INFLUENCE OF RAT CENTRAL THALAMIC NEURONS ON FORAGING BEHAVIOR IN A HAZARDOUS ENVIRONMENT

The basolateral amygdala (BL) is a major regulator of foraging behavior. Following BL inactivation, rats become indifferent to predators. However, at odds with the view that the amygdala detects threats and generate defensive behaviors, most BL neurons have reduced firing rates during foraging and at proximity of the predator. In search of the signals determining this unexpected activity pattern, this study considered the contribution of the central medial thalamic nucleus (CMT), which sends a strong projection to BL, mostly targeting its principal neurons. Inactivation of CMT or BL with muscimol abolished the rats’ normally cautious behavior in the foraging task. Moreover, unit recordings revealed that CMT neurons showed large but heterogeneous activity changes during the foraging task, with many neurons decreasing or increasing their discharge rates, with a modest bias for the latter. A generalized linear model revealed that CMT neurons encode many of the same task variables as principal BL cells. However, the nature (inhibitory vs. excitatory) and relative magnitude of the activity modulations seen in CMT neurons differed markedly from those of principal BL cells but were very similar to those of fast-spiking BL interneurons. Together, these findings suggest that, during the foraging task, CMT inputs fire some principal BL neurons, recruiting feedback interneurons in BL, resulting in the widespread inhibition of principal BL cells.


INTRODUCTION
Behavioral experiment 100 Surgery. Figure 1A summarizes the experimental timeline of the behavioral experiments. Under 101 aseptic conditions and deep isoflurane anesthesia, rats were placed in a stereotaxic apparatus with non-102 puncture ear bars (Kopf, Tujunga, CA). Rats received atropine sulfate (0.05 mg/kg, i.m.) to facilitate 103 breathing and their scalp was injected with a local anesthetic (bupivacaine, 0.1-0.3 mL of a 0.125% solution, 104 s.c.) in the area to be incised. Ten minutes later, we made a 0.5 cm scalp incision and a craniotomy above 105 the brain region of interest. Under stereotaxic guidance, rats were implanted with guide cannulae (0.48 mm 106 o.d., 0.32 mm i.d.; World Precision Instruments, Sarasota, FL) aimed at CMT (only one cannula) or BLA 107 (one cannula per hemisphere). The cannulae were attached to the skull with dental cement and anchoring 108 screws. They contained a dummy cannula to prevent blocking. At the conclusion of the surgery, rats were 109 administered an analgesic with a long half-life (ketoprofen, 2 mg/kg, s.c., daily for three days). They were 110 allowed a 7-day recovery period during which they were habituated to handling daily. We used the 111 following stereotaxic coordinates (in mm relative to bregma): BLA (AP -2.4; ML ±5.00, DV -8.00; Angle 112 from midsagittal line: 0°); CMT (AP -2.28, ML 0.4; DV -7.40, 20° mediolateral angle). 113 Foraging apparatus. The foraging apparatus (Fig. 1B1) was rectangular in shape and made of black 114 polycarbonate. It had walls 60 cm high, and was divided in two compartments, both 60 cm wide. The first 115 compartment was a small, dimly-lit nest (30 cm in length; 10 Lux) with a water bottle. The second was an 116 elongated and brightly lit foraging arena (245 cm in length; 200 Lux). The two compartments were divided 117 by a sliding door. 118 Behavioral procedures. Habituation (Days 1-2): Rats were first habituated to the nesting area (3.5 119 h/day for two days). During these sessions, the gateway to the nesting area was closed and rats could 120 consume up to 6 g of food. 121 Foraging without the predator (Days 3-9): After habituation to the nest, rats underwent seven 122 consecutive days of foraging training without the predator. After 60-90 s in the nest (no food), the door was 123 opened and the rat was allowed to explore the foraging arena and search for a food pellet. On the first trial, 124 the food pellet was placed 25 cm from the nest. After each successful trial, the distance was increased in 125 steps of 25 cm up to 75 cm. Upon successful retrieval of the food pellet and reentry into the nest, the door 126 was closed. One minute after the animal finished consuming the pellet, the gateway was reopened and 127 another trial began. Each day, rats were required to complete at least three successful trials. 128 Foraging with predator (Days 10-11): During the last two days of the experiment, on each trial, we 129 placed a robotic predator at the end of the foraging arena opposite to the nest, but facing it. The predator 130 (Mindstorms, LEGO Systems, Billund, Denmark) was 14 cm tall, 17 cm wide, and 34 cm long. Each time 131 rats approached within ∼25 cm of the food pellet, the predator surged forward (80 cm at 60 cm/s), snapped 132 its jaw repeatedly (~9 times), and returned to its original position. On the first trial of each experimental 133 day, a food pellet was placed 75 cm from the nest. If the rat successfully retrieved the pellet, the distance 134 was increased in steps of 25 cm until the rat failed. When the rats failed at 75 cm, the distance was decreased 135 in steps of 25 cm until the rat retrieved the pellet. Trials were separated by 1-2 min intervals during which 136 rats were in the nest with the door closed.  ' position and velocity by taking advantage of the shifting  152  distribution of light intensity across frames. Moreover, we analyzed the video files frame-by-frame to  153  identify when rats started waiting at the door threshold (operationally defined as when their snout extended  154 passed the door into the foraging arena), when they started foraging (operationally defined as the last frame 155 of stillness prior to moving entirely out of the nest), retrieved a food pellet, escaped, and retreated into the 156 nest (Fig. 1B2). We also noted whether rats succeeded or failed each trial. The start of the "escape" phase 157 was defined as when rats, after approaching the food pellet, abruptly turned around to run back to the nest. 158 This behavior was observed whether the predator was present or not and whether the trial was successful 159 or not. 160 Histology. One day after completion of the behavioral tests, rats received a bilateral infusion of 161 fluorescent muscimol, as described above. Two hours later, they were overdosed with isoflurane and 162 perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. were allowed 2-3 weeks to recover from the surgery. 176 Foraging task. The foraging apparatus was identical to that used in the first experiment. So were the 177 training stages and behavioral scoring procedures. However, after rats were trained on the foraging task in 178 the absence of the predator (days 3-4), on each recording day (days 5-7), we conducted alternating trial 179 blocks with (n = 10-20) or without (n = 10-15) the predator, for a total of 100-120 trials per day. 180 Histology rates and spike waveforms were considered in the analyses. Statistical tests were two tailed. Different 220 procedures were used to assess statistical significance depending on the data type, as detailed below. 221 Behavior. In the inactivation experiment, we used a mixed-model ANOVA followed by post-hoc 222 paired-samples t-tests to examine the effect of with infusion site (BL, CMT, Control) as between-subject 223 variable and treatment (vehicle, muscimol). In the unit recording experiments, to compare the incidence of 224 a behavior in two conditions, we computed Chi-square tests for independence with a threshold alpha of 225 0.05. To compare the duration of a behavior in two conditions, we computed a Student's t-test. 226 Task-related changes in firing rates. To determine whether individual neurons showed statistically 227 significant task-related variations in firing rates, we computed Kruskal-Wallis one-way ANOVAs and 228 applied a Bonferonni correction of α for the number of neurons considered (0.05/716 or 0.000014). For 229 those cells with a significant ANOVA, we then computed Tukey-Kramer post-hoc tests with a threshold α 230 of 0.05. To assess the influence of the predator (presence/absence) and prior trial outcome (success/failure) 231 on firing rates, we computed a Friedman test followed by post-hoc Wilcoxon tests with Bonferroni-232 corrected α for the number of comparisons. 233 GLM normalized peak firing rate modulations. The absolute peak modulation associated with each 234 variable was normalized to baseline firing rates according to this equation: (Peak-235 Baseline)/(Peak+Baseline). We kept the sign of the normalized peak to distinguish cells inhibited or excited 236 by each variable. Normalized peak modulations with values ≤0.001 were set to zero because considered 237 non-informative. The average modulation by a task feature was assessed by averaging the absolute value 238 of all peak unit modulations. We assessed the relationship between the modulations associated with the 239 variables across cell types by computing a rank-based (Spearman) correlation. To compare the model fit to 240 observed spiking, we used the coefficient of determination (R 2 ) as follows: 241 242 where " represents the observed unit spiking and " is the model-estimated firing at different time points i, 245 and + is the overall average of the observed unit spiking. 246 247

249
Experiment 1: Muscimol infusions in BLA and CMT 250 Rats were trained to leave a nest-like compartment to retrieve a food pellet in a brightly lit and 251 elongated arena (Fig. 1B). On some trials, rats were confronted with a mechanical predator, which was 252 located at the other end of the foraging arena, facing rats as they approached the food pellet. Trials started 253 when the gateway to the foraging arena opened. After a variable delay, rats moved to the gateway and 254 paused at the door threshold (Waiting phase). Eventually, they left the nest to retrieve a food pellet placed 255 12.5 to 150 cm from the nest (Foraging phase). Upon retrieving the food pellet (or failing to do so), rats 256 abruptly changed direction and ran to the nest (Escape phase). Upon nest reentry (Nest phase), the gateway 257 was closed. On predator trials, each time rats came within ∼25 cm of the food pellet, the predator surged 258 forward, snapped its jaw repeatedly, and returned to its original position. 259 We compared behavior in the foraging task, 15-30 min after bilaterally infusing the same volume 260 of vehicle (ACSF, 0.1 µl) or fluorescent muscimol (0.9 nanomoles) in BLA or CMT. We formed a third 261 (control) group out of CMT rats with incorrect cannula placements, resulting in the following samples: 10 262 BLA rats, 8 CMT rats, and 11 control rats. See figure 1C for examples of fluorescent muscimol infusions 263 and figure 2 for schemes illustrating cannula tip locations in the three groups. Vehicle and muscimol 264 infusions were performed on different days, 24 h apart. To minimize the influence of habituation to the 265 predator, the order of the infusions was counterbalanced in all groups. 266 Our behavioral protocol adhered closely to that used by Choi and Kim (2010), with one exception: 267 the nest and foraging arena were separated by a sliding door, whose opening signaled the start of a trial. In 268 brief, after habituation to the behavioral apparatus (Days 1-2) and training on the foraging task in the 269 absence of the predator (Days 3-9), rats received a vehicle or drug infusion 15-30 min before giving them 270 the opportunity to retrieve a food pellet placed in the foraging arena, 75 cm from the nest (Days 10-12). 271 When rats advanced within ~25 cm of the food, the predator surged forward (80 cm at 60 cm/s), snapped 272 its jaw repeatedly, and returned to its original position. If the rat successfully retrieved the food pellet, the 273 distance was increased in steps of 25 cm until the rat failed. When the rat failed at 75 cm, the distance was 274 decreased to 50 cm, 25 cm, then 12.5 cm until the rat retrieved the pellet. Trials were separated by 1-2 min 275 intervals during which rats were in the nest with the door closed. 276 The dependent variables we monitored were the maximal foraging distance (Fig. 1D)

310
Experiment 2: Unit recordings in CMT during the foraging task 311 We recorded unit activity in the CMT while rats (n=7) performed the foraging task. Figure 3A  312 summarizes the experimental timeline. On each recording day, we conducted alternating trial blocks with 313 (n=10-20) or without (n=10-15) the predator, for a total of 100-120 trials per day. On each trial, the 314 distance between the nest and food pellet was varied randomly between 12.5 and 150 cm. Thus, on predator 315 trials, the proximity of the food pellet to the predator varied randomly on a trial-by-trial basis. Rats showed 316 signs of increased apprehension on predator trials. Specifically, the proportion of aborted trials, that is trials 317 in which after hesitating at the door threshold, rats retreated into the nest instead of initiating foraging, was 318 ~3.5 times more frequent in the presence (2.1%) than absence (0.6%) of the predator (Chi-square test for  319 independence, χ 2 =8.40, 28 sessions from seven rats, p=0.003). Moreover, when rats did initiate foraging in 320 the presence of the predator, the retrieval interval increased by 265±34% ( Task-related activity of CMT neurons 327 A total of 716 single units, histologically determined to have been located within CMT (Fig. 3C), 328 were recorded while rats performed the foraging task. We begin by describing the activity of CMT neurons 329 irrespective of whether the predator was present or absent. We will return to the impact of the predator in a 330 subsequent section. 331 Figure 4 shows four representative examples of CMT neurons with obvious task-related activity. 332 For each cell, we provide spike rasters centered on the onset of particular task events and below them, the 333 corresponding average ± SEM firing rate. As the duration of each behavioral phase varied from trial to trial, 334 we rank-ordered the trials based on the timing of events that occurred just before (door opening, left column) 335 or after the behavior of interest (escape, middle column; nest reentry, right column). These events are 336 indicated by blue tick marks. Finally, the cyan lines indicate the average speed of the rats (upward and 337 downward deflections indicate movements away from vs. toward the nest, respectively). 338 As shown in figure 4, the task-related activity of CMT neurons was extremely variable with some 339 cells showing transient increases in firing rates in relation to most task events (Fig. 4A), others exhibiting 340 persistent firing rate reductions from the start of the trials until rats returned to the nest (Fig. 4B), and others 341 displaying more differentiated activity profiles (Fig. 4C,D).      A better appreciation of cell-to-cell variations in task-related activity can be gained by inspecting 377 figure 5A1, which shows the activity of all cells with a significant Kruskal-Wallis ANOVA (n=633), z-378 scored based on variations in firing rates during the baseline period. Cells were rank ordered from least 379 (top, blue) to most active (bottom, yellow) based on their activity during the foraging phase and the same 380 order was kept in the other phases. Figure 5A2 plots their average (± SEM) firing rates. Comparing the 381 color distributions in the different phases (Fig. 5A1) reveals that activity during foraging is a poor predictor 382 of that in other task phases. In fact, rank correlations between z-scored activity in the various phases are 383 consistently low, in the -0.2 to 0.2 range (Fig. 5B1). The lack of consistent association between CMT 384 activity in the different phases is highlighted in figure 5B2, which plots the ranks of all significant cells in 385 the waiting, foraging, escape, and nest phases, but color coded based on their activity during waiting. 386 Nevertheless, as a group, CMT cells increased their firing rates (0.5-0.7 z score) in all phases of the foraging 387 task until rats returned to their nest (Fig. 5A2).

388
In an attempt to identify subsets of CMT neurons with distinct profiles of task-related activity, we 389 used unsupervised clustering (kmeans function in MATLAB). Using raw firing rates, the optimal number 390 of identified clusters by gap statistics was always near the maximum number of tested clusters. Separately 391 averaging the cells' activity within each cluster failed to reveal distinct patterns of task-related activity 392 (not shown). Similarly inconclusive results were obtained when clustering was carried out on normalized 393 firing rates (z-scored based on variations during the baseline period). 394 395

404
Influence of the predator and prior trial outcome 405 As mentioned above, rats showed signs of increased apprehension on trials carried out in the 406 presence of the predator. That is, they waited longer before initiating foraging and foraged more tentatively, 407 especially if they had failed to retrieve the food on the prior trial. To examine the neuronal correlates of 408 these behavioral variations, we compared the z-scored activity of CMT cells on no predator vs. predator 409 trials, rank ordering the cells based on their activity in the foraging phase of no predator trials and keeping 410 the same ordering in the other conditions (Fig. 6A). Predator trials were subdivided in two groups, 411 depending on whether rats had failed or succeeded to retrieve the food pellet on the prior trial (failed trials 412 were rare in the absence of the predator). As there were large differences in phase duration across trial types, 413 we plotted the data of figure 6A as a function of relative time, by distributing a fixed number of samples 414 evenly across each of the phases of interest. 415 This analysis revealed that not only were the relative activity levels of CMT cells different across 416 different phases on the same trial type (Fig. 5A1), they also differed in the same phase of different trial 417 types (Fig. 6A1). Moreover, the average z-scored activity of CMT cells was lower on predator than no 418 predator trials, particularly if rats had failed the prior predator trial (Fig. 6A2) difference between the two subtypes of predator trials. The difference across trial types can be attributed to 425 changes in the kurtosis of the CMT firing rate distributions as opposed to changes in skewness (Fig. 6C). 426 Predator presence and failure in the preceding trial decreased the peakedness of the firing rate distributions. 427 Overall, the above analyses indicate that CMT neurons exhibit large but variable firing rate 438 fluctuations during the foraging task. However, identifying which factors drive these variations in not 439 trivial. Indeed, as foraging trials unfold, a number of temporally overlapping variables fluctuate besides the 440 task phases, such as the rats' speed and position. To help us quantify the relative influence of these factors, 441 we fit the activity of each CMT unit with a group least absolute shrinkage and selection operator (Lasso) 442 generalized linear model (GLM). This type of GLM exploits variations in the timing and duration of 443 relevant variables to determine the neurons' encoding preference. Specifically, we considered the rats' 444 speed, position, distance from the food pellet, the influence of task phases (baseline, door opening, waiting 445 at the door, foraging, predator activation, escape, nest entry), trial types (with or without predator), and 446 prior trial outcome (failure, success). Critically, this type of GLM promotes dimensionality reduction of 447 correlated data and permits sparsity in the identification of the factors linked to neuronal activity (Breheny 448 and For instance, the beta values of CMT cells for 'door opening' and 'nest entry' show much more variability 466 than for 'speed' and 'distance from food' (Fig. 8A). Yet, the peakedness of CMT distributions (Fig. 8A) is 467 generally higher than that of BL distributions (Fig. 8B). Moreover, CMT distributions are generally more 468 symmetric than BL distributions, which are typically skewed to the left, betraying a preponderance of 469 negative beta values.   These contrasting features are also manifest in figure 9, which plots for CMT cells (n=716; Fig.  487 9A), principal BL neurons (n=599; Fig. 9B), and fast-spiking BL cells ( Fig. 9C; n=71; also recorded in 488 Amir et al., 2015), their average absolute modulation (Fig. 9A1, B1, C1) as well as cumulative excitatory 489 (blue) and inhibitory (red) distributions (Fig. 9A2, B2, C2) for all the variables considered in the GLM. 490 Variables were rank ordered from the highest to lowest absolute modulations, in the three cell types 491 separately. Although the variables with the lowest absolute modulations are nearly identical in the three cell 492 types (trial type, prior trial, position, distance from food, speed), there are major differences between the 493 other variables. First, the magnitude of beta values is 2-3 times higher in principal BL neurons than in CMT 494 or fast-spiking BL cells (compare axis range in Fig. 9B1 vs. Fig. 9A1, C1). This difference is likely related 495 to the much higher spontaneous firing rates of CMT and fast-spiking BL neurons compared to principal BL 496 cells (in the nest with door closed: CMT, n=716, 6.09 ± 6.30 Hz; fast-spiking BL cells, n=71, 23.66 ± 13.66 497 Hz; principal BL cells, n=599, 0.48 ± 1.23 Hz; Kruskal-Wallis ANOVA, df=2, χ 2 =891.65, p<0.0001). As a 498 result, task-related changes in firing rates are proportionally much larger in principal BL cells. Second, 499 whereas inhibitory firing rate modulations are generally higher than excitatory ones in principal BL cells 500 (Fig. 9B2), CMT and fast-spiking BL cells show no such imbalance (Fig. 9A2, C2). Third, while the 501 variables with the highest modulations were similar in CMT and fast-spiking BL neurons (door opening, 502 food retrieval, nest entry, predator activation), they differed markedly from those dominating the activity 503 of principal BL neurons, particularly escape and foraging. 504 Consistent with the qualitative similarities between the GLM results obtained in CMT and fast-505 spiking BL neurons, the average absolute beta values associated with the 12 variables were highly correlated 506 in these two cell types (Fig. 9C1, inset; Spearman r=0.96, p<0.0001). And so were the positive betas 507 associated with these variables (Fig. 9C2, inset; Spearman r=0.82, p=0.002). Opposite to this, a negative 508 correlation was found between the positive beta values of CMT cells and the negative beta values of 509 principal BL neurons (Fig. 9A2, inset; Spearman r=-0.69, p=0.015), raising the possibility that CMT inputs 510 influence BL neurons by activating fast-spiking BL interneurons. 511

512
Converging evidence indicates that BL is a major regulator of foraging behavior. In the foraging 513 task for instance, BL inactivation causes rats to become indifferent to the predator (Choi and Kim, 2010). 514 However, at odds with the widely held view that the amygdala detects threats and generate defensive 515 behaviors, unit recordings revealed that most BL neurons have reduced firing rates at proximity of the 516 predator (Amir et al., 2015(Amir et al., , 2019a. In search of the signals determining this unexpected activity pattern, 517 the present study considered the possible contribution of CMT, which sends a very strong projection to BL 518 (Vertes et al., 2015;Amir et al., 2019b). 519 In support of the hypothesis that CMT regulates foraging behavior, we observed that inactivation of 520 CMT with muscimol increased foraging distances and decreased waiting times, similar to the effects of 521 BLA inactivation. Moreover, many CMT neurons showed large changes in activity during the foraging 522 task. However, in most task phases, our sample was split between neurons with decreased or increased 523 discharge rates, with a modest bias for the latter. In addition, while GLM analyses revealed that CMT 524 neurons encode many of the same task variables as principal BL cells, the nature and relative importance 525 of the activity modulations seen in CMT neurons differed markedly from those of principal BL cells. By 526 contrast, the GLM revealed striking similarities between the beta values of CMT cells and fast-spiking BL 527 interneurons. Below, we consider the significance of these findings for the regulation of BL activity by 528 CMT neurons. 529 530 CMT neurons have large but heterogeneous activity correlates in the foraging task 531 Consistent with relay neurons in other thalamic nuclei (Steriade, 1993), CMT neurons fired tonically 532 (median firing rate: ~5 Hz) during quiet wakefulness. In the foraging task, most CMT neurons (88%) 533 displayed significant changes in firing rates in relation to one or more task events. However, in each task 534 phase, the firing rate of many CMT neurons increased while others showed the opposite. Moreover, activity 535 changes developing during any particular task phase were poor predictors of those occurring in other phases. 536 At a group level, this heterogeneity resulted in modest task related activity. Specifically, average 537 CMT firing rates increased ~0.5 z-scores from baseline to waiting, remained elevated at or slightly above 538 this level (0.7 z-score) through foraging and escape, only returning to, and eventually below, baseline levels 539 after reentry into the nest. Moreover, in the presence of the predator, especially if the rat failed the preceding 540 trial, the overall increase in the firing rates of CMT cells was attenuated. As such, the variability of the 541 predator's influence (present vs. absent, and success vs. failure) did not impact the heterogeneous activity 542 of CMT neurons, only their overall relative firing rates. 543 544 CMT neurons encode multiple task features 545 As trials unfold in the foraging task, multiple overlapping variables potentially contribute to alter 546 neuronal activity. These include factors such as the rats' speed, position in the apparatus, proximity to the 547 food and predator, as well as the rats' recent experience in the task. Disentangling the relative influence of 548 these multiple factors is especially difficult around the time of predator activation, when rats suddenly 549 switch from foraging to escape. Since peri-event histograms of firing rates cannot dissociate these 550 overlapping factors, we used a GLM, allowing us to assess the influence of each variable while factoring 551 out that of the others by taking advantage of variations in the timing and duration of relevant variables. 552 As for BL neurons in the foraging (Amir et al., 2019a) and risk-reward interaction tasks (Kyriazi et  553 al., 2018, 2020), this approach revealed that CMT cells encode many task features. That CMT neurons 554 concomitantly represent multiple types of information is consistent with the functionally diverse inputs they 555 receive. Indeed, CMT is the recipient of afferents from the superior colliculus ( interneurons and the consequent inhibition of a high proportion of principal cells. 582 Two GLM results are consistent with this model. First, we found a nearly perfect correlation 583 between the firing modulations of CMT and fast-spiking BL interneurons (Fig. 7C, insets). Second, the 584 modulation of CMT and principal BL neurons were inversely correlated (Fig. 7A, inset). Also supporting 585 this model, fluctuations in the population activity of CMT neurons during the foraging task were opposite 586 to that of principal BL neurons. That is, whereas the average firing rate of CMT cells increased during 587 foraging and escape, most principal BL neurons show the converse (Amir et al., 2015(Amir et al., , 2019a. Inverse firing 588 rate fluctuations were also observed upon nest reentry, when the firing rate of CMT cells decreased whereas 589 that of principal BL neurons increased (Amir et al., 2015(Amir et al., , 2019a. Finally, whereas CMT neurons showed 590 significantly decreased firing rate elevations on predator relative to no predator trials, principal BL neurons 591 showed the opposite (Amir et al., 2015). 592 593 Relation between CMT unit activity and the effects of CMT inactivation. 594 If the above model is correct and the CMT-driven inhibition of principal BL cells is a necessary 595 condition for rats to initiate foraging, how come CMT inactivation results in increased risk-taking? There 596 are two non-exclusive possible explanations. First, besides BL, CMT projects to the dorsal striatum, nucleus 597 accumbens, and multiple cortical areas (anterior cingulate, prelimbic, orbital and insular cortices; Vertes et 598 al., 2012). Thus, it is possible that CMT inactivation affects foraging behavior indirectly, through a 599 disfacilitation of these other sites. Second, since CMT projects to BL and most of CMT's cortical targets 600 also project to BL (McDonald, 1998), CMT inactivation is expected to cause a major reduction in the 601 excitatory drive to BL neurons. In turn, this "functional deafferentation" may cause a reduction in the firing 602 rate of BL neurons, of comparable magnitude to that seen upon the initiation of foraging. 603 604 Limitations of the foraging task 605 A limitation of the present study is the use of a pseudo-predator, which imperfectly reproduces the 606 features of an actual predator, particularly with respect to its olfactory cues. Although the pseudo-predator 607 does not fully capture the presentation of natural threats, it did alter the rats' behavior as would be expected 608 from an actual predator. That is, rats showed signs of increased apprehension on predator trials: they waited 609 longer before initiating foraging, failed to retrieve the food on a higher proportion of trials, and foraged 610 more tentatively, especially if they had failed the prior trial. Critically, we could detect changes in the 611 activity of CMT neurons in relation to these behavioral variations. Hence, it appears that the foraging task 612 captures essential features of prey-predator interactions, justifying its use here and the interpretation of the 613 results. 614 615 Relation to previous work on the regulation of motivated behaviors by thalamic inputs to the amygdala 616 The role of the thalamus in motivated behavior has received comparatively little attention, possibly 617 because it is thought to support general functions like arousal or the transfer of sensory information 618 (Bradfield et  . Combined with our results, these findings raise the possibility that 628 multiple thalamic inputs regulate foraging behavior via projections to different amygdala nuclei. 629