memory Systems and Extinction FIGURE 5 | Response extinction of DLS-dependent response learning. (A,B) For animals in the response extinction group, the number of perseverative trials decreased (A) and latency increased (B) indicating the effectiveness of response extinction training. In Experiment 1, following acquisition of the place learning control group. Therefore, this increase in latency from the task, animals given latent or response extinction displayed last acquisition day to the probe day may not be readily greater latency and fewer perseverative trials than animals given attributed to the latent extinction protocols. In addition, latent no extinction. Interestingly, animals given response extinction extinction and no extinction control groups did not show displayed higher latencies than animals given latent extinction, a decrease in number of perseverative trials across the 2 suggesting response extinction may have had greater efficacy days. than latent extinction in the place learning task. However, there A finding secondary to the differential effects of the extinction was no difference in number of perseverative trials between protocols, but of considerable relevance to classical learning latent and response extinction groups. It is possible that, relative theories, pertains to the initial acquisition curves in the place to latent extinction, response extinction was more efficient at and response learning tasks. During most days of initial slowing the running approach response, but not necessarily more acquisition, the first few trials were accompanied with greater effective at extinguishing the location of food reward. latencies and more errors than the last few trials of the In Experiment 2, following acquisition of a response learning previous training day (see Figures 1C,D, 4C,D). However, task, animals given response extinction displayed higher latencies this rise in latency and inaccuracy on the first few training and fewer perseverative trials than animals given no extinction, trials of a given day became progressively less pronounced indicating the effectiveness of response extinction in this on subsequent training days. The present finding is consistent task. In contrast, animals given limited or extended latent with early principles in learning theory pertaining to decay extinction did not differ in latency or perseveration from animals theory (e.g., Ebbinghaus, 1913; Thorndike, 1913). Thorndike given no extinction, suggesting that these latent extinction (1913) proposed that following acquisition, a memory begins protocols were not effective at producing extinction in the to fade as a function of its disuse over time (i.e., decay). response learning task. Even though latencies in the limited However, some traces of the memory survive this decay, and and extended latent extinction groups showed a slight increase thus relearning not only proves faster than initial learning, but from the last acquisition day to the probe day, a comparable also results in a stronger memory that is less sensitive to memory increase was also observed for animals in the ‘‘no extinction’’ decay. Although the precise mechanisms of memory decay have Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 26 Goodman and Packard Multiple Memory Systems and Extinction FIGURE 6 | Extinction probe trials in the DLS-dependent response learning task. (A) There were no differences between groups in perseveration during the first few trials of the last acquisition day (i.e., training day 8). Only the response extinction group displayed a decrease in number of perseverative trials from the last acquisition day to the probe day. On the probe day, the response extinction group displayed lower perseveration than all other groups. The latent extinction groups (limited and extended) did not differ in perseveration from the no extinction control group on the probe day. (B) There were no between-group differences in latency on the last training day. All groups increased latency from the last acquisition day to the probe day. On the probe day, the response extinction group had higher latency than all other groups. Latency was not higher in the latent extinction groups (limited and extended), relative to the no extinction group. Results indicate that response extinction was effective and latent extinction was ineffective at extinguishing memory of DLS-dependent response learning. The principal finding that latent extinction was effective in the place learning task but not the response learning task may be related to differences between the memories acquired in each task. That is, latent extinction might only be effective when the to-be-extinguished memory contains certain critical features. The tasks selected for the present experiments depended on distinct neural systems, and solving each task hinged on different learning requirements. The hippocampus-dependent place learning task presumably required animals to encode the spatial location of the food reward to guide behavior to the correct arm, whereas the DLS-dependent response learning task only required that animals encode a left body-turn response at the maze choice point. Although animals being trained in the response learning task could also encode the spatial locations of the food reward, this information was not necessary for acquisition and ongoing performance in this task. In fact, FIGURE 7 | Thorndike’s hypothetical model of memory decay and extensive evidence indicates that spatial information might recovery. The segmented linear curve indicates memory performance (y axis) interfere with acquisition in the response learning task (for over the course of five learning sessions (x axis). The vertical lines on the x axis indicate four periods of disuse (i.e., periods of time between sessions in which reviews, see Poldrack and Packard, 2003; Packard and Goodman, the memory is not retrieved). Performance decreases (i.e., decays) following 2013). each period of disuse. However, relearning during a subsequent session Latent extinction in maze learning tasks might only be results in a stronger memory that is less sensitive to decay. Therefore, decay effective when the spatial location of the reinforcer is a becomes progressively less pronounced following each subsequent period of critical part of the to-be-extinguished memory. Previous studies disuse (From Thorndike, 1913, p. 283; axis labels added). examining latent extinction have typically employed maze tasks, such as the straight alley maze, that could be solved adequately been disputed (McGeoch, 1932), the general predictions of using either spatial or non-spatial learning strategies. In ‘‘dual- Thorndike’s model (see Figure 7) resemble the acquisition curves solution’’ tasks such as these, animals typically employ spatial obtained in the present study. It is possible that some decay (or, learning strategies when the learning environment constitutes more generally, forgetting) occurred in between daily training a heterogeneous visual surround, whereas animals employ sessions, but that with each subsequent session of relearning response learning strategies when the task is conducted in a the memory became more firmly ingrained and less sensitive to homogeneous visual surround (for reviews, see Restle, 1957; decay. Packard and Goodman, 2013). Interestingly previous studies Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 27 Goodman and Packard Multiple Memory Systems and Extinction have indicated that latent extinction was only effective in Latent extinction may involve spatial memory processing heterogeneous visual surrounds conducive to allocentric spatial insofar as confining an animal to a previously rewarded spatial learning (e.g., Seward and Levy, 1949; Denny and Ratner, 1959; location without food (i.e., latent extinction) might allow the Dyal, 1962). Latent extinction was not effective in homogenous animal to acquire a new memory in which the spatial location visual surrounds that prevented the use of allocentric spatial becomes associated with absence of food. Thus, for latent learning (e.g., Bugelski et al., 1952; Scharlock, 1954; Denny and extinction to be successful, a rat must be confined to the Ratner, 1959). These previous findings are consistent with the previously rewarded spatial location. Confining a rat to an empty suggestion that in maze learning tasks, latent extinction might be goal box located in a different room (Iwahara et al., 1953) or selectively effective at extinguishing allocentric spatial memory. a different spatial location in the same room (Clifford, 1964) The finding that latent extinction might only be successful does not produce extinction. This proposed mechanism for latent at extinguishing certain types of memory could be attributed to extinction is consistent with its dependance on hippocampal the distinct learning mechanisms through which latent extinction function, i.e., in addition to acquiring information about food operates. Unlike response extinction, latent extinction does not rewarded locations, the hippocampus is similarly involved in conform to classical models of extinction that suggest the animal linking spatial locations with the absence of food reward (Gaskin must make the previously acquired response for extinction to and White, 2006). occur (e.g., Hull, 1943, 1952). Proponents of the Hullian S-R This putative spatial mechanism could also explain why view of learning have suggested that latent extinction, although latent extinction was effective in the place learning task, but it may not be readily explained by Hull’s traditional response- not the response learning task. In the place learning task, inhibition theory of extinction, could still be accounted for memory performance was presumably guided by a learned through a Hullian fractional anticipatory response mechanism association in which a spatial location had been associated (Hull, 1931; Spence, 1951). According to this view (Moltz, 1957), with the food reward. Thus, if the same spatial location were an unobservable component of the consumatory goal response subsequently associated with the absence of food reward, which is elicited by cues throughout the maze during initial acquisition putatively occurs during latent extinction, we should expect of the task, and this partially guides behavior to the correct goal memory performance in the place learning task to decline. In location. When an animal is confined to the goal box during contrast, memory performance in the response learning task latent extinction, this fractional goal response is elicited and over was presumably not guided by the spatial locations of the food time, becomes extinguished to the goal box cues. To the extent reward, and therefore associating spatial locations with the that the goal box cues might resemble earlier sections of the maze, absence of food reward should not affect later retrieval of extinction of the fractional goal response will generalize to other the previously acquired response. parts of the maze, resulting in increased latency and incorrect Given the effectiveness of typical response extinction across turns during extinction probe trials. Several cogent arguments both place and response learning tasks, it is tempting to have been raised indicating the inadequacy of this potential S- speculate that response extinction might depend on a distinct R mechanism in explaining latent extinction (Gleitman et al., learning mechanism. Previous evidence from our laboratory 1954; Treisman, 1960). In addition, this putative mechanism is indicates that in contrast to latent extinction, the effectiveness not supported by the present findings. If latent extinction were of response extinction in the straight alley maze is not impaired to operate by extinguishing a fractional response in the goal box following hippocampal inactivation (Gabriele and Packard, that generalizes to other parts of the maze, then it would be 2006). Rather, response extinction in the straight alley maze is reasonable to predict that latent extinction would be effective attenuated following lesion or temporary inactivation of the DLS across both place and response learning tasks, which presently (Dunnett and Iversen, 1981; Thullier et al., 1996; Gabriele, 2008). was not observed. Considering that the DLS is a chief neural substrate implicated Previous evidence from our laboratory suggests that latent in S-R learning and memory processes (Packard and Knowlton, extinction may involve spatial memory mechanisms (Gabriele 2002), one possibility is that during response extinction the and Packard, 2006). Temporary inactivation of the dorsal DLS forms S-R associations between visual cues in the learning hippocampus with bupivacaine blocks the effectiveness of latent situation (i.e., the stimuli) and the inhibition of a behavior (i.e., extinction in the straight alley maze (Gabriele and Packard, the response). Several investigators have proposed similar S- 2006). Considering that a principal function of the hippocampus R mechanisms to account for extinction across maze learning, involves spatial memory formation (O’Keefe and Nadel, 1978; operant lever pressing, and Pavlovian conditioning paradigms Morris et al., 1982), it is possible that hippocampal inactivation (Guthrie, 1935; Hull, 1943; Rescorla, 1993; Delamater, 2004). blocked latent extinction by disrupting hippocampus-dependent Importantly, the learned inhibition of behavior during response spatial memory processing. That latent extinction might depend extinction could potentially explain the effectiveness of this in part on spatial memory processing is largely consistent protocol in both place learning and response learning tasks. with previous behavioral evidence. As mentioned previously, Aside from the direct involvement of multiple memory latent extinction is selectively effective in heterogeneous visual systems, another potential mechanism underlying the selective environments conducive to spatial memory formation, but not effectiveness of latent extinction pertains to the immediate homogenous visual environments that prevent spatial memory differences between the two tasks. Although the place and formation (Seward and Levy, 1949; Bugelski et al., 1952; response learning tasks were identical in terms of their Scharlock, 1954; Denny and Ratner, 1959; Dyal, 1962). motivational, sensory, and motoric requirements, it was Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 28 Goodman and Packard Multiple Memory Systems and Extinction necessary that the tasks differed slightly in some respects so that explanation for the selective effectiveness of latent extinction each task invoked a different memory system. We cannot rule across these learning tasks. Future studies utilizing a wider variety out the possibility that slight differences between the two tasks of spatial and non-spatial memory tasks are required to further (e.g., in the place learning task, animals received food in one examine this hypothesis. location; in the response learning task, animals received food in two locations) may have partially influenced the effectiveness of AUTHOR CONTRIBUTIONS latent extinction. In sum, the present findings indicate that whereas response JG contributed ideas, manuscript writing, and conducted extinction successfully extinguished memory in hippocampus- research. MGP contributed ideas and manuscript writing. dependent place learning and DLS-dependent response learning tasks, latent extinction was selectively effective in the place ACKNOWLEDGMENTS learning task and not the response learning task. The suggestion that the principal learning mechanisms underlying latent The authors would like to thank Gizelle Leal and Chelsey extinction involve an acquired association between the spatial Thibodeaux for their assistance on this project. This study was location and the absence of food reward may provide an supported by Texas A&M University PESCA grant (MGP). REFERENCES Gabriele, A., Setlow, B., and Packard, M. G. (2009). Cocaine self-administration alters the relative effectiveness of multiple memory systems during extinction. Asem, J. S. A., and Holland, P. C. (2015). Dorsolateral striatum implicated in Learn. Mem. 16, 296–299. doi: 10.1101/lm.1253409 the acquisition, but not expression, of immediate response learning in rodent Gaskin, S., and White, N. M. (2006). Cooperation and competition between the submerged T-maze. Neurobiol. Learn. 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No use, distribution or Animal Learn. Behav. 21, 327–336. doi: 10.3758/bf03197998 reproduction is permitted which does not comply with these terms. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 314 | 30 REVIEW published: 09 November 2015 doi: 10.3389/fnbeh.2015.00298 The Role of the Medial Prefrontal Cortex in the Conditioning and Extinction of Fear Thomas F. Giustino and Stephen Maren* Department of Psychology and Institute for Neuroscience, Texas A&M University, College Station, TX, USA Once acquired, a fearful memory can persist for a lifetime. Although learned fear can be extinguished, extinction memories are fragile. The resilience of fear memories to extinction may contribute to the maintenance of disorders of fear and anxiety, including post-traumatic stress disorder (PTSD). As such, considerable effort has been placed on understanding the neural circuitry underlying the acquisition, expression, and extinction of emotional memories in rodent models as well as in humans. A triad of brain regions, including the prefrontal cortex, hippocampus, and amygdala, form an essential brain circuit involved in fear conditioning and extinction. Within this circuit, the prefrontal cortex is thought to exert top-down control over subcortical structures to regulate appropriate behavioral responses. Importantly, a division of labor has been proposed in which the prelimbic (PL) and infralimbic (IL) subdivisions of the medial prefrontal cortex (mPFC) regulate the expression and suppression of fear in rodents, respectively. Here, we critically review the anatomical and physiological evidence that has led to this proposed dichotomy of function within mPFC. We propose that under some conditions, the PL and IL act in concert, exhibiting similar patterns of neural activity in response to aversive conditioned stimuli and during the expression or inhibition of conditioned fear. Edited by: This may stem from common synaptic inputs, parallel downstream outputs, or cortico- Onur Gunturkun, Ruhr University Bochum, Germany cortical interactions. Despite this functional covariation, these mPFC subdivisions may Reviewed by: still be coding for largely opposing behavioral outcomes, with PL biased towards fear Cyril Herry, expression and IL towards suppression. University of Bordeaux, France Miguel Angel Fullana, Keywords: prelimbic, infralimbic, freezing, fear, extinction Hospital del Mar, Spain *Correspondence: Stephen Maren INTRODUCTION maren@tamu.edu Pavlovian fear conditioning is a form of learning that serves as a robust model to explore Received: 29 August 2015 the neurobiological underpinnings of disorders of fear and anxiety, including post-traumatic Accepted: 26 October 2015 stress disorder (PTSD). In a typical rodent experiment, an innocuous conditioned stimulus Published: 09 November 2015 (CS; e.g., an auditory tone) is paired with an aversive unconditioned stimulus (US; e.g., a mild Citation: electric footshock). After one or more conditioning trials, presentation of the CS alone comes Giustino TF and Maren S (2015) The to elicit a conditioned fear response (CR) that includes freezing behavior (i.e., immobility Role of the Medial Prefrontal Cortex except that necessary for respiration), changes in heart rate and respiration, and potentiated in the Conditioning and Extinction of Fear. acoustic startle (Davis, 1992; LeDoux, 2000; Maren, 2001). Importantly, these fear CRs can Front. Behav. Neurosci. 9:298. be extinguished by repeated presentations of the CS in the absence of the US. In rodents doi: 10.3389/fnbeh.2015.00298 and humans alike, CRs to an extinguished CS tend to return under a number of conditions Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 31 Giustino and Maren PFC and fear including the passage of time (spontaneous recovery), when nuclei; the basolateral complex of the amygdala (BLA; consisting the CS is presented outside the extinction context (renewal), of the lateral, basal and basomedial nuclei) and the central or with exposure to an unsignaled US (reinstatement; Bouton, nucleus of the amygdala (CeA; consisting of lateral and medial 2000, 2002; Hermans et al., 2006; Maren et al., 2013; Vervliet components) play critical roles in the acquisition of both fear et al., 2013; Goode and Maren, 2014). These recovery or and extinction memories. It has been suggested that inhibitory relapse phenomena suggest that extinction does not erase fear neurons within the amygdala play a role in regulating fear memories, but generates a new safety memory that inhibits the output. These include: (1) the intercalated cell masses (ITCs) expression of fear. In addition, extinction learning itself is a positioned between the BLA and CeA (Nitecka and Ben-Ari, fragile process, dependent on many factors including timing 1987; McDonald and Augustine, 1993; Paré and Smith, 1993; relative to conditioning (Maren and Chang, 2006; Myers et al., Royer et al., 1999; Lee et al., 2013; Duvarci and Pare, 2014); 2006; Maren, 2014) and stress (Maren and Holmes, 2015). (2) local inhibitory interneurons within the BLA (Spampanato While learned fear serves an adaptive purpose aiding survival, et al., 2011; Wolff et al., 2014); and (3) inhibitory interneurons in pathological fear states are thought to underlie various stress CeL that project to CeM (Ciocchi et al., 2010; Haubensak et al., and trauma-related disorders such as PTSD, which has a lifetime 2010). prevalence of nearly 8% in the general population (Kessler How one structure supports the formation and storage et al., 1995, 2005). Not surprisingly, this number increases to of opposing memories is not fully understood, although it as high as 30% in combat-exposed veterans (Koenen et al., appears that distinct cell populations within the BLA may 2008), amplifying the need for more effective therapies. PTSD preferentially encode low and high fear states (Goosens et al., has been described as the only mental health disorder with a 2003; Hobin et al., 2003; Herry et al., 2008; Senn et al., 2014). known cause (i.e., a traumatic experience; Pitman et al., 2012) For example, lesions of the lateral amygdala (LA), a locus for and is characterized by heightened arousal and resistance to CS and US convergence, or the CeA disrupt fear conditioning extinction learning (Rauch et al., 2006). Many have argued (LeDoux et al., 1990; Goosens and Maren, 2001; Wilensky et al., that PTSD may, at least in part, be a disorder of the fear 2006). Similarly, reversible inactivation of the BLA prevents circuitry (Shin and Handwerger, 2009) and an enhanced the acquisition and expression of conditioned fear (Helmstetter understanding of learned fear is relevant to the psychological and Bellgowan, 1994; Muller et al., 1997), suggesting a large processes underlying this disorder (Liberzon and Sripada, 2008; degree of overlap between the subnuclei of the amygdala. Studies VanElzakker et al., 2014). It is possible that PTSD patients using overtraining procedures have demonstrated that amygdala exhibit exaggerated fear conditioning, resistance to extinction, lesions disrupt fear memories, not the ability of animals to or both; ultimately, they exhibit persistent fear CRs (Pitman, emit conditioned fear responses (Maren, 1998, 1999). Single-unit 1988). recordings have demonstrated learning-related changes in short- Due to the prevalence and debilitating nature of stress and latency (less than 15 ms) CS-evoked responses in the LA after trauma-related disorders, there has been a surge in interest fear conditioning, suggesting that these changes are mediated in understanding the neural processes subserving learned fear by direct thalamo-amygdala projections (Quirk et al., 1995; and its subsequent extinction (Quirk and Mueller, 2008; Milad Maren, 2000). Moreover, these conditioning-induced changes and Quirk, 2012; Maren et al., 2013). A triad of brain regions, in spike firing are specifically related to the associative nature including the amygdala, hippocampus and medial prefrontal of the CS, indicating that the LA is a crucial site of plasticity cortex (mPFC) has been heavily studied in relation to fear (Maren for fear memories independent of freezing behavior (Goosens and Quirk, 2004; Herry et al., 2010; Dejean et al., 2015). While it et al., 2003). In contrast, the CeA is primarily thought of is well accepted that the amygdala and hippocampus play a role as an output station, relaying information to the brain stem, in conditioned fear and extinction, a dichotomy of function has hypothalamus and periaqueductal gray (PAG) to initiate fear been proposed within the mPFC in which the prelimbic (PL) and responses such as freezing (Paré et al., 2004). Whereas the CeL infralimbic (IL) cortices regulate the expression and suppression is necessary for fear acquisition, CRs are mediated by CeM of fear, respectively (Quirk and Mueller, 2008; Sotres-Bayon and output (Ciocchi et al., 2010; Haubensak et al., 2010). Curiously, Quirk, 2010; Milad and Quirk, 2012; Maren et al., 2013). Here, we while the LA encodes CS-US information, there are no direct critically review the anatomical and physiological evidence that connections between the LA and CeA to directly mediate fear has led to this proposed dichotomy of function within mPFC, output, suggesting that the BL or BM or both may act as an comparing results from rodents with those in humans. interface (Amano et al., 2011). Interestingly, post-conditioning lesions of the basal nuclei block fear expression while leaving THE FEAR CIRCUIT learning intact (Anglada-Figueroa and Quirk, 2005; Amano et al., 2011). Selective inactivation of either BM or BL alone It is well established that both the acquisition and extinction of was not sufficient to mimic this effect, whereas inactivation of fear memories requires synaptic plasticity within the amygdala, both BM and BL was sufficient. This implies that some level however a comprehensive discussion of the amygdala circuitry of functional overlap exists between these two regions (Amano is beyond the scope of this review (Fanselow and LeDoux, et al., 2011). 1999; LeDoux, 2003; Maren and Quirk, 2004; Herry et al., Additionally, several studies have shown that BLA synaptic 2010; Pape and Pare, 2010; Lee et al., 2013; Duvarci and plasticity is crucial for the acquisition of extinction (Falls et al., Pare, 2014). The amygdala is a node of highly interconnected 1992; Lu et al., 2001; Herry et al., 2006, 2008; Kim et al., 2007; Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 32 Giustino and Maren PFC and fear Sotres-Bayon et al., 2007). Upon extinction learning, LA neurons Fear regulation must be tightly controlled and this is thought typically show a reduction in CS-evoked neural activity (Quirk to depend on the mPFC. Two subdivisions of mPFC in rodents, et al., 1995; Repa et al., 2001). However, a distinct population of and their human homologs, have been identified as having LA cells maintain CS-evoked responding throughout extinction distinct roles within the fear circuit. The prelimbic cortex (PL) learning (Repa et al., 2001). Interestingly, after extinction, is thought to regulate fear expression, whereas the infralimbic patterns of CS-evoked neural activity in LA are mediated by the cortex (IL) mediates fear suppression (Quirk and Beer, 2006; context and reflect the level of freezing (i.e., larger responses Sotres-Bayon and Quirk, 2010; Milad and Quirk, 2012; Riga et al., occur when fear renews; Hobin et al., 2003). In summary, there 2014). A similar division of labor has been proposed in humans, is compelling evidence to support the notion that the amygdala indicating that the neural mechanisms of extinction learning may is a crucial locus for the acquisition and extinction of learned be conserved across species (Phelps et al., 2004; Schiller et al., fear with both ‘‘fear’’ and ‘‘extinction’’ neurons existing within 2008; Sehlmeyer et al., 2009; Milad and Quirk, 2012; Vervliet the same subnuclei whose CS-evoked activity strongly correlates et al., 2013). Below we review the extant literature that has led with the level of fear expression (Quirk et al., 1995; Repa et al., to this proposed dichotomy of function. 2001; Goosens et al., 2003; Herry et al., 2008; Senn et al., 2014). The hippocampus has also been identified as a key mediator ANATOMY OF THE RODENT mPFC of learned fear. Given the role of the hippocampus in encoding contextual and spatial information it is not surprising this Initially, the PFC was defined by a granular layer IV; this region plays a substantial role in the fear circuit. Numerous criterion excluded lower level mammalian species, including studies have shown that hippocampal lesions dampen fear to rodents (Brodmann, 1909). This classification was challenged a context previously associated with a shock US (Selden et al., by Rose and Woolsey, who suggested that projections from 1991; Kim and Fanselow, 1992; Phillips and Ledoux, 1992). the mediodorsal nucleus of the thalamus were the defining Importantly, hippocampal lesions produce larger deficits when feature of the PFC. This re-definition of the PFC was inclusive made soon after context conditioning, suggesting that recent of all mammalian species (Rose and Woolsey, 1948) and it is memories rely more heavily on the integrity of the hippocampus now generally accepted that rodents have a PFC with some (Maren et al., 1997; Anagnostaras et al., 1999). Interestingly, homology to that of higher-order species (Uylings and van hippocampal lesions do not necessarily interfere with context Eden, 1990; Uylings et al., 2003). These homologies are based conditioning when damage is made prior to training (Maren on several criteria including cytoarchitectonics, connectivity et al., 1997; Frankland et al., 1998), although deficits in the patterns, electrophysiological properties, protein expression, and acquisition of contextual fear can be obtained with single-trial changes in behavior following damage (Campbell and Hodos, procedures (Wiltgen et al., 2006). Collectively, these results 1970; Uylings and van Eden, 1990; Uylings et al., 2003). Indeed, suggest that the hippocampus is required for forming and the rodent PFC like that in humans plays a role in an array of storing memories of the context, but not necessarily context- complex behaviors (Heidbreder and Groenewegen, 2003; Kesner US associations (Young et al., 1994). These findings support and Churchwell, 2011). the notion that the hippocampus plays a key role in both the acquisition and expression of conditioned fear to a particular Laminar Organization and Cell Types context. In rodents, the mPFC is identified as the agranular portion As mentioned above, the extinction of fear is highly of the frontal lobe and is divided into three subdivisions: the context-dependent; that is, fear returns or ‘‘renews’’ when the anterior cingulate (ACC), the PL and the IL. Here, we will CS is presented outside the extinction context. Considerable primarily focus on PL and IL. The rodent PFC exhibits laminar evidence indicates that the renewal of fear is mediated by organization with deep and superficial layers (Caviness, 1975; the hippocampus (Bouton, 2000, 2002; Bouton et al., 2006; Yang et al., 1996; Uylings et al., 2003; van de Werd et al., Hermans et al., 2006; Maren et al., 2013; Vervliet et al., 2013; 2010), although a granular layer IV is less well defined when Goode and Maren, 2014). For example, many studies have compared to humans and non-human primates (Krettek and shown that hippocampal inactivation dampens fear renewal Price, 1977b; Uylings and van Eden, 1990; Uylings et al., 2003). when the CS is presented outside of the extinction context PL and IL are neighboring structures, with PL lying just dorsal (Holt and Maren, 1999; Corcoran and Maren, 2001; Hobin to IL, which can be distinguished based on cytoarchitectonic et al., 2006; Maren and Hobin, 2007; Zelikowsky et al., features and laminar organization. For example, layer V of PL 2012). In addition, disconnections of the hippocampus from is less well organized compared to more dorsal regions (i.e., the amygdala or prefrontal cortex impair renewal (Orsini ACC), whereas layer VI cells are arranged in a horizontal fashion et al., 2011), amygdala neurons engaged during fear renewal in both rats and mice (van de Werd et al., 2010). Due to receive hippocampal and prelimbic input (Knapska et al., the relatively large size of PL, layer II and III appear broad 2012) and individual hippocampal neurons expressing Fos after compared to neighboring subdivisions. Interestingly, there is fear renewal preferentially project to both the amygdala and evidence for a changing organization along the dorsal-ventral prefrontal cortex (Jin and Maren, 2015). These data suggest axis of PL, which may transition into IL (Heidbreder and that the hippocampus integrates contextual information during Groenewegen, 2003; Perez-Cruz et al., 2007; van de Werd et al., conditioning and likely regulates the context dependent recall of 2010). This distinction is mainly based on the expansion of fear after extinction learning. layer II at the expense of layers III and V along this axis. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 33 Giustino and Maren PFC and fear In contrast to PL, IL layer II neurons innervate layer I at a influencing firing rate and action potential synchronization much higher rate, making IL layer II appear broad (Krettek (Cobb et al., 1995). Interestingly, PV expression in mPFC and Price, 1977b; van de Werd et al., 2010). While the more is generally similar between PL and IL, suggesting the superficial layers II and III are easily discernible from a lighter mechanisms for modulating mPFC output are similar layer V in PL, IL layers are less distinct (Krettek and Price, between these two brain regions (Gabbott et al., 1997; van 1977b). In general, IL layers II-VI have a relatively homogenous de Werd et al., 2010). As with the vast array of principal layout in terms of cell size and density, with smaller cell neurons, the differential contribution of specific subtypes of bodies compared to PL (van Eden and Uylings, 1985; van de interneurons within and between mPFC layers within the Werd et al., 2010). The contribution of different layers and fear circuit are questions of high interest that remain to be functional changes along the dorsal-ventral axis of PL and IL resolved. are largely unknown, but may be differentially engaged in the fear circuit, similar to the findings noted above regarding distinct Inputs populations within amygdala nuclei regulating opposing fear It is well established that PL and IL receive excitatory states. inputs from regions including, but not limited to, the Cortical processing of information requires complex midline thalamus, BLA, hippocampus and contralateral mPFC interactions between a number of distinct cell types that fall into (Krettek and Price, 1977a; Little and Carter, 2012, 2013). The two broad categories: principal cells (80–90%) and interneurons posterior portion of the amygdala strongly projects to both (10–20%; DeFelipe and Fariñas, 1992; Gabbott et al., 2005). PL and IL with sparse innervation from the anterior regions Neurons are typically classified based on unique characteristics (Krettek and Price, 1977a). Some studies however, have shown including cell size and shape, dendritic arborization, molecular strong connectivity from anterior regions, especially from the markers, and connectivity. Pyramidal cells are typically thought BLA (Sarter and Markowitsch, 1984; McDonald, 1987). BLA to communicate to long-distance targets and are found in projections synapse on layers II–VI with a small percentage of layers II-VI (DeFelipe and Fariñas, 1992), although there are these projections targeting PVINs (Gabbott et al., 2006). Thus, noted differences in the firing properties, cell body size and BLA projections can functionally modulate mPFC output via dendritic morphology within and across layers (Yang et al., feed-forward inhibitory mechanisms. In addition, dorsal and 1996; Barthó et al., 2004; Molnár and Cheung, 2006; Wang ventral hippocampus (CA1/subiculum) exhibit robust excitatory et al., 2006; Otsuka and Kawaguchi, 2008; Brown and Hestrin, projections to PL and IL (Swanson, 1981; Jay et al., 1989; Jay and 2009; Dembrow et al., 2010; van Aerde and Feldmeyer, 2015). In Witter, 1991; Azuma and Chiba, 1996; Hoover and Vertes, 2007). addition, a number of molecular markers have been identified These projections have been reported to terminate in all layers to categorize specific subclasses of pyramidal cells (Gong et al., of mPFC, although this may shift in density along the dorsal- 2003; Hevner et al., 2003; Gray et al., 2004; Molnár and Cheung, ventral axis (Jay et al., 1989; Jay and Witter, 1991). In addition, 2006; Watakabe et al., 2007). The complexity and organization of a population of ventral CA1 neurons innervates IL layers I and V cortical pyramidal neurons makes the PFC well suited to regulate and these same hippocampal neurons also synapse on entorhinal several functions and an array of behaviors (Heidbreder and neurons, which may be important for integrating contextual and Groenewegen, 2003; Kesner and Churchwell, 2011). spatial information (Swanson, 1981). Similar to the amygdala, Similar to pyramidal cells, interneurons of the cortex are some hippocampal projections may preferentially target mPFC separated into several classes based on unique physiological, interneurons, inhibiting mPFC output to downstream targets morphological, and immunocytochemical markers (Kawaguchi (Sotres-Bayon et al., 2012). In summary, PL and IL receive many and Kubota, 1993, 1997; Kawaguchi, 1995; Gupta et al., similar input patterns, suggesting that these two subdivisions of 2000; Ascoli et al., 2008; Povysheva et al., 2008). While mPFC integrate incoming information from multiple sources to sparse in number relative to pyramidal cells, interneurons drive appropriate behavioral responding. nonetheless serve to modulate cortical function. Broad classes of interneurons, based on the heterogeneous expression Outputs of calcium-binding proteins and neuropeptides such as The regulation of fear is thought to rely heavily on the integrity parvalbumin (PV), somatostatin, vasoactive intestinal of the mPFC, which functions to exert top down control polypeptide and cholecystokinin, have been observed in over subcortical structures, coding for appropriate behavioral most layers of rodent PFC, although this distribution may responses. The most widely accepted view is that PL and IL not be uniform (DeFelipe, 1993; Kawaguchi and Kubota, project broadly to the same region (e.g., the amygdala) but 1993, 1997; Kawaguchi, 1995; Gabbott et al., 1997). These to distinct populations of cells that ultimately dictate CRs. To distinct classes of interneurons exhibit unique firing patterns, this end, PL and IL both strongly innervate the BLA and these synapsing on specific morphological subregions of pyramidal glutamatergic projections originate from layers II, V and VI cells. For example, somatostatin-positive interneurons typically (DeFelipe and Fariñas, 1992; Pinto and Sesack, 2000, 2008; innervate pyramidal cell dendrites to modulate the gain of Gabbott et al., 2005; Hoover and Vertes, 2007). In terms of their inputs terminating within those subregions (Kawaguchi and potential functional opposition, PL projections terminate in the Kubota, 1997; Gupta et al., 2000; Freund and Katona, 2007). BLA whereas IL projects to the ventral region of the LA, the In contrast, fast-spiking parvalbumin-positive interneurons basomedial nucleus, and the lateral central nucleus (McDonald (PVINs) target the perisomatic region of pyramidal cells, thereby et al., 1996; McDonald, 1998; Vertes, 2004). Although many Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 34 Giustino and Maren PFC and fear have proposed that IL projections to the ITCs gate CeA output it was shown that these animals were capable of maintaining (Royer et al., 1999; Royer and Paré, 2002; Likhtik et al., 2005), long-term fear, suggesting that dmPFC is not necessary for recent data challenge this possibility (Cassell and Wright, 1986; memory formation and retention or fear expression (Holson, Gutman et al., 2012; Pinard et al., 2012; Strobel et al., 2015). 1986). More recent work, however, has shown that pre- Pinard et al. (2012) have suggested that if this indeed is the training ACC lesions impair fear acquisition, while leaving pathway mediating fear inhibition, it must work via sparse fear expression intact in laboratory rats, although this deficit connections. These weak connections may partially explain could be overcome with additional training (Bissière et al., why extinction learning is not always robust and prone to 2008). In a separate study, Morgan et al. (1993) demonstrated relapse. Similar results using diffusion tensor imaging and that pre-conditioning mPFC lesions (encompassing ACC, PL, structural tract-tracing techniques in mice further demonstrate and IL) did not have an appreciable effect on the rate of largely indistinguishable amygdalar projections from PL and acquisition or level of fear expression to either context or IL (Gutman et al., 2012), although little is known about the cued fear conditioning. However, these animals took longer to functional aspects of PL innervation of the ITCs. One possibility reach extinction criterion, suggesting that mPFC neural activity is that IL mediated excitation of the ITCs is disynaptic, acting plays a role in extinction learning (Morgan et al., 1993). In through the BLA (Strobel et al., 2015). In addition, PL and IL a follow up study, selective PL lesions (damage was mainly have direct projections the PAG (Hardy and Leichnetz, 1981; restricted to dorsal PL) produced a general increase in both Beitz, 1982; Sesack et al., 1989; Floyd et al., 2000; Vianna cued and context fear during acquisition and extinction phases, and Brandão, 2003; Hoover and Vertes, 2007). Floyd et al. suggesting that dmPFC lesions yield a general increase in fear (2000) have suggested that rostral PL/IL preferentially innervate (Morgan and LeDoux, 1995). The authors suggest that these the ventrolateral PAG, whereas more caudal portions of PL/IL findings revealed a differential contribution of PL vs. IL to innervate the dorsolateral PAG. It remains possible that mPFC the expression of conditioned fear. However, based on the projections can bypass the amygdala to directly influence freezing extent of the lesions presented in each study, an alternative behavior. In summary, recent anatomical evidence suggests that interpretation is that behavioral differences reflected gross PL and IL display overlapping connections, especially to the differences in functions mediated by the dorsal-ventral axis amygdala and very weakly innervate the ITCs. The majority of mPFC and not specifically PL vs. IL. In support of this, of these findings are from behaviorally naïve animals however. some studies have reported decreased freezing and differential It would be advantageous to explore the functional outcome cardiovascular responses to a CS as a function of the dorsal- of these overlapping projections throughout stages of aversive ventral extent of mPFC lesions, suggesting that the functional learning. contribution of mPFC may differ along this axis rather than being exclusively confined to PL vs. IL (Frysztak and Neafsey, 1991, mPFC Intrinsic Connectivity 1994). A key question in mPFC function revolves around cortico- On the basis that animals with mPFC damage display cortical interactions, which originate from superficial layers II extinction impairments (Morgan et al., 1993), a subsequent study and III (Hoover and Vertes, 2007). While this has not been sought to directly compare the effects of damage restricted to studied extensively in fear, in slice preparations IL has higher different mPFC subregions and better define their contribution frequency local field potential (LFP) components than PL, and to extinction learning. It was found that while vmPFC lesions these differ when the two regions are disconnected—implying (encompassing IL and to some extent PL) do not impair some level of functional connectivity regulating basal activity extinction learning per se, they disrupted extinction recall. (van Aerde et al., 2008). In addition, optogenetic activation of Importantly, this effect was not observed in sham operated IL inhibits PL pyramidal cells in vivo (Ji and Neugebauer, 2012). animals or animals with lesions that spared the majority of This feed-forward inhibition may be a necessary component of IL. The authors suggest that IL neural activity in particular is extinction learning, although this has not been tested. Difficulty involved in the consolidation of extinction learning (Quirk et al., arises when addressing these questions simply due to the physical 2000). proximity of PL and IL, and the trouble of restricting infusions Many of these studies have formed the basis for the proposed solely to one region. dichotomy of function in the mPFC in which PL regulates fear expression and IL fear suppression. However, these findings are EARLY EVIDENCE FOR A DIVISION largely discrepant in nature with reports indicating increases, OF LABOR decreases, or no changes in learning following mPFC damage. Moreover, of particular interest, Holson (1986) and Morgan and Lesion Studies LeDoux (1995) demonstrate that dorsal PL lesions produce a One of the first studies to examine the role of mPFC in generalized increase in fear expression, indicating that an intact defensive behaviors showed that damage to this structure had dorsal PL may actually function to suppress fear, which is at no effect on flight, biting or reactivity to handling in wild odds with the current view. In addition, while Quirk et al. (2000) rats, although these lesions primarily encompassed more dorsal suggest that IL neural activity is importantly involved in the regions than PL and IL (i.e., ACC; Divac et al., 1984). In contrast consolidation of extinction memories, similar experiments have to this report, dmPFC lesions (encompassing ACC/dorsal PL) in not replicated these effects insofar as mPFC lesions do not yield laboratory rats increased reactivity to an aversive stimulus and deficits in either conditioned inhibition or extinction learning Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 35 Giustino and Maren PFC and fear under some conditions (Gewirtz et al., 1997; Garcia et al., 2006). activation enhanced fear acquisition and expression (Bissière Thus, it appears the mPFC is not necessary for the formation et al., 2008). Interestingly, in a study in which rats were or retrieval of extinction memories under some circumstances trained in a contextual bi-conditional discrimination task (in and this may be partially influenced by factors such as the strain context A, one CS is paired with shock while a second CS is of the animals used in these experiments (Chang and Maren, not, and this contingency is reversed in a second context) PL 2010). inactivation interfered with both the encoding and expression As noted above, it has been shown that both behavioral of appropriate CS responding. This suggests that PL may and autonomic responses to a CS are differentially modulated integrate contextual information to inform both learning and as a function of the dorsal-ventral extent of mPFC damage responding to conditioned stimuli (Sharpe and Killcross, 2014). (Frysztak and Neafsey, 1991, 1994). These findings leave open Moreover, PL inactivation disrupts both recent and remote the possibility that cell populations with overlapping function contextual fear memories after brief memory retrieval, indicating exist in PL and IL. A more general interpretation of these lesion that PL signaling may be involved in reconsolidation. This studies may be that the observed functional differences are a reconsolidation blockade also prevented reinstatement, further product of the lesion technique and size. It is possible that the showing that PL activity may subserve the reactivation of fear behavioral effects reflect a shift in function along the dorsal- memories and contribute to their long-term maintenance (Stern ventral axis, although this may not be solely interpreted as a et al., 2014), expanding the role of PL in the fear circuit. In functional opposition between PL and IL. It is worth noting that summary, PL signaling appears to be a key component encoding PL shows changes in laminar organization and cytoarchitectonic the acquisition and expression of learned fears and this may vary features along this axis which transitions into IL (Heidbreder and based on specific task parameters. Groenewegen, 2003; Perez-Cruz et al., 2007; van de Werd et al., While the PL appears to be involved in the expression of 2010). Hippocampal input to the mPFC is not uniform along this fear, it is widely believed that IL is involved in the suppression axis (Jay et al., 1989; Jay and Witter, 1991) and these differences of fear during extinction learning and retrieval. IL inactivation may influence the behavioral outcome of localized damage. increases freezing to conditioned tones while impairing within- Overall, despite the controversies around the conclusions one session extinction and retrieval in both rats and mice (Sierra- can draw from these lesion studies, they have been instrumental Mercado et al., 2006, 2011; Laurent and Westbrook, 2009; to our understanding of the fear circuit and have led to a rapid Morawska and Fendt, 2012; Sangha et al., 2014). Additionally, increase in additional studies examining the mPFC in fear. conditioned tones paired with IL electrical stimulation enable low levels of freezing in rats that had not been previously extinguished, suggesting that IL activation is sufficient to mimic Pharmacological and Microstimulation extinction training (Milad and Quirk, 2002; Milad et al., 2004). Studies Interestingly, IL stimulation paired with presentation of a CS in In an attempt to further characterize the role of PL and IL anesthetized rats mimics the behavioral experience of extinction in fear, many studies have used pharmacological agents to training (Park and Choi, 2010). These effects are frequency- temporarily inactivate the mPFC during behavioral tasks. These dependent: high-frequency IL stimulation immediately after methods allow for circuit manipulation at discrete time points. fear memory retrieval reduces freezing at a later time point, For example, intra-PL infusion of the Na+ channel blocker whereas low-frequency stimulation impairs extinction learning tetrodotoxin prior to fear conditioning does not disrupt the (Maroun et al., 2012; Shehadi and Maroun, 2013). This may acquisition of conditional fear, but reduces fear expression to reflect IL potentiation vs. depression with high- and low- a CS or context previously paired with shock (Corcoran and frequency stimulation, respectively. In line with these studies, Quirk, 2007). Consistent with PL activity being necessary for IL activation, via infusion of the GABA-A receptor antagonist fear expression, inactivation of PL, with the GABA-A receptor picrotoxin, rescues extinction learning in extinction-deficient agonist muscimol, prior to extinction training also impairs fear mice (Fitzgerald et al., 2014). Others have shown that IL expression (Laurent and Westbrook, 2009; Sierra-Mercado et al., activation prior to an extinction session dampens the expression 2011). However, this manipulation has no long-term effect on of fear (Chang and Maren, 2011) and subsequently enhances extinction recall, suggesting PL inactivation does not interfere extinction recall (Thompson et al., 2010; Chang and Maren, with the acquisition of extinction (Laurent and Westbrook, 2011). 2009; Sierra-Mercado et al., 2011). Collectively, these findings Extinction learning produces a labile suppression of fear that suggest that PL activity underlies fear expression, but not is susceptible to relapse when a previously extinguished cue learning per se. is presented outside the extinction context (i.e., fear renewal; There is some evidence to support the idea that PL signaling Bouton, 2000, 2002; Bouton et al., 2006; Hermans et al., plays a role in aversive learning, beyond its role in fear 2006; Maren et al., 2013; Vervliet et al., 2013; Goode and expression, however, and this may extend to more dorsal Maren, 2014). This process is likely mediated by hippocampal- regions, including ACC. For example, PL microstimulation prefrontal circuits (Corcoran and Maren, 2001; Maren et al., increases fear expression while preventing successful extinction 2013). In addition, the timing of extinction trials relative (Vidal-Gonzalez et al., 2006), implying that PL signaling shunts to conditioning is also a key factor governing the long- extinction learning by elevating fear. In addition, transient term success of extinction training (Maren and Chang, 2006; inactivation of rostral ACC impairs fear learning whereas Myers et al., 2006; Maren, 2014). Extinction trials delivered Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 36 Giustino and Maren PFC and fear soon after conditioning often result in a failure to retain the idea that mPFC IEG expression may be partly modulated this memory long-term, which may reflect impaired mPFC by context, PL and IL exhibited opposing patterns of Fos signaling. Using an immediate extinction paradigm, intra-IL expression in a renewal paradigm in which an extinguished picrotoxin abolished conditioned freezing during extinction CS is presented in the extinction context (low fear) and in a training and promoted a faster reduction of conditioned different context (high fear). PL showed robust increases in responding the following day (Chang and Maren, 2011). In Fos expression during fear renewal whereas presentation of the a separate study, IL electrical stimulation paired with CS extinguished CS in the extinction context induced increased presentations limited the spontaneous recovery of fear the Fos expression in IL (Knapska and Maren, 2009). Similarly, following day, rescuing the immediate extinction deficit (Kim in a separate set of studies, levels of Zif268 were greater in et al., 2010). Collectively, these findings support the idea PL upon contextual fear recall (Stern et al., 2014), whereas that IL signaling promotes extinction learning and suppresses increased IL Zif268 expression has been reported in animals conditional fear. recalling a remote cued fear memory; this effect was not Overall, the findings discussed above generally lend support to observed in PL (Fitzgerald et al., 2015b). In addition, prefrontal a division of labor in which PL and IL are functionally opposed. levels of Arc mRNA expression show context specificity, with However, due to the physical proximity of PL and IL, it is higher levels in BA, LA and IL of extinguished rats (Orsini difficult to restrict infusions or electrical stimulation to only et al., 2013). Further supporting a role for IL in extinction one subdivision. Moreover, pharmacological manipulations lack learning, extinction-deficient mice display reduced Fos and cell specificity, affecting both principal cells and interneurons in Zif268 expression in IL, implying that reduced IL activity may a similar fashion. Additionally, electrical stimulation results in underlie this behavioral deficit (Hefner et al., 2008). In summary, ortho- and antidromic signaling which clouds the interpretation these data suggest that PL and IL IEG expression displays of directionality and localization of these effects. Given these context specificity with PL being primarily activated in a high experimental limitations, it is not surprising that there is evidence fear state whereas IL is activated in a low fear state. These that challenges the dichotomous role of PL and IL in fear findings indicate that the mPFC may integrate contextual cues expression and suppression, respectively. For example, if PL to process the meaning of the CS and inform conditioned activity underlies fear expression to associative stimuli, then responding. PL activation at any time point of associative fear learning The above IEG studies mainly suggest opposing roles for PL should increase freezing behavior whereas inactivation should and IL in the expression or suppression of fear, respectively, impair freezing. Curiously, PL inactivation does not affect while having little influence on learning per se. However, it freezing under some conditions (Bravo-Rivera et al., 2014; has been shown that both PL and IL exhibit increased levels Sharpe and Killcross, 2015) suggesting that ongoing freezing of Fos after conditioning, implying that PL and IL activity behavior is not solely dependent on PL activity and that other may underlie new learning. Interestingly, conditioning induced neural structures can compensate in its absence. Similarly, greater activation of PL and IL compared to extinction learning, if IL is a necessary component of fear suppression, then and Fos expression following each session was indistinguishable IL activation should serve to promote extinction learning between brain regions (Morrow et al., 1999; Herry and Mons, and subsequently reduce fear responding while inactivation 2004). This conditioning-induced increase in Fos expression may should have the opposite effect. Interestingly, some studies partly be a response to the unconditioned footshock, rather than have reported facilitated extinction learning with IL inactivation associative learning per se. However, an antisense oligonucleotide in both aversive and appetitive conditions (Akirav et al., against c-fos mRNA, injected simultaneously into both PL and 2006; Mendoza et al., 2015) making it possible that cell IL 12 h prior to conditioning, attenuated fear responses during populations within IL exist that can bi-directionally modulate an extinction session (Morrow et al., 1999). Thus, PL and IL extinction learning. These findings challenge existing models appear to be involved in the acquisition of conditioned fear and of PL and IL function in fear and leave open the possibility to a lesser extent, are activated following extinction learning. that there is some functional overlap between PL and IL that It is worth noting that this effect was seen with simultaneous allows one structure to compensate for the other under some manipulations to PL and IL (Morrow et al., 1999), implying conditions. that there is some level of functional overlap between the two regions. However, the authors did not manipulate PL or IL mPFC NEURAL CORRELATES OF FEAR alone, leaving the possibility that the decreased fear responding AND EXTINCTION during extinction may be preferentially driven by one of these two regions. In support of the idea that PL and IL may covary Immediate Early Genes at times, a separate study has shown that Fos and Zif268 Immediate early genes (IEGs) such as c-fos, Arc and Zif268 expression were similar after the retrieval of both a recent are activated in response to cellular stimulation, providing an and remote contextual fear memory (Frankland et al., 2004). indirect measure of neural activation and have been implicated These studies suggest that PL and IL can fluctuate similarly in learning and memory (Davis et al., 2003; Plath et al., during the acquisition, extinction and expression of conditional 2006). Interestingly, patterns of mPFC gene expression may fear. be context-dependent, possibly as a result of feed-forward As mentioned previously, animals subjected to extinction information being integrated from the hippocampus. In line with trials soon after conditioning often spontaneously recover high Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 37 Giustino and Maren PFC and fear levels of freezing the following day which may result from output (Li et al., 2011). Recent evidence, however, has suggested impaired mPFC function (Maren and Chang, 2006; Maren, that IL exhibits low levels of connectivity to the ITCs (Gutman 2014). In support of this hypothesis, rats extinguished 15 min et al., 2012; Pinard et al., 2012; Strobel et al., 2015) bringing after conditioning displayed a general decrease in Fos expression question to this proposed mechanism of extinction learning. in both PL and IL when compared to animals extinguished These findings have prompted an updated hypothesis that posits 24 h after conditioning (Kim et al., 2010; but see Stafford disynaptic projections from IL to the ITCs via the BLA serve et al., 2013). This suggests that some basal level of activity in to engage inhibitory processes involved in extinction (Strobel both regions is necessary for extinction learning. Additionally, et al., 2015). These disynaptic projections may be necessary others have shown that the spontaneous recovery of fear after for IL to overcome the inter-ITC inhibitory network in order extinction is associated with reduced Fos and Zif268 induction to promote extinction learning and reduce fear. Overall, these in both PL and IL of rats (Herry and Mons, 2004). Collectively, data support a role for IL excitability in successful extinction these studies further demonstrate that neuronal activity in PL learning. and IL are positively correlated under some conditions. The Given that the PL has been implicated in the acquisition and observed similarities may stem from similar synaptic inputs expression of conditioned fear, it follows that this should be and cortico-cortical interactions, although this remains an open reflected in single-unit activity in awake, behaving animals. It has question. been reported that sustained spike firing in the PL during aversive CSs correlates with ongoing freezing behavior (Burgos-Robles et al., 2009). Consistent with this, extinction-deficient 129/S1 Electrophysiology mice show elevated CS-evoked responses in PL, although this Single-Unit Recordings effect was also mirrored in IL (Fitzgerald et al., 2014). In contrast, Electrophysiological methods also provide insight into the others have reported that the expression of freezing behavior function of PL and IL neurons during the conditioning and is associated with robust CS-evoked responses in IL (Chang extinction of fear. Using in vivo single-unit recordings in et al., 2010; Fitzgerald et al., 2015b). Interestingly, Chang et al. awake, behaving rats, Milad and Quirk (2002) provided the (2010) also found that, in contrast to IL, CS-evoked PL activity first evidence that CS-evoked responses in IL correlate with was attenuated during fear expression, revealing a reciprocal successful extinction recall. This study showed that IL neurons relationship between PL and IL activity in the opposite direction preferentially responded to a CS when rats successfully retrieve to that predicted by prevailing models. In a recent study, we an extinction memory, but not during conditioning or the examined the pattern of spontaneous firing in simultaneously initial extinction session. This effect was specific to IL, as it recorded PL and IL neurons immediately after fear conditioning was not seen in neurons recorded in PL or the medial orbital (Fitzgerald et al., 2015a). In this post-conditioning period, rats cortex. The authors suggested that extinction consolidation exhibit sustained and high levels of fear that persisted for the may enhance IL activity and this subsequently reduces fear duration of the 1 h recording session. During this transition from the following day (Milad and Quirk, 2002). In agreement with a low-fear to a high-fear state, spontaneous firing rates some this, successful extinction correlates with high-frequency IL neurons in PL and IL were transiently excited in the minutes bursting (Burgos-Robles et al., 2007), and under conditions in following conditioning, but returned to basal levels soon after, which extinction fails (i.e., immediate extinction) IL bursting despite ongoing freezing behavior. Interestingly, spontaneous is diminished (Chang et al., 2010). These in vivo findings have firing rates of other neurons in IL were persistently suppressed been complemented by in vitro studies, which have also provided over the duration of the post-conditioning period (Fitzgerald support that IL signaling is altered upon extinction learning. et al., 2015a). Collectively, these data suggest that PL spike For example, in slice preparations, the intrinsic excitability of firing alone is unlikely to mediate sustained freezing behavior; IL neurons was decreased for up to 4 h after conditioning indeed, the expression of fear may be due, at least in part, to and this can be reversed with extinction training (Santini suppression of IL activity (Chang et al., 2010; Fitzgerald et al., et al., 2008; Cruz et al., 2014). This reversal suggests the 2015a). acquisition of extinction induces a ramping upward of spike Interestingly, similar to IEG studies, there is evidence for firing during the consolidation phase, although this inhibition positively correlated single-unit activity in PL and IL after returned in rats that spontaneously recovered fear (Cruz et al., the conditioning or extinction of fear. For example, during 2014). the expression of conditioned fear (high fear), spontaneous How extinction learning and recall are precisely computed firing rates are suppressed in both IL and PL, although at the circuit level is not fully understood, although this was IL suppression was more robust (Fitzgerald et al., 2015a). previously thought to be mediated by a direct IL→ITC pathway Additionally, Holmes et al. (2012) reported no differences in (Royer et al., 1999; Royer and Paré, 2002; Pape and Pare, 2010; PL vs. IL CS-evoked responses throughout extinction learning Duvarci and Pare, 2014). In support of this idea, the ITCs as well as extinction retrieval. In a separate study, comparable are strongly responsive to IL stimulation in anesthetized rats conditioning-induced increases in CS-evoked activity were (Amir et al., 2011). Interestingly, at basal levels of activity, observed in the PL and IL of extinction-deficient 129/S1 mice ITC neurons actively inhibit each other; however, with brief (Fitzgerald et al., 2014). This provides further evidence that IL stimulation the ITCs display increased firing rates which PL and IL may covary in their response properties at the diminishes CeA output, a potential mechanism for reduced fear single-neuron level, at least under some conditions. Other Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 38 Giustino and Maren PFC and fear experiments have found that PL and IL neurons exhibit similar OPTOGENETICS AND CHEMOGENETICS: firing patterns in response to CSs or contexts associated with CAUSAL MECHANISMS OF FEAR shock (Baeg et al., 2001) or in relation to the types of behavioral responses animals emit (e.g., freeze or move) in response to The acquisition and retrieval of memories depend on complex aversive CSs (Halladay and Blair, 2015). Hence, single-unit patterns of neural activity from distinct neuronal populations activity in IL and PL fluctuates similarly under a number of defined by their genetic markers. Whereas much of the conditions, which is not surprising given their similar afferent above evidence convincingly demonstrates a role of mPFC inputs. in fear, electrophysiology is only correlative and inactivation methods lack cellular specificity. As such, the fear-related causal Local Field Potentials mechanisms of precise neural activity and the contribution In addition to single-unit recordings, LFP recordings suggest a of different cell types remain largely unknown. Optogenetics high degree of synchrony between the mPFC, amygdala, and and chemogenetics are virally-mediated techniques allowing for hippocampus throughout different stages of aversive learning. cell and circuit specific manipulations to selectively excite or LFPs are generated by finely tuned synaptic input patterns, suppress specific neuronal populations. Briefly, optogenetics and recent studies have focused on LFPs at the circuit level requires the expression of exogenous light-sensitive ion channels as a mechanism by which distant brain regions effectively to modulate neuronal activity with high temporal precision communicate. The coupling and synchronization of brain (Boyden et al., 2005; Fenno et al., 2011). One chemogenetic regions within the fear circuit are likely involved in memory approach makes use of Designer Receptors Exclusively Activated formation and retrieval. Importantly, theta oscillations act to by Designer Drugs (DREADDs), which are synthetic G- coordinate regional synchronization, providing a means of protein coupled receptors that respond selectively to the timely and efficient transmission of information. For example, systemic injection of an inert ligand, clozapine N-oxide the BLA and mPFC show enhanced theta synchrony during (CNO; Dong et al., 2010; Urban and Roth, 2015). These sleep after conditioning, which plays a role in memory technologies provide an in vivo mechanism to control cellular consolidation (Popa et al., 2010). In line with this, increased physiology in intact neural circuits and delineate the causal BLA-mPFC theta synchrony has been observed in animals contribution of specific neuronal subtypes to learning and that successfully learned to differentiate between safe and memory. aversive conditions (Likhtik et al., 2014). During learned Recently, optogenetic methods have been used to explore safety, BLA firing activity was entrained to theta input from plasticity in prefrontal projections to the amygdala after mPFC, suggesting that the BLA is selectively tuned to mPFC fear conditioning. Combining optogenetics and ex vivo input, a potential mechanism underlying memory recall and electrophysiology, Arruda-Carvalho and Clem (2014) have thus behavioral responding (Likhtik et al., 2014). mPFC shown that in behaviorally naïve mice, the synaptic connectivity projections excite BLA neurons, indicating that inhibition of of IL and PL projections onto BLA principal neurons were CeM output may be mediated by an active gating mechanism similar. However, fear conditioning led to a decrease in downstream of BLA (Likhtik et al., 2005). The directionality inhibitory-excitatory balance in PL, but not IL. These data of this effect supports the role of mPFC in regulating suggest that a PL→BLA pathway is crucial for encoding fear amygdala activity, although it is well known that amygdala memories and may be engaged when encountering the CS at a output influences mPFC function as well (Senn et al., 2014) later time point to promote a high fear state (Arruda-Carvalho and inactivation of BLA decreases PL activity (Sotres-Bayon and Clem, 2014). et al., 2012). One study has shown that in male mice, As discussed above, extinction learning is thought to involve PL and IL display opposing patterns of theta power across feed-forward inhibition that blunts CeA output via the ITCs extinction, which may reflect new learning. Given their physical (Royer et al., 1999), with IL synaptic transmission regulating proximity and similar input it is somewhat surprising that this pathway via the BLA. The direct role of mPFC, however, LFPs would be drastically different between the two regions. had not previously been tested, including differences between Interestingly, this effect was not seen in females as they PL and IL. One possibility is that, while weak in number, displayed heightened freezing and persistently increased mPFC direct IL projections to the ITCs increase in strength with theta in both PL and IL (Fenton et al., 2014). In addition, extinction training to inhibit the CeA, or IL projections to the PL gamma power is elevated in extinction-deficient mice BLA are modulated which ultimately influences ITC output. compared to mice that successfully extinguished (Fitzgerald If so, the synaptic strength of this pathway may be causally et al., 2014). Moreover, other work has reported theta synchrony linked to both the acquisition and recall of extinction. Using of an expanded network involving CA1-LA-IL during the ex vivo electrophysiology and the excitatory optogenetic virus retrieval of conditioned fear. Theta synchronization declined channelrhodopsin restricted to principal cells under control of with extinction training, but was partially restored upon the CAMKII promoter, Cho et al. (2013) demonstrated that extinction recall (Lesting et al., 2011). In summary, LFPs may extinction learning reduced synaptic efficacy in BLA projecting importantly affect the fear circuit at a global level and theta mPFC neurons. Interestingly, mPFC synaptic transmission to interactions might provide a mechanism for the fine-tuned ITCs was unchanged and thus the overall balance in the mPFC- organization of neural pathways underlying memory formation BLA pathway shifted towards inhibition following extinction. and recall. This effect may stem from monosynaptic connections to BLA Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2015 | Volume 9 | Article 298 | 39