Short-Term Fasting Specifically Impairs Long-Term Memory but not Short-Term Memory of Cued and Context Fear

To investigate the role of short-term fasting on the acquisition of conditioned fear, male C57BL/6N mice were fasted overnight for 16 h before fear conditioning and their performance was compared with non-fasted littermates (Figure 1a). Short-term fasting resulted in a mean reduction of body weight by 17.4% (Supplementary Figure 1). Acquisition of conditioned fear was unchanged in fasted and non-fasted mice (Figure 1b, two-way ANOVA for repeated measurements: time—F (4,80) =60.74, P<0.0001, treatment—F (1,20) =0.24, P>0.05, interaction F (4,80) =2.51, P>0.05). Context fear memory, however (Figure 1c, 3 min context testing on day 2: t (20) =3.21, P<0.01), tested 24 h after fear acquisition and refeeding by exposing the mice to the original conditioning chamber (context A), was significantly reduced, whereas context fear acquisition was unchanged (Supplementary Figure 2). Owing to reduced context fear expression, context extinction appeared to be facilitated in fasted mice compared with fed mice (Supplementary Figure 2, two-way ANOVA for repeated measurements: time—F (14,280) =6.77, P<0.0001, treatment—F (1,20) =9.84, P<0.01, interaction F (14,280) =0.45, P>0.05). Similarly, freezing to the CS under fed conditions, tested 48 h after fear acquisition by exposing mice to the auditory stimulus alone (CS test, long-term memory (LTM); Figure 1a+d) in a different chamber (context B), was significantly lower in mice that were fasted before fear acquisition (Figure 1d; t (20) =3.20, P<0.01). However, short-term memory (STM) tested (Figure 1a+d, CS test STM, under fed conditions) in separate groups of mice (Figure 1a) was similar in fasted and non-fasted mice, as demonstrated by CS-induced freezing 30 and 150 min after fear acquisition (Figure 1d; t (10) =0.67, P>0.05 and t (10) =1.26, P>0.05, respectively). As the effect of fasting on fear memory developed slowly, we performed fear acquisition after 16 h of fasting and 24 h of refeeding, further demonstrating that fasting did not affect fear acquisition (Supplementary Figure 3). Collectively, these data suggest impaired consolidation or recall of conditioned fear in mice that were fasted before fear acquisition, whereas learning and STM remain unaffected.

Figure 1 Acute short-term fasting before fear conditioning inhibits formation of context- and conditioned stimulus (CS)-induced long-term fear memory (LTM) while not affecting short-term memory (STM). (a) Fasting started 16 h before and continued during fear acquisition. All mice had access to food ad libitum after fear acquisition and before and during fear testing. (b) Both fasted and fed mice exhibited similar baseline freezing and acquisition of conditioned fear. (c) Reduced context freezing of mice that were fasted before and during fear acquisition. (d) Mice that were fasted before and during fear acquisition displayed reduced CS-induced freezing 48 h after fear conditioning, whereas STM tested 30 and 150 min after fear acquisition was similar to controls (repeated two-way analysis of variance (ANOVA) for acquisition, Student’s t-test for context and CS testing, **P<0.01; LTM—food, n=11, fasting: n=11; STM 30 min—food: n=6, fasting: n=6; STM 150 min: food: n=6, fasting: n=6). PowerPoint slide Full size image

Short-Term Fasting Facilitates Extinction Learning and Promotes Extinction Recall

To understand if fasting-induced inhibition of fear depends on memory consolidation and to investigate the effect of fasting on fear extinction, a separate group of mice was tested for fear recall after 16 h of fasting. Fasting was initiated here either immediately or 24 h after fear acquisition (Figure 2a). There was no change in CS-induced freezing during fear recall in mice that were fasted immediately or 24 h after fear acquisition compared with non-fasted controls, suggesting that fasting induced-changes develop slowly, over time (eg, 16 h), and have to be present within the consolidation window to affect long-term fear memory. Interestingly, freezing to the CS upon fear recall was similar in animals that were fasted after fear acquisition and non-fasted controls (Figure 2c; t (16) =0.38, P>0.05), suggesting equal expression of fear in fasted and non-fasted mice. Fear extinction learning, however, was significantly facilitated in fasted mice (Figure 2d; two-way ANOVA for repeated measurements, time—F (24,432) =9.03, P<0.0001, treatment—F (1,18) =5.44, P<0.05 and interaction F (24,432) =3.55, P<0.0001). More importantly, extinction recall, tested under fed conditions 24 h after fear extinction training, by exposing the mice to 5 CS in context B was still reduced in those mice that were fasted before extinction learning (Figure 2e; t (16) =3.61, P<0.01). Taken together, these data indicate that 16 h of acute fasting does not alter learning in general, but rather modulates fear memory by specifically influencing the emotional valance of learning processes. Thus, fasting inhibits the consolidation of an acquired fear memory but promotes the acquisition and consolidation of fear extinction.

Figure 2 Short-term fasting before fear extinction improves extinction learning. (a) Following fear conditioning, mice were fasted for 16 h before and during extinction training. Extinction recall was tested 24 h later with food available ad libitum. (b) Following fear acquisition, mice were divided into two equal groups, one that was fasted and one with food available. (c) No difference in conditioned stimulus (CS) induced freezing was observed between fasted and non-fasted mice, (d) but facilitated fear extinction in mice that were fasted 16 h before and during fear extinction learning, and (e) reduced CS-induced freezing in extinction recall of mice that were fasted before and during extinction learning (repeated two-way analysis of variance (ANOVA) for acquisition and extinction learning, Student’s t-test for CS-induced freezing and extinction recall testing, *P<0.05, **P<0.01; Food: n=10, fasting: n=10). PowerPoint slide Full size image

Genetic Deletion of the Y4 Receptor Reduces Appetite and Impairs Fear Extinction

If survival circuits, such as feeding and fear, were indeed influencing each other, we hypothesized that mice with altered feeding behavior or genetic ablation of feeding-related genes would also display specific changes in fear extinction behavior (Gutman et al, 2008; Verma et al, 2012). Y4 receptors are expressed in the CNS and are activated by PP that is released from the pancreas in response to feeding. Interestingly, Y4KO mice display decreased body weight and reduced food intake, both suggesting chronic suppression of the hunger circuit (Lin et al, 2004; Sainsbury et al, 2002). To investigate the relation of a feeding-related gene and satiety to fear extinction, we subjected Y4KO mice to Pavolvian fear conditioning (Figure 3a). Acquisition (Figure 3b, two-way ANOVA for repeated measurements, time—F (4,48) =27.29, P<0.0001, genotype—F (1,12) =2.54, P>0.05, interaction F (4,48) =1.28, P>0.05) and recall of conditioned fear (Figure 3c; t (13) =0.54, P>0.05) were unchanged in Y4KO mice compared with controls. Fear extinction, however, was significantly impaired in Y4KO mice (Figure 3d and e; two-way ANOVA for repeated measurements, time—F (14,182) =0.82, P>0.05, genotype—F (1,13) =26.90, P<0.001, interaction F (14,182) =3.08, P<0.001, and fear recall—t (13) =3.86, P<0.01), suggesting that genetic alterations in the feeding circuit considerably alter fear behavior. Sensitivity to the electric foot-shock was not different from controls (Supplementary Figure 4). These results suggest that modification of the feeding circuit has a significant impact on fear processing. In particular, dysregulated appetite correlated with impaired fear extinction.

Figure 3 Impaired fear extinction in Y4 receptor knockout (KO) mice was rescued by repeated fasting episodes before extinction learning. (a) Y4KO mice and wild-type controls were subjected to fear acquisition, conditioned stimulus (CS) testing 24 h later, and to a total of four extinction sessions on days 2–6, followed by extinction recall on day 7. (b) No difference in fear acquisition and (c) fear recall, but (d) impaired extinction learning (note the apparent reduction of freezing levels in Y4KO mice during CS1 and 3, that was, however, due to jumping behavior, thus probably indicating an increased stress reaction) and (e) extinction recall in Y4KO mice compared with wild-type controls. (f) After fear acquisition, one group of Y4KO mice was subjected to repeated cycles of fasting before and during extinction learning followed by extinction recall under fed conditions. (g) Following fear acquisition, Y4KO mice were divided into two equal groups, one that was fasted before extinction training and one that was not, (h) no difference in fear expression between fasted and non-fasted Y4KO mice, (i) rescued fear extinction in fasted Y4KO mice compared with fed Y4KO controls, (j) successive reduction of freezing in extinction recall on testing days 3, 5, and 7 tested under fed conditions and reduced reinstatement on day 21 in Y4KO mice that were fasted before extinction training, suggesting permanent suppression of fear (repeated two-way analysis of variance (ANOVA) for acquisition and extinction learning, Student’s t-test for CS-induced freezing and extinction recall and reinstatement testing, *P<0.05, **P<0.01, ***P<0.001; WT vs Y4KO—WT: n=9, Y4KO: n=6 and Y4KO fasted vs non-fasted—Y4KO fasting: n=8, Y4KO food: n=8). KO, knock out. PowerPoint slide Full size image

Short-Term Fasting Rescues Impaired Fear Extinction in Y4KO Mice

Next, we tried to rescue the specifically impaired fear extinction in Y4KO mice by subjecting them to three cycles of extinction-recall sessions, each consisting of 16 h fasting before extinction training followed by extinction recall tested under fed conditions 24 h later (Figure 3f and Supplementary Figure 1 for reduction of body weight). CS-induced freezing in context B on the testing day was similar in fasted and fed Y4KO mice (Figure 3h; t (16) =0.20, P>0.05), indicating equal acquisition and expression of conditioned fear. Extinction learning, however, was significantly enhanced in Y4KO mice that were subjected to 16 h fasting compared with fed Y4KO mice (Figure 3i; two-way ANOVA for repeated measurements, time—F (24,168) =0.72, P>0.05, treatment—F (1,7) =7.46, P<0.05, interaction F (7,168)= 1.01, P>0.05). More importantly, this extinction memory was preserved upon refeeding, as demonstrated by the reduced freezing behavior during extinction recall in context B (Figure 3j; t (7) =1.63, P>0.05; t (7) =2.55, P<0.05; t (7) =4.84, P<0.01 for extinction recalls 1, 2, and 3, respectively), suggesting that impaired fear extinction can be rescued by modulation of the feeding circuit.

To investigate whether this reduction of fear was permanent, we subjected these mice 2 weeks after the last extinction trial to a reinstatement paradigm, consisting of one unsignaled foot-shock in context A and testing of CS-induced freezing in context B on days 20 and 21, respectively (Figure 3f). Importantly, CS-induced freezing during reinstatement testing was still reduced in Y4KO mice that were repetitively fasted before extinction learning (Figure 3j; t (7) =3.89, P<0.01), suggesting a long-lasting, stress-resistant suppression of fear.

Short-Term Fasting Specifically Activates Medial Intercalated Neurons and Facilitates Feed-Forward Inhibition from the Basolateral to the Centromedial Amygdala

We next investigated the underlying synaptic correlates linking feeding and fear extinction circuits. Following fear acquisition, Y4KO mice were fasted for 16 h, subjected to extinction training, and brains were processed for immunohistochemistry 90 min after the end of fear extinction training. Compared with non-fasted Y4KO mice (Figure 4a–c), expression of the immediate-early gene cFos was increased in those Y4KO mice that were fasted before extinction training (Figure 4d–f), specifically in the medial intercalated cells (mITCs), a brain nucleus associated with fear extinction (Busti et al, 2011; Likhtik et al, 2008) (Figure 4g; t (14) =2.60, P<0.05). Thus, short-term fasting activates specific neuronal populations in extinction-related brain areas.

Figure 4 Fasting in Y4KO mice results in activation of the medial intercalated cell (mITC) and enhanced basolateral amygdala (BLA) to centromedial amygdala (CEm) feed-forward inhibition. (a–c) Expression of immediate-early gene cFos in the ITC of a fasted Y4KO compared with (d–f), a non-fasted Y4KO control reveals (g) increased activation of ITC neurons, (h) experimental setup for ex vivo electrophysiology indicating home-cage controls, wild-type (WT) and Y4KO undergoing fear acquisition on day 1, extinction on day 2, and electrophysiology on day 3 under fed conditions and experimental group with a 16 h fasting period before and during extinction training, (i) enhanced feed-forward inhibition from BLA to CEm via mITC in WT mice after successful fear extinction, (j) lack of increased feed-forward inhibition in fed Y4KO after extinction training corresponding to impaired fear extinction and (k) rescue of impaired fear extinction in Y4KO mice by fasting facilitates enhanced feed-forward inhibition between BLA and CEm. (l) Example traces from WT and Y4KO mice of the individual groups with increasing stimulation intensities (n=WT home cage 4 mice, 12 cells; WT food+Ext 4 mice, 15 cells; Y4KO home cage 5 mice, 13 cells; Y4KO food+Ext 4 mice, 14 cells; Y4KO fasting+Ext 4 mice, 12 cells; *P<0.05, two-way analysis of variance (ANOVA) for repeated measurements for electrophysiology, and Y4KO food: n=8, Y4KO fasting: n=8; Student’s t test for c-Fos immunohistochemistry). KO, knock out. PowerPoint slide Full size image

As shown previously in rats, fear extinction results in enhanced feed-forward inhibition from the BLA to the CEm, mediated by increased activity of mITC neurons (Amano et al, 2010). To determine whether facilitated fear extinction of fasted mice corresponded to alterations in synaptic neurotransmission, we performed whole-cell patch-clamp recordings to measure BLA to CEm feed-forward inhibition in acute amygdala slices of mice 24 h after two extinction trainings. Stimulation of the BLA at intensities ranging from 100 to 500 μA consistently evoked an IPSP in CEm neurons with an initial, brief EPSP component (Figure 4l). Compared with untrained C57BL/6N mice (WT home cage), the amplitude of IPSPs was increased 24 h after successful fear extinction (Figure 4i; two-way ANOVA F (2,36) =4.93, P<0.01). Bath application of the AMPA receptor antagonist, DNQX, abolished both the IPSP and EPSP (Supplementary Figure 5A), whereas application of GABA receptor antagonist, picrotoxin, completely blocked the IPSP. These data confirm that fear extinction results in enhanced feed-forward inhibition in an amygdala microcircuit connecting the BLA with the CEm.