Past experience shapes the neural circuits recruited for future learning

Abstract

Experimental research controls for past experience, yet prior experience influences how we learn. Here, we tested whether we could recruit a neural population that usually encodes rewards to encode aversive events. Specifically, we found that GABAergic neurons in the lateral hypothalamus (LH) were not involved in learning about fear in naïve rats. However, if these rats had prior experience with rewards, LH GABAergic neurons became important for learning about fear. Interestingly, inhibition of these neurons paradoxically enhanced learning about neutral sensory information, regardless of prior experience, suggesting that LH GABAergic neurons normally oppose learning about irrelevant information. These experiments suggest that prior experience shapes the neural circuits recruited for future learning in a highly specific manner, reopening the neural boundaries we have drawn for learning of particular types of information from work in naïve subjects.

Extended Data Fig. 1. Extended data 1. Histological verification of Cre-dependent NpHR and eYFP in GAD+ neurons and fiber placement in the LH for all experiments.

Top row: Unilateral representation of the bilateral viral expression in the LH, −2mm to −3mm posterior to bregma. Bottom row: Approximate location of fiber tips in LH, indicated by black squares, −2mm to −3mm posterior to bregma.

Extended Data Fig. 2. Extended data 2. Rats in our NpHR learners group showed a persistent increase in conditioned fear to the contextual cues, which extinguished before tone presentations in the extinction test.

During conditioning, our NpHR learners group showed high levels of fear to the background contextual cues (left; see Figure 3 in main text for more information). To reduce these levels of context fear before the test, 24 hours after conditioning, rats received a context extinction session where they were placed in the experimental chambers without any stimuli. Here, we found that our NpHR learners group maintained higher level of context fear relative to our eYFP learners group (middle). This context extinction was effective in reducing contextual fear as all rats showed low levels of freezing at the beginning of the next test session, where we presented the tone under extinction to examine fear that had acquired to these stimuli (right). A mixed-design repeated-measures ANOVA on levels of freezing to the contextual cues across the context and tone extinction sessions showed a main effect of time (F_14,140=4.614, p=0.000), and a significant session x time x group interaction (F_14,140=1.697, p=0.032). This interaction was owed to a between-group difference in freezing during context extinction that revealed itself most prominently towards the end of the scoring period (group: F_1,10=5.939, p=0.035), that was not seen in the tone extinction test (group: F_1,10=0.007, p=1.000). Further, there was a significant difference in freezing exhibited by the NpHR group when comparing the context extinction session with the tone test (n=6 rats; F_1,10=8.071, p=0.018), that was not present in the eYFP control group (n=6 rats; F_1,10=1.161, p=0.307). Finally, a one-way ANOVA showed there was no between-group difference in freezing to the context immediately before tone presentations in the tone test after context extinction had taken place (F_1,10=1.943, p=0.194). Error bars = SEM.

Extended Data Fig. 3. Extended data 3. Responding during conditioning in the second-order conditioning experiment (see Figure 4 main text).

Rates of responding are represented as time spent in the food port (%; ±SEM). Rats (n=12 eYFP; n=8 NpHR) learnt to distinguish between A2 and B2 during conditioning, with no difference in the rates of learning between groups (note: patch cords were placed on rats in session 6 of conditioning to habituate them to the cords prior to pairings of A1→A2 and B1→B2, which is why there is a dip in responding). A repeated-measures ANOVA revealed a main effect of stimulus (F_1,18=12.383, p=0.002) and session (session: F_6,108=10.799, p=0.424), with no interactions by group (stimulus x group: F_1,18=1.151, p=0.298; session x group: F_6,108=1.008, p=0.424; stimulus x session: F_6,108=5.440, p=0.000; stimulus x session x group: F_6,108=0.233, p=0.965).

Extended Data Fig. 4. Extended data 4. Responding during conditioning in the sensory-preconditioning experiment (see Figure 5 main text).

Rates of responding are represented as time spent in the food port (%; ±SEM). Rats (n=12 eYFP, n=8 NpHR) learnt to distinguish between A2 and B2 during conditioning, with no difference in the rates or ultimate levels of learning between groups A repeated-measures ANOVA revealed a main effect of stimulus (F_1,18=3.553, p=0.076), and a stimulus x session interaction (F_2,36=8.281, p=0.001; stimulus × group: F_1,18=0.13, p=0.911), with follow-up comparisons, granted by the significant stimulus x session interaction, showing an increase in learning about A2 across sessions (F_2,17=8.860, p=0.002), that was not present with relation to B2 (F_2,17=1.953, p=0.172).

Figure 1.. Inhibition of LH GABA neurons was achieved by infusion of a Cre-dependent AAV virus carrying halorhodopsin (NpHR) into the LH of GAD-Cre rats.

Top panels indicate optogenetic technique used to inactivate neurons carrying the Cre-dependent NpHR virus. Rats were first infused with AAV5-Ef1α-DIO-NpHR- eYFP or AAV5-Ef1α-DIO-eYFP into the LH. During this surgery, 200-μm fiber optics were implanted above the LH. Bottom panel shows LH virus expression selective to GABAergic neurons . See Extended Data 1 for individual virus expression and fiber placement. Scale bar = 1mm.

Figure 2.. LH GABAergic neurons are necessary to encode fear memories after reward learning.

Responding is shown as mean level of freezing (%; ±SEM) Top: LH GABAergic neurons were inhibited by light (green rectangle) during the tone or light, and not during shock presentation, in both naïve rats (Exp 1; left) or a separate group of rats that had experienced reward learning (Exp 2; right). Bottom left: (A) shows freezing during conditioning in naïve rats, where inhibition of LH GABA neurons in the NpHR group (n=4 rats) had no impact on learning about fear relative to the eYFP control group (n=4 rats; trial: F_2,12=5.812, p=0.017; trial x group: F_2,12=0.187, p=0.831; group: F_1,6=0.377, p=0.562). Similarly, (B) demonstrates that there were no differences in levels of freezing between groups during extinction (group: F_1,6=0.206, p=0.666). Bottom right: (C) in a separate group of rats that had prior experience with rewards, inhibition of LH GABAergic neurons in our NpHR group (n=7 rats) significantly attenuated fear learning during conditioning relative to eYFP controls (n=6 rats; trial: F_2,22=17.886, p=0.000; trial x group: F_2,22=9.133, p=0.001; group: F_1,11=29.615, p=0.000). (D) shows this difference was maintained in an extinction test with LH GABA neurons intact (group: F_1,11=5.553, p=0.038). Data were analyzed with a repeated-measures ANOVA, which utilize a two-sided test.

Figure 3.. LH GABAergic neurons are recruited to encode fear memories only in rats that experience contingencies between cues and rewards.

Responding is shown as mean level of responding (%; ±SEM) during Exp 3. Top: LH GABAergic neurons were inhibited (green rectangle) during fear learning in rats with a prior history of reward learning. (A) Prior to aversive learning, rats were trained to associate a light with food delivery in context A, with no differences between NpHR learners (n=6 rats) and eYFP learners (n=6 rats; session: F_4,40=11.171, p=0.000; session x group: F_4,40=0.174, p=0.951; group: F_1,10=1.498, p=0.249). (B) During fear learning, we found that NpHR learners showed less learning about the tone, and greater learning to the context (simple main effect after group interaction: F_1,20=4.831, p=0.040), in contrast to eYFP reward learners (F_1,20=0.773, p=0.390). (C) The deficit in learning about the tone was maintained in extinction test, after extinction to the contextual cues, with LH GABAergic neurons intact (group: F_1,10=6.085, p=0.033). Bottom: LH GABAergic neurons were inhibited (green rectangle) during fear learning in rats that received light presentations without reward. (D) Prior to aversive learning, NpHR naïve (n=6 rats) and eYFP naïve (n=6 rats) group received presentations of the light and did not acquire an appetitive response across days (session: F_4,40=0.498, p=0.737; session x group: F_4,40=0.951, p=0.445; group: F_1,10=0.263, p=0.620). (E) During fear learning, NpHR naïve rats without LH GABAergic activity showed no difference in freezing to the tone and contextual cues (F_1,20=0.435, p=0.517), similarly to eYFP naïve rats (F_1,20=0.048, p=0.828). (F) There was no difference in the expression of fear to the tone during the subsequent extinction test when LH GABAergic neurons were intact (group: F_1,10=0.303, p=0.594). Data were analyzed with a repeated-measures ANOVA, where analyses of simple-main effects were warranted after a significant interaction was determined and did not necessitate controls for multiple comparisons. In the case of an expected interaction, one-tailed tests were used to warrant investigation of further simple-main effects.

Figure 4.. Computational modeling of the fear conditioning data supports a role for LH GABA neurons in learning about the shock-predictive cue after experience with rewards.

We tested whether a computational model of reinforcement learning could predict our specific pattern of data seen in Figure 2 and 3. We modeled LH GABA neurons blocking the update of learning attributed to the shock-predictive cue after experience with rewards. We used the data from Exp 1 (A) to set the parameters for our remaining experiments. This model predicted the differences seen in context learning between Exp 2 (B) and Exp 3 (C). The model can account for the low levels of context fear seen in Exp 2 in two ways: as reward learning also occurred in the fear context, the context could either receive little attention as the rats have received a lot of exposure to it (model 1), or the context acquires appetitive value (model 2). The higher levels of contextual fear occur in Exp 3 as learning to the cue is blocked by LH GABA neuronal inhibition, and is relayed instead to the background contextual cues (present when LH GABA neurons are not inhibited, and unique to the fear conditioning procedures). The model supports the validity of our context-specific design in Exp 3, as it suggests generalization between the fear and reward contexts was low, and that was unlikely the reason for LH GABA involvement in fear conditioning after reward learning.

Figure 5.. LH GABAergic neurons oppose learning of cue-cue associations after reward learning.

Responding is represented as the time spent in the food port (%; ±SEM) during stimulus presentation when rats were learning the second-order contingencies (orange box; left panels early trials, right panels late trials), and during the probe test (gray box; left panels index group means, right panels indicate individual rat responses). Scatterplots represent rats’ individual responses, where equivalent rates of responding to the cues mean responses should congregate on the diagonal. Rats first learnt to associate A2 with food, and to differentiate that from B2, which did not predict food. Then, during second-order learning, rats were presented with A1→A2 and B1→B2 pairs. During this time, light (532nm, 16mW, green rectangle) was delivered into the brain during A1, resulting in inhibition of LH GABA neurons in our NpHR group (n=8 rats) but not our eYFP group (n=12 rats). Finally, we conducted a probe test, where we presented A1 and B1 alone and without reward. Across both learning and the probe test, rats in our NpHR group showed an enhancement of responding to A1 relative to B1 (stimulus: F_1,18=10.576, p=0.004; stimulus x group: F_1,18=4.657, p=0.045), which was maintained in the probe test without LH GABA neuronal inhibition (stimulus x session: F_2,36=0.368, p=0.695). Follow up analyses showed a significant difference in responding to A1 and B1 in our NpHR group (F_1,18=12.195, p=0.003) that was not present in the eYFP group (F_1,18=0.748, p=0.05). This demonstrated that inhibition of LH GABA neuronal activity enhanced learning about cue-cue relationships after experience with reward learning. Data were analyzed with a repeated-measures ANOVA, with simple-main effects following a significant interaction, not necessitating control for multiple comparisons.

Figure 6.. LH GABAergic neurons oppose learning of cue-cue associations in naïve rats.

Responding is represented as time spent in the food port (%; ±SEM) during stimulus presentation in the probe test (gray box). Left panels index group mean responses, middle panels show responding by trial, and the right panel shows individual rats’ responses. Rats first learnt novel cue pairs, A1→A2 and B1→B2, where we delivered light into the brain during A1, inhibiting LH GABAergic neurons in our NpHR group (n=8 rats) and not our eYFP group (n=12 rats) during A1 presentation. Then, A2 was paired with food and B2 was presented without consequence. Pairing A2 with reward allowed us to test what rats had learnt during learning of A1→A2 and B1→B2 in the first phase. (A) shows mean levels of responding by group for all rats, demonstrating more appetitive responding towards A1, showing they had learnt the A1→A2 association and so inferred A1 predicts food after it’s associate A2 was paired with reward. However, rats without LH GABA neuronal activity in the first phase showed an enhancement of this effect, demonstrating that inhibition of LH GABA neurons increased the association between A1→A2. This was confirmed with statistical analyses, showing a main effect of stimulus (F_1,18=15.438, p=0.001), as well as a significant cue x group interaction (F_1,18=5.691, p=0.028), where simple main effects analyses afforded by the interaction demonstrated this was due to a significant difference between responding to A1 and B1 in the NpHR group (F_1,18=16.615, p=0.001), that was not present in the eYFP control group (F_1,18=1.489, p=0.238), (B) shows the difference between A1 and B1 across trial in the analyses represented in A, and (C) shows rats’ individual responses to A1 and B1 in the probe test; to the extent that responding to these cues is equivalent, cues should congregate around the diagonal. Data were analyzed with a repeated-measures ANOVA, with simple-main effects following a significant interaction, not necessitating control for multiple comparisons.

Figure 7.. LH GABAergic neurons are necessary for the downregulation of processing of explicitly irrelevant cues.

Responding is represented as the time spent in the food port (%; ±SEM) during stimulus presentation when S1 and S2 were paired with food reward. Plots to the left indicate group means by trial, while plots on the right show individual rats’ responses. To the extent that responding to the cues is equivalent, points should congregate on the diagonal. Rats first received pre-exposure to S1. Here, we optogenetically inhibited LH GABA neurons during S1 in our NpHR group (n=9 rats). Then, rats received S1 and S2 paired individually with food. During this session, our control group (eYFP; n=10 rats), showed slower learning about S1 relative to novel S2 (top), demonstrating that our control group downregulated processing of S1 during pre-exposure. However, rats in our NpHR group did not show this effect, demonstrating equivalent rates of learning about S1 and S2 across conditioning (bottom). This shows that LH GABA neurons are necessary to downregulate processing of the irrelevant S1, consistent with the idea that LH GABA neurons usually oppose learning and processing of information that does not predict something motivationally significant. This was confirmed with statistical analyses. A repeated-measures ANOVA showed no main effect of cue (F_1,17=2.246, p=0.152), but a significant interaction between cue and group (F_1,17=6.333, p=0.022), due to a significant difference between responding for S1 and S2 in the eYFP group (F_1,17=8.508, p=0.010) that was not present in the NpHR group (F_1,17=0.492, p=0.492). There was no significant between-group difference in responding to S1 (F_1,17=0.001, p=0.980), or S2 (F_1,17=2.263, p=0.151). Finally, there was no between-group difference in overall levels of responding during the cues (F_1,17=0.667, p=0.425). Data were analyzed with a repeated-measures ANOVA, with simple-main effects following a significant interaction, not necessitating control for multiple comparisons.