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Post-traumatic stress disorder (PTSD) is characterized by an impairment in the ability to neutralize fear responses to cues associated with trauma. Human and animal studies point to differences in the involvement of certain areas of the frontal cortex as key mediators that determine the success of fear suppression, but the interplay of neural circuits that determine the differential involvement of these areas is not clear. To better understand how individual differences in extinction recall are reflected in differences in neuronal circuit activity, we labeled projections from the rat sublimbic cortex (IL) with retrograde tracers and compared neuronal projections inside and outside of IL projection neurons. We analyzed these data, grouping them according to the degree of preservation of fading memory in rats. We found that in IL-projecting cells, neurons in the posterior parathalamus exhibited increased activity in rats, which exhibited good extinction recall. In addition to IL-projecting cells, increased Fos activity was observed in selected areas of the rat claustrum and ventral hippocampus with good resolution. Our results indicate that differences in extinction recall are associated with specific patterns of neural activity within and outside of IL projections.
Fear conditioning occurs when a neutral stimulus is associated with an aversive unconditioned stimulus (UCS), such that the originally neutral stimulus, now the conditioned stimulus (CS), elicits a conditioned fear response (CR) in the absence of the UCS. Reversal of conditioned fear was driven by a decrease in CR to CS due to repeated presentation of CS in the absence of UCS1. Previous research has shown that post-traumatic stress disorder (PTSD) is associated with an inability to remember the extinction of conditioned fear responses2. The cornerstone of cognitive behavioral therapy for the treatment of post-traumatic stress disorder is exposure therapy based on the extinction of learned fear responses3,4. Therefore, studies of individual differences in rodent extinction fear and underlying neural mechanisms may help elucidate differences in human responses to trauma and treatment of post-traumatic stress disorder. Despite progress in identifying the neural mechanisms that distinguish successful from unsuccessful extinction memories, much remains to be discovered.
Rodent models are useful in this work because there are significant individual differences in recollection of rodent extinction7,8,9,10. Previous work investigating the neural mechanisms of fear extinction at the population level has shown that activation of the infralibic cortex (IL) is required for extinction recall (refs 11, 12, 13, but see 14), and some studies have found a decrease in rodent activity in IL exhibiting poor memory about extinction compared to rodents, which are well feared. However, the mechanisms by which ILs are differentially involved in facilitating fear elimination in rodents compared to those showing weaker extinction are unclear.
One possibility is that differences in fear extinction memory between individuals are the result of differential activation of specific afferent ILs. Anatomical studies18 have shown that various cortical and subcortical areas of the brain send dense projections to IL, which in turn sends efferent projections to many areas of the brain. Population-level studies have shown that IL projections to the amygdala are important for acquiring fear extinction20,21,22 and IL input from the basolateral amygdala (BLA) is also associated with extinction learning. There is less research on the involvement of IL-centered circuits in extinction recall, although recent work suggests that both the ventral and dorsal hippocampus are involved in IL prediction. The efferent projections of the IL to the recombining nucleus of the thalamus, apparently, also participate in the memory of fear extinction.
These previous studies are beginning to paint a picture of the interaction of neural circuits involved in extinction recall, but there is very little data on whether activity in IL-centered neural circuits influences individual differences in extinction recall. Here, we sought to determine whether differences in fear extinction memory between individuals are associated with changes in IL input activation in specific brain regions. In particular, we assessed the activation of IL afferent cells in the paraventricular nucleus of the thalamus (PVT), clavicle (CLA), BLA, and ventral hippocampus (vHPC). These brain regions were chosen both because they send dense projections to IL and because there is reason to suspect that they may be involved in the expression of fear extinction 18 . For example, a recent study showed that PVT, a region known to be involved in fear acquisition and reproduction, is required for extinction reproduction. In addition, previous studies have shown increased basal amygdala and vHPC activity in rats expressing extinction memory. Finally, the analysis of the claustrum is more exploratory given that none of the previous work has assessed its role in extinction. However, recent work suggests that it plays a role in the contextual conditioning of fear29.
Viral GFP-conjugated retrograde tracers were injected into the IL of rats prior to behavioral testing, and Fos activity in IL afferents was measured during extinction replay, fear recall, and in rats not subjected to behavioral testing. Our results indicate that projections from the posterior paraventricular thalamus to IL show increased activity in rats that successfully recall extinction. In addition to IL predictions, neural activity in certain areas of the clavicle and ventral hippocampus was increased in rats that regressed well. Our results show that patterns of intrinsic and extrinsic neural activity projected onto IL are associated with individual differences in fear extinction memory.
Fifty-four adult male Sprague-Dawley rats (300-325 g on arrival) obtained from Charles River Laboratories (Raleigh, NC) were used as subjects. The rats were housed in pairs, with free access to food and water, on a 12-hour light/dark cycle (lights on at 7 am). Two groups of rats (n = 28 and n = 26) were used for these experiments. After exclusion of death, surgical error, lack of GFP expression at the target site, poor tissue quality, and behavioral problems (explained in Methods), the extinction recall group included 21 rats and the fear recall group included 7 rats, house cages. the group consisted of 7 rats (35 rats were included in the final analysis). All procedures were approved by the Stony Brook University Institutional Animal Care and Use Committee and were in accordance with the ARRIVE Guidelines (https://arriveguidelines.org) and the NIH Guidelines for the Care and Use of Laboratory Animals.
Rats were treated two days before surgery. Rats were anesthetized with ketamine (87 mg/kg) and xylazine (10 mg/kg), placed in a stereotaxic apparatus (Stoelting, Woodale, IL) and received unilateral injections of AAVrg-CAG-GFP (Addgene, 30) into IL. (balance left and right injections). For injection, a 22-gauge cannula was lowered into place (AP: + 3.00, ML: ± 0.6, DV: – 5.2). Insert a 28G inner cannula (connected to an infusion pump via PE 20 tubing) into the guide cannula to deliver 0.6 µl of virus at a rate of 0.15 µl per minute and leave in place for 5 minutes after the infusion is intact. . After suturing, the rats were injected with meloxicam (1 mg/kg) and as soon as they were able to move they were returned to their cages. The rats were kept in their cages for about 7 weeks to allow for virus recovery and retrograde transport. Three rats died under anesthesia, resulting in 51 rats (94%) recovering successfully from surgery.
All procedures were performed in 32 cm × 25 cm × 21 cm conditioning chambers (Clever Systems Inc., Reston, VA) housed in 45.7 cm × 43.2 cm × 43.2 cm sound-absorbing isolation boxes (Clever Sys. . Inc.). ). During the extinction learning and extinction recall sessions, the context was changed to be different from the original conditional context. Condition A (fear generation) included 28-volt incandescent, household light bulbs (Chicago Micro Lighting, UK), while condition B (extinction training, extinction recall test, and fear recall test) included infrared LED lamps (Univivi IR Illuminator, Shenzhen). , China; U48R). In addition, while Context A has an anti-vibration slatted floor with stainless steel and plexiglass walls, Context B contains painted metal inserts placed on the floor and walls. The Context B shape was also modified by placing a 33.5 cm x 21.3 cm curved metal insert into a standard conditioning chamber. In addition, in context A the chambers were wiped with 5% acetic acid, while in context B the chambers were wiped with 5% ammonium hydroxide. Finally, in context B, the rats were brought into the test room in buckets rather than being rolled around in cages on carts. Behavioral sessions were recorded with the top camera, and the video signal from each camera was fed into software (FreezeScan 2.00, Clever Sys. Inc., Reston, VA) that evaluated fading behavior based on pixel changes. The parameters were chosen so that the behavior of the fading estimated by the computer closely matches the behavior of trained observers estimated manually. A value indicating the percentage of freeze time rolled up in a 30-second interval.
All behavioral programs are performed during the light portion of the light/dark cycle. Rats were treated 5 days prior to the start of behavioral procedures and transported to the behavioral room for the last three days of treatment. On the first day of behavioral testing, a group of extinction recall rats were placed in a fear conditioned reflex, then placed in context A, given a 6 min unstimulated acclimatization period, and then given both combinations of 4 kHz, 76 dB, 30 s. tone and overall termination, 1.0 mA, kick 1 s (2 min ITI). For all behavioral trainings, the rats were returned to the cages 2 min after the last presentation of the stimulus. The following day, rats from the extinction-recall group were placed in context room B and performed 20 sound presentations (2 min ITI) as extinction training after a 6 min habituation period. The next day, rats in the extinction reproduction group were exposed to 4 tones in context B after a 6-minute acclimatization period as an extinction test. Rats in the extinction memory group were perfused 60 minutes after the behavioral session. A group of control rats evoking fear memories were subjected to the same procedure on the first day of the fear conditioned response in context A. Forty-eight hours later, the rats were placed in context room B and subjected to 4 audio presentations (2-minute ITI) as a recall test. fear after a 6-minute acclimatization period. Rats were perfused 60 minutes after the behavioral session. A group of domestic control rats remained in their home cages throughout the experiment and were perfused on the same day as the experimental rats. Each of the two groups of rats was divided into two series, and the number of animals in each group was balanced between series. One rat in the fear memories group was excluded from the analysis because it did not show signs of fear conditioning (freezing less than 15% of the time during the fear memories test). See Figure 2A for a diagram of the behavioral timeline.
Rats were overdosed with Fatal Plus solution (100 mg/kg), then perfused with ice-cold 10% PBS followed by 10% buffered formalin. The brain was removed and stored in a 30% sucrose solution in formalin at 4°C for about 1 week. Then the brain was frozen and cut into a 40 µm thick cryostat. Sections were stored sequentially in 10% PBS at 4°C. Then, immunofluorescence was performed on free-floating sections containing the brain region of interest. Sections were washed 3 times in 10% PBS for 5 min each. The sections were then incubated in 5% blocking solution of normal goat serum for 2 hours at room temperature, and then washed three more times for 5 minutes each in 10% PBS. Sections were then incubated overnight at 4° C. in primary antibodies (c-Fos, #2250, 1:500) (Cell Signaling, Danvers, MA) diluted in 1% BSA in 10% PBS. The next day, sections were washed in 10% PBS for 30 min at 4°C, then 3 times for 5 min in 10% PBS and incubated with a secondary antibody (Alexa Fluor 594 goat anti-rabbit, red conjugate, 1:500). ) (Invitrogen, Carlsbad, CA) at room temperature for 2 hours. After 3 additional washes in 10% PBS for 5 minutes, sections were placed on glass slides and sealed with Fluoromount-G (Invitrogen). See representative images of immunostaining in Figure 3G.
A fluorescent microscope using an Infinity3 digital camera (Lumenera, Ottawa, Ontario, Canada) and a light engine (Lumencor, Beaverton, OR) connected to a Zeiss microscope was used to acquire images from each region of the brain of interest, including sections containing IL without immunofluorescence. performed to confirm correct placement of the injection site. The images used for cell counting were obtained at 20x magnification. For each tissue section, take one image with a filter that allows visualization of GFP and one image with a filter that allows visualization of the Alexa Fluor red conjugate in the secondary antibody, and imaging software (Infinity Analyze, version 3) was used for image overlay. Acquire all images of all areas of the brain using the same exposure time and gain settings. Six rats were excluded from the analysis because the main transmission of the virus occurred outside the IL (88% hit rate). Eight more rats were excluded because, despite the virus attacking IL, they did not show sufficient GFP expression in all of the target brain regions of interest. In addition, one rat was excluded due to poor tissue quality.
Adjust brightness and contrast to reduce background noise in image J (NIH) using the same procedure for each image. Cell counts for total retrograde labeled cells, total Fos-labeled cells, and total double-labeled cells were performed manually by the experimenter, who did not identify the animals, using the Image J cytometer plug-in. Cell counts were normalized to cells/mm2. To analyze Fos expression in IL-projecting cells, the number of double-labeled cells was normalized to the total number of retrograde-labeled cells. For mBLA, mvHPC and pvHPC analysis, cell counts from multiple 20x images were summed and normalized to cells/mm2. For analysis of the rest of the brain regions, a 20x image or a portion of a 20x image was analyzed and normalized to cells/mm2. The vHPC analysis included CA1, CA2 and subdominant vHPC regions. Figure 1 shows the brain regions analyzed with images delineating the anterior-posterior border of the plane.
Abbreviations and location of brain regions of interest. Explanation of abbreviations and locations of brain regions given in the manuscript. Public domain brain map taken from Swanson (2004) Brain Map: Structure of the Rat Brain, 3rd Edition, licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/ 4.0) . /), available for download at https://larrywswanson.com.
Freeze time percentage is averaged over a 30 second tone playback period, excluding intermediate intervals. Extinction recall rates were calculated by expressing the percentage of fade time during extinction recall as the percentage of fade during the first 4 extinction training trials (fade during four extinction recall tones/fade during the first four extinction training tones*100). Low scores indicate good fade memory, and high scores indicate poor fade memory. Rats were sorted by extinction recall score, with rats in the top third of extinction recall score classified as “bad extinction rats” and rats in the last two thirds of extinction recall score were classified as “good”. Memory fading rats.
Nonparametric tests are used because the data often violates assumptions about normal distribution and/or homogeneity of variances. Spearman’s rank correlation was used to determine if there was a significant association between extinction recall scores and Fos markers and dual markers in brain regions of interest in all rats subjected to the extinction recall test. The Mann-Whitney U-test was used to determine if there was a difference between two independent groups. The Kruskal-Wallis test is used to determine if 2 or more groups differ from each other, and the Dunn multiple comparison test is used when the Kruskal-Wallis statistic is significant. Fading during extinction learning was assessed using repeated measures analysis of variance with the group as the inter-subject factor and the test as the intra-subject factor. Results were considered significant when p < 0.05 for all statistical tests. Results were considered significant when p < 0.05 for all statistical tests. Результаты считались значимыми при p < 0,05 для всех статистических тестов. Results were considered significant at p < 0.05 for all statistical tests.当所有统计检验的p < 0.05 时，结果被认为是显着的。当所有统计检验的p < 0.05 时，结果被认为是显着的。 Результаты считались значимыми при p < 0,05 для всех статистических тестов. Results were considered significant at p < 0.05 for all statistical tests.
Figure 2 shows the experimental timeline (Figure 2A) and the frequency distribution of all rats subjected to extinction (Figure 2B). Rats in the good and poor extinction groups differed significantly in these calculated extinction recall scores (U = 0, p < 0.001) (Fig. 2C). Rats in the good and poor extinction groups differed significantly in these calculated extinction recall scores (U = 0, p < 0.001) (Fig. 2C). Крысы в ​​группах с хорошим и плохим угашением значительно различались по этим рассчитанным показателям припоминания угашения (U = 0, p <0,001) (рис. 2C). Rats in the good and poor extinction groups differed significantly in these calculated extinction recall rates (U=0, p<0.001) (Figure 2C).在这些计算的灭绝回忆分数中，良好和不良灭绝组中的大鼠存在显着差异（U = 0，p <0.001）（图2C）。 U = 0，p <0.001）（图2C， В этих рассчитанных показателях припоминания угашения крысы в ​​группах с хорошим и плохим угашением значительно различались (U = 0, p <0,001) (рис. 2C). In these calculated extinction recall rates, rats in the good and poor extinction groups differed significantly (U = 0, p < 0.001) (Figure 2C). There was no significant difference in freezing time between groups with good extinction, poor extinction, and fear recall during the base period of the fear conditioned reflex session (X2(2) = 2.746, p = 0.253) (Figure 2D). In addition, during the presentation of the first tone of the fear conditioned reflex, there was no significant difference in the freezing time between groups with good extinction, poor extinction and fear recall (X2(2) = 1.107, p = 0.575), as well as during fear during the second tones. During the conditioning session, there was a significant difference in freezing time between the groups with good extinction, poor extinction, and fear recall (X2(2) = 2.214, p = 0.331) (Figure 2D). There was also no significant difference in fade time between good and poor extinction groups during the baseline extinction training period (U = 45.00, p = 0.799) (Figure 2D). Next, there was a significant main effect of trial block (5 tones per block) on time spent freezing during the extinction training session (F (2.884, 54.80) = 8.331, p < 0.001), indicating that extinction learning occurred (Fig. 2D). Next, there was a significant main effect of trial block (5 tones per block) on time spent freezing during the extinction training session (F (2.884, 54.80) = 8.331, p < 0.001), indicating that extinction learning occurred (Fig. 2D ). Затем наблюдался значительный основной эффект пробного блока (5 тонов на блок) на время, затрачиваемое на замирание во время тренировки угашения (F (2,884, 54,80) = 8,331, p <0,001), что указывает на то, что обучение угашению происходило (рис. 2D). There was then a significant main effect of the trial block (5 tones per block) on the time taken to freeze during extinction training (F(2.884, 54.80) = 8.331, p < 0.001), indicating that extinction learning was taking place ( Fig. 2D). ).接下来，在消退训练期间，试块（每块5 音）对冻结时间有显着的主效应（F（2.884，54.80）=8.331，p < 0.001），表明发生了消退学习（图2D） ）。接下来，在消退训练期间，试块（每块5 音）对冻结时间有显着的主效应（F（2.884，54.80）=8.331，p < 0.001），表明发生了消退学习（图2D） ） 。 Затем, во время обучения угашению, пробные блоки (5 тонов на блок) оказали значительное основное влияние на время замирания (F (2,884, 54,80) = 8,331, p <0,001), что указывает на то, что обучение угашению происходило (рис. 2D). Then, during extinction learning, trial blocks (5 tones per block) had a significant main effect on fade time (F(2.884, 54.80) = 8.331, p < 0.001), indicating that extinction learning was taking place (Fig. .2D). However, the extinction group (F(1, 19) = 3.091, p = 0.095) had no significant effect on the fade time throughout the extinction training period, and there was no interaction between the trial block and the extinction group (F(4, 19)) . 76) = 1.890, p = 0.121) (Fig. 2D). During the testing session, there was a significant difference among the good extinction, poor extinction, and fear recall groups in time spent freezing during the baseline period (X2 (2) = 8.569, p = 0.014) such that the fear recall group froze significantly more than the good extinction group (Mean Rank Diff. = 10.57, p = 0.017), but not the poor extinction group (Mean Rank Diff. = − 3.714, p > 0.999) (Fig. 2D). During the testing session, there was a significant difference among the good extinction, poor extinction, and fear recall groups in time spent freezing during the baseline period (X2 (2) = 8.569, p = 0.014) such that the fear recall group froze significantly more than the good extinction group (Mean Rank Diff. = 10.57, p = 0.017), but not the poor extinction group (Mean Rank Diff. = − 3.714, p > 0.999) (Fig. 2D). During the testing session, there was a significant difference between the good extinction, poor extinction, and fear recall groups in time spent freezing during the baseline period (X2(2) = 8.569, p = 0.014), such that the fear recall group froze significantly . больше, чем в группе хорошего вымирания (средняя разница рангов = 10,57, p = 0,017), но не в группе плохого вымирания (средняя разница рангов = -3,714, p> 0,999) (рис. 2D). greater than in the good extinction group (mean rank difference = 10.57, p = 0.017) but not in the bad extinction group (mean rank difference = −3.714, p > 0.999) (Figure 2D).在测试期间，良好消退组、不良消退组和恐惧回忆组在基线期冻结时间方面存在显着差异（X2 (2) = 8.569，p = 0.014），因此恐惧回忆组冻结显着超过良好的灭绝组（平均秩差= 10.57，p = 0.017），但不是差的灭绝组（平均秩差= – 3.714，p > 0.999）（图2D）。在 测试 期间 ， 良好 消退组 、 消 退组 和 恐惧 在 基线期 冻结 时间 方面 存在 显着 差异 （（x2 (2) = 8.569 ， p = 0.014） ， 恐惧 回忆组 冻结 显着 良好 的 组 组 组 组 组 组 组 组（平均秩差= 10.57，p = 0.017），但不是差绝组（平均秩差= – 3.714，p > 0.999）（图2D）。 В течение периода тестирования наблюдалась значительная разница между группой с хорошим угашением, группой с плохим угашением и группой с припоминанием страха с точки зрения времени замирания на исходном уровне (X2 (2) = 8,569, p = 0,014), поэтому припоминание страха группа замерзает значительно чаще, чем группа с хорошим вымиранием (средняя разница рангов = 10,57, p = 0,017), но не группа с плохим вымиранием (средняя разница рангов = -3,714, p> 0,999) (рис. 2D). During the testing period, there was a significant difference between the good extinction group, the poor extinction group, and the fear recall group in terms of freezing time at baseline (X2(2) = 8.569, p = 0.014), so the fear recall group freezes significantly more frequently than the group with good extinction (mean rank difference = 10.57, p = 0.017) but not the group with poor extinction (mean rank difference = -3.714, p > 0.999) (Figure 2D). The good extinction group, the poor extinction group, and the fear recall group also had significantly different fade times during the tone presentation of the test session (X2(2) = 14.93, p = 0.001), so the good extinction group had significantly less time. freezing time than in the weak extinction group (mean rank difference = 9.286, p = 0.044) and the fear memory group (mean rank difference = 13.86, p = 0.001) (Fig. 2D).
Individual differences in recollection of extinction. (A) Outline of surgical and behavioral procedures. (B) Frequency distribution showing individual differences in vanishing memory scores. (C) Evidence that groups designed based on calculated extinction recall scores represent two different phenotypes. (D) Mean percentage of time rats freeze for bad extinction, good extinction, and fear recall in 30 second cells of a conditioned fear reflex session, in tones 20, 30 s, collapsed into 5 blocks during the extinction learning session (4 tones ). each), and in four tones in sessions of memories of fading and memories of fear. Error bars represent the standard deviation of the mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. *р < 0,05, **р < 0,01, ***р < 0,001, ****р < 0,0001. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. *p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001。 *p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001。 *р < 0,05, **р < 0,01, ***р < 0,001, ****р < 0,0001. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
A retrograde indicator was injected into the IL (Fig. 3A) and the number of GFP+ cells along the anterior-posterior axis of the region of interest was determined (Fig. 3B-F). There was a significant difference in the number of GFP+ cells between anterior, middle and posterior PVT (X2(2) = 8.200, p = 0.017), so mPVT showed significantly more GFP+ cells than aPVT (mean rank) Diff. = 18.37, p = 0.035) and pPVT (mean rank Diff. = 17.71, p = 0.045) (Fig. 3C). Although several animals did not detect any GFP+ cells in pCLA and therefore could not map activity in this region, there was no significant difference between anterior, middle, and posterior CLA (X2(2) = 5.596, p = 0.061). Number of GFP+ cells (Figure 3D). Then, since some GFP+ cells were found in aBLA or avHPC in several rats, only the middle and back of these areas were analyzed. The middle and posterior BLA (U=393, p=0.009) differed significantly in the number of GFP+ cells, so pBLA showed more IL projections than mBLA (Figure 3E). Similarly, there was a significant difference between mid and posterior vHPCs, so pvHPCs showed more IL projections than mvHPCs (U = 403.5, p = 0.014) (Figure 3F). Figure 3G is an exemplary image showing Fos, aavRG-GFP, and double-labeled cells.
Quantify IL afferents in the entire brain region of interest. (A) Schematic representation of the distribution of aavRG-CAG-GFP in whole rat IL. (B) Representative images of retrograde markers at different anteroposterior locations in the brain region of interest. Quantification of retrograde labeling along the anteroposterior axis (C) paraventricular thalamus, (D) clavicle, (E) basolateral tonsil, and (F) ventral hippocampus. (G) Representative images showing retrograde aavRG labeling, Fos labeling, and double aavRG and Fos labeling in aPVT. Error bars represent the standard deviation of the mean. *p < 0.05, **p < 0.01. *p < 0.05, **p < 0.01. *р < 0,05, **р < 0,01. *p < 0.05, **p < 0.01. *p < 0.05，**p < 0.01。 *p < 0.05，**p < 0.01。 *р < 0,05, **р < 0,01. *p < 0.05, **p < 0.01. Scale bar 100 µm. The public domain brain map in panel A is reproduced from Swanson (2004) Brain Map: Rat Brain Structure, 3rd Edition, licensed under the Creative Commons Attribution-NonCommons 4.0 International License (https://creativecommons.org/licenses/by-nc). /4.0/) available for download at https://larrywswanson.com.
Global and IL projection-specific Fos activity was analyzed in aPVT, mPVT and pPVT in all rats. There was no significant difference between good extinction, poor extinction, fear recall, and home cell groups in Fos expression in aPVT (X2(3) = 3.888, p = 0.274) (Fig. 4A), nor was there a significant correlation between Fos in aPVT between expression and extinction recall (rs = 0.092, p = 0.691) (Figure 4B) or between Fos expression in aPVT IL afferents and extinction recall (rs = 0.143, p = 0.537) (Figure 4D). However, in aPVT IL afferents, Fos expression differed significantly between good extinction, poor extinction, fear recall, and home cell groups (X2(3) = 15.05, p = 0.002), so the fear recall group showed relatively good extinction. regression (mean rank difference = 11.54, p = 0.003), poor regression (mean rank difference = 10.57, p = 0.034), and home cell (mean rank difference = 12.79, p = 0.005) groups (Fig. 4C). Further, there was no significant difference between good extinction, poor extinction, fear recall, and home cell groups for Fos expression in mPVT (X2(3) = 2.272, p = 0.518) (Fig. 4E) and for Fos expression in mPVT. . Significant correlation with extinction recall (rs = 0.168 p = 0.468) (Figure 4F). Although there was a significant difference between the good, bad, fear recall, and home cell groups in Fos expression in IL afferent mPVT cells (X2(3) = 9.252, p = 0.026), post hoc comparison did not reveal any or two. Significant differences between groups (Figure 4G). Furthermore, there was no significant correlation between Fos expression in IL-afferent mPVT cells and extinction recall (rs = 0.174, p = 0.450) (Figure 4H). Next, there was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the pPVT (X2 (3) = 13.89, p = 0.003), such that the good extinction group (Mean Rank Diff. = 14.96, p = 0.010), but not the poor extinction (Mean Rank Diff. = 12.86, p = 0.113) or fear recall group (Mean Rank Diff. = 2.571, p > 0.999), displayed more Fos expression than the home cage group (Fig. 4I). Next, there was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the pPVT (X2 (3) = 13.89, p = 0.003), such that the good extinction group (Mean Rank Diff. = 14.96, p = 0.010), but not the poor extinction (Mean Rank Diff. = 12.86, p = 0.113) or fear recall group (Mean Rank Diff. = 2.571, p > 0.999), displayed more Fos expression than the home cage group (Fig. 4I). Далее, наблюдалась значительная разница между группами с хорошим угасанием, плохим угашением, отзывом страха и домашней клеткой в ​​экспрессии Fos в pPVT (X2 (3) = 13,89, p = 0,003), так что группа с хорошим угашением (средний ранг Diff. = 14,96, p = 0,010), но не в группе плохого угашения (средняя ранговая разница = 12,86, p = 0,113) или группы воспоминаний о страхе (средняя ранговая разница = 2,571, p > 0,999), демонстрировалась более выраженная экспрессия Fos, чем в группе группа домашних клеток (рис. 4I). Further, there was a significant difference between good extinction, poor extinction, fear recall, and home cell groups in Fos expression in pPVT (X2(3)=13.89, p=0.003), so that the good extinction group (mean rank Diff. = 14.96, p = 0.010), but not in the poor extinction group (mean rank difference = 12.86, p = 0.113) or the fear memory group (mean rank difference = 2.571, p > 0.999), showed more pronounced Fos expression than in the home cell group (Figure 4I).其次 ， ppvt 中 fos 表达 好 消 、 差消 、 恐惧 和 家笼组 之间 存在 显着 差异 （x2 （3） = 13.89 ， p = 0.003） 使得 消组 （（mean Rank Difff. = 14.96, p = 0.010)，但不是较差的消退(Mean Rank Diff. = 12.86, p = 0.113) 或恐惧回忆组(Mean Rank Diff. = 2.571, p > 0.999)，比家庭笼组（图4I）。 = 14.96, p = 0.010)，但不是较差的消退(Mean Rank Diff. = 12.86, p = 0.113) ）。 Second, there were significant differences in Fos expression in pPVT between the good, bad, fear recall, and home cell groups (X2(3) = 13.89, p = 0.003), making the good intake group (mean rank difference = 14.96). , p = 0,010), но не хуже по угашению (средняя разница рангов = 12,86, p = 0,113) или группе отзыва страха (средняя разница рангов = 2,571, p > 0,999), чем в группе домашней клетки (рис. 4I). , p = 0.010), but no worse in extinction (mean rank difference = 12.86, p = 0.113) or fear recall group (mean rank difference = 2.571, p > 0.999) than the home cell group (Figure 4I). . However, there was no significant correlation between pPVT Fos expression and extinction recall (rs = 0.051, p = 0.825) (Figure 4J). Finally, there was a significant difference in Fos expression in pPVT IL afferents between groups with good extinction, poor extinction, fear memories, and in home cells (X2(3) = 12.34 p = 0.006), so good Fos expression in IL- was worse than in the extinction groups (mean rank difference = 12.54, p = 0.014) and in the home cell (mean rank difference = 12.89, p = 0.049) (Fig. 4K) and significantly correlated with IL afferents in within pPVT Between activation and revocation of extinction, better extinction recall was associated with greater activation of these IL afferents (rs = -0.438, p = 0.047) (Figure 4L).
Fos activity was increased in IL afferents of the posterior paraventricular thalamus (PVT) in rats, which showed good regression. (A) There were no significant differences between groups in Fos expression in aPVT. (B) There was no significant correlation between Fos expression and extinction recall in aPVT. (C) The fear recall group showed increased Fos expression in IL afferents compared to all other groups. (D) There was no significant correlation between Fos expression in IL afferents and extinction recall in aPVT. (E) There were no significant intergroup differences in Fos expression in mPVT. (F) There was no significant correlation between Fos expression and extinction memory in mPVT. (G) Fos expression in afferent IL cells in mPVT did not differ significantly between groups. (H) There was no significant correlation between Fos expression in IL afferents and extinction recall in mPVT. (I) A well extinct group, but no other group, showed increased Fos activity in pPVT compared to the home cage group. (J) There was no significant correlation between Fos expression and extinction recall in pPVT. (K) The good extinction group showed increased Fos expression in IL afferent cells compared to the weak extinction group and the home cell group. (L) There is a significant correlation between Fos expression in IL afferents and extinction recall, so good extinction recall is associated with greater Fos expression in IL afferents. Error bars represent the standard deviation of the mean. *p < 0.05, **p < 0.01. *p < 0.05, **p < 0.01. *р < 0,05, **р < 0,01. *p < 0.05, **p < 0.01. *p < 0.05，**p < 0.01。 *p < 0.05，**p < 0.01。 *р < 0,05, **р < 0,01. *p < 0.05, **p < 0.01.
The global and IL projection-specific Fos activity in aCLA and mCLA of rats was then analyzed in all groups. There was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the aCLA (X2 (3) = 8.455, p = 0.036) such that the fear recall group (Mean Rank Diff. = 14.50, p = 0.049), but neither the poor (Mean Rank Diff. = 10.21, p = 0.373) nor good extinction (Mean Rank Diff. = 4.607, p > 0.999) groups, displayed more Fos expression than the home cage group (Fig. 5A). There was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the aCLA (X2 (3) = 8.455, p = 0.036) such that the fear recall group (Mean Rank Diff. = 14.50, p = 0.049), but neither the poor (Mean Rank Diff. = 10.21, p = 0.373) nor good extinction (Mean Rank Diff. = 4.607, p > 0.999) groups, displayed more Fos expression than the home cage group ( Fig. 5A). Между группами с хорошим угашением, плохим угашением, припоминанием страха и домашними клетками наблюдалась значительная разница в экспрессии Fos в aCLA (X2 (3) = 8,455, p = 0,036), так что группа припоминания страха (среднее ранговое различие = 14,50, p = 0,049), но ни плохая (средняя ранговая разница = 10,21, p = 0,373), ни группа с хорошим вымиранием (средняя ранговая разница = 4,607, p > 0,999) не демонстрировали большей экспрессии Fos, чем группа в домашней клетке ( Рис. 5А). There was a significant difference in aCLA Fos expression between the good extinction, poor extinction, fear recall and home cells groups (X2(3) = 8.455, p = 0.036), so that the fear recall group (mean rank difference = 14.50, p = 0.049), but neither the poor (mean rank difference = 10.21, p = 0.373) nor the good extinction group (mean rank difference = 4.607, p > 0.999) showed more Fos expression than the home cell group (Fig. .5A). aCLA 中Fos 表达的良好消退、消退差、恐惧回忆和家庭笼组之间存在显着差异(X2 (3) = 8.455, p = 0.036)，因此恐惧回忆组(Mean Rank Diff. = 14.50，p = 0.049），但无论是差（平均秩差= 10.21，p = 0.373）还是良好灭绝（平均秩差= 4.607，p > 0.999）组，都显示出比家庭笼组更多的Fos 表达（图5A)。 Acla 中 fos 表达 的 消退 、 消退差 、 回忆 和 家庭 笼组 之间 存在 显着 差异 差异 差异 差异 差异 (x2 (3) = 8.455, p = 0.036) ， 因此 恐惧 回忆组 回忆组 回忆组 回忆组 (mean Rank Diff. = 14.50 ， P = P = P = P = P = 14. 0.049） ， 但 无论是 差 平均 秩差 秩差 = 10.21 ， p = 0.373） 良好 灭绝 （平均 秩差 秩差 = 4.607 ， p> 0.999） 组 都 出 比 家庭 笼组 更 多 的 表达 图 图 图 5a) 。 Была значительная разница между группами с хорошим угашением, плохим угашением, отзывом страха и домашней клеткой по экспрессии Fos в aCLA (X2(3) = 8,455, p = 0,036), поэтому группа отзыва страха (среднее ранговое различие = 14,50) , p = 0,049), но группы с плохим (средняя разница рангов = 10,21, p = 0,373) и с хорошим вымиранием (средняя разница рангов = 4,607, p > 0,999) показали более высокую экспрессию Fos, чем группа с домашней клеткой (рис. 5A). There was a significant difference between good extinction, poor extinction, fear recall and home cell groups in aCLA Fos expression (X2(3) = 8.455, p = 0.036), so fear recall group (mean rank difference = 14.50), p = 0.049), but groups with poor (mean rank difference = 10.21, p = 0.373) and good extinction (mean rank difference = 4.607, p > 0.999) showed higher Fos expression than the home cell group (Fig. 5A). . There was no significant correlation between global Fos expression in (rs = 0.036, p = 0.876) (Figure 5B) or Fos expression in IL aCLA afferent cells (rs = -0.282, p = 0.215) and extinction recall (Figure 5B). .5D), there was also no significant difference between good extinction, poor extinction, fear recall, and home cell groups in Fos expression in aCLA IL afferents (X2(3) = 6.722, p = 0.081) (Figure 5C). . ). Next, there was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the mCLA (X2 (3) = 10.12, p = 0.018) such that the good extinction group (Mean Rank Diff. = 12.93, p = 0.038), but neither the poor extinction (Mean Rank Diff. = 5.143, p > 0.999) nor fear recall groups (Mean Rank Diff. = 14.00, p = 0.063) displayed significantly more Fos expression in the mCLA relative to the home cage group (Fig. 5E). Next, there was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the mCLA (X2 (3) = 10.12, p = 0.018) such that the good extinction group (Mean Rank Diff . = 12.93, p = 0.038), but neither the poor extinction (Mean Rank Diff. = 5.143, p > 0.999) nor fear recall groups (Mean Rank Diff. = 14.00, p = 0.063) displayed significantly more Fos expression in the mCLA relative to the home cage group (Fig. 5E). Затем наблюдалась значительная разница между группами с хорошим угашением, плохим угашением, воспоминаниями о страхе и домашней клеткой в ​​экспрессии Fos в mCLA (X2 (3) = 10,12, p = 0,018), так что группа с хорошим угашением (средняя разница рангов . = 12,93, p = 0,038), но ни группы плохого угашения (средняя ранговая разница = 5,143, p > 0,999), ни группы отзыва страха (средняя ранговая разница = 14,00, p = 0,063) не показали значительно большей экспрессии Fos в mCLA. There was then a significant difference between good extinction, poor extinction, fear memories and home cell groups in mCLA Fos expression (X2(3) = 10.12, p = 0.018), so that the good extinction group (mean difference rank = 12.93, p = 0.038), but neither the poor extinction groups (mean rank difference = 5.143, p > 0.999) nor the fear recall groups (mean rank difference = 14.00, p = 0.063) showed significantly greater Fos expression in mCLA. compared to the home cage group (Figure 5E).接下来 ， 在 mcla 中 的 fos 表达 ， 良好 消退组 、 不良 消 退组 、 恐惧 回忆组 家庭 笼组 之间 显着 差异 （x2 （3） = 10.12 ， p = 0.018） ， 良好 （（（（（（（（（ Mean Rank Diff . = 12.93, p = 0.038)，但在mCLA 中，弱消退（平均秩差= 5.143，p > 0.999）和恐惧回忆组（平均秩差= 14.00，p = 0.063）均未显示出更多的Fos 表达相对于家庭笼组（图5E）。 = 12.93, p = 0.038) ， 在 在 mcla 中 ， 弱消退 （平均 秩差 秩差 秩差 = 5.143 ， p> 0.999） 和 回忆组 （平均 秩差 = 14.00 ， p = 0.063） 未 显示 出 更 多 的 fos 表达 表达 的 fos 表达 表达 的 fos 表达相对于家庭笼组（图5E）。 Далее, в экспрессии Fos в mCLA наблюдалась значительная разница между группой с хорошим угасанием, группой с плохим угасанием, группой с отзывом о страхе и группой с домашней клеткой (X2(3) = 10,12, p = 0,018), так, группа хорошего угашения (средняя разность рангов = 12,93, p = 0,038), но в mCLA ни слабое угасание (средняя разница рангов = 5,143, p > 0,999), ни группа отзыва страха (средняя разница рангов = 14,00, p = 0,999) = 0,063) показали лучшую экспрессию Много Fos по сравнению с группой с домашней клеткой (Рисунок 5E). Further, there was a significant difference in Fos expression in mCLA between the good extinction group, the poor extinction group, the fear feedback group, and the home cell group (X2(3) = 10.12, p = 0.018), thus, the good extinction (mean rank difference = 12.93, p = 0.038), but in mCLA neither weak extinction (mean rank difference = 5.143, p > 0.999) nor fear recall group (mean rank difference = 14.00, p = 0.999) = 0.063) showed better Multi Fos expression compared to the home cell group (Figure 5E). However, global Fos expression in mCLA (rs = 0.321, p = 0.156) (Fig. 5F) or in afferent IL mCLA cells (rs = -0.121, p = 0.602) and extinction recall (Fig. 5H), no significant difference between groups with good extinction, poor extinction, fear recall, and a home cell for Fos expression in IL mCLA afferent cells (X2(3)=4.923, p=0.178) (Figure 5G).
Fos activity was elevated in the mid-claustrum in rats with good extinction memory. (A) The fear recall group, but not the other groups, showed increased Fos activity compared to the home cell group in aCLA. (B) There was no significant correlation between Fos expression in aCLA and extinction recall. (C) Fos expression in IL afferent aCLA cells did not differ significantly between groups. (D) There was no significant correlation between Fos expression in IL afferents and extinction recall in aCLA. (E) The well extinct group, but not the other groups, showed increased Fos activity in mCLA compared to the home cell group. (F) There was no significant correlation between Fos expression and extinction recall in mCLA. (G) Fos expression in IL mCLA afferent cells did not differ significantly between groups. (H) There was no significant correlation between Fos expression in IL afferents and extinction recall in mCLA. Error bars represent the standard deviation of the mean. *p < 0.05. *p < 0.05. *р < 0,05. *p < 0.05. *p < 0.05。 *p < 0.05。 *р < 0,05. *p < 0.05.
Then, global and IL projection-specific Fos activity in mBLA and pBLA was analyzed in all groups of rats. There was no significant difference between good extinction, poor extinction, fear recall, and home cell groups in Fos expression in mBLA (X2(3)=0.944, p=0.815) (Figure 6A). There was also no significant difference between groups with good regression, poor regression, fear recall, and home cell Fos expression in IL mBLA afferent cells (X2(3)=0.518, p=0.915) (Figure 6C). In addition, there was no significant correlation between global Fos expression in mBLA (rs = 0.126, p = 0.588) (Figure 6B) and Fos expression in IL mBLA afferent cells (rs = 0.200, p = 0.385) (rs = 0.200, p = 0.385). p = 0.385). Figure 6D) and extinction recall. There was also no significant difference in good extinction, poor extinction, fear memory, and home cell groups in Fos expression in pBLA (X2(3) = 4.246, p = 0.236) (Fig. 6E), and there was also no significant difference in pBLA Good. extinction, poor extinction, fear recall, and home cell groups in Fos expression in IL afferent cells (X2(3)=1.954, p=0.582) (Figure 6G). Finally, global Fos expression in pBLA (rs = 0.070, p = 0.762) (Fig. 6F) and Fos expression in pBLA IL afferent cells (rs = 0.122, p = 0.597) and extinction recall (Fig. 6H).
Individual differences in extinction reproduction were not mapped to differences in Fos expression in the basolateral amygdala. (A) There were no significant intergroup differences in Fos expression in mBLA. (B) There was no significant correlation between Fos expression and extinction recall in mBLA. (C) Fos expression in IL mBLA afferent cells did not differ significantly between groups. (D) There was no significant correlation between Fos expression in IL afferent cells and extinction recall in mBLA. (E) There were no significant intergroup differences in Fos expression in pBLA. (F) There was no significant correlation between Fos expression and extinction recall in pBLA. (G) Fos expression in afferent IL pBLA cells did not differ significantly between groups. (H) There was no significant correlation between Fos expression in IL afferent cells and extinction recall in pBLA. Error bars represent the standard deviation of the mean.
Finally, global and IL projection-specific Fos activity was analyzed in mvHPC and pvHPC in all rats. There was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the mvHPC (X2 (3) = 8.056, p = 0.045) such that the good extinction (Mean Rank Diff. = 13.29, p = 0.031), but neither poor extinction (Mean Rank Diff. = 6.857, p > 0.999) nor fear recall (Mean Rank Diff. = 8.000, p = 0.864) groups showed more Fos expression than the home cage group (Fig. 7A). There was a significant difference among the good extinction, poor extinction, fear recall, and home cage groups in Fos expression in the mvHPC (X2 (3) = 8.056, p = 0.045) such that the good extinction (Mean Rank Diff. = 13.29 , p = 0.031), but neither poor extinction (Mean Rank Diff. = 6.857, p > 0.999) nor fear recall (Mean Rank Diff. = 8.000, p = 0.864) groups showed more Fos expression than the home cage group (Fig. 7A). Между группами с хорошим угасанием, плохим угашением, отзывом страха и домашними клетками наблюдалась значительная разница в экспрессии Fos в mvHPC (X2 (3) = 8,056, p = 0,045), так что хорошее угасание (средняя ранговая разница = 13,29) , p = 0,031), но ни в группах с плохим угасанием (средняя ранговая разница = 6,857, p > 0,999), ни в группе с отзывом страха (средняя ранговая разница = 8,000, p = 0,864) экспрессия Fos была выше, чем в группе с домашней клеткой (рис. 7А). There was a significant difference in mvHPC Fos expression between groups with good extinction, poor extinction, fear recall, and home cells (X2(3) = 8.056, p = 0.045), so good extinction (mean rank difference = 13.29), p = 0.031), but neither in the poor extinction group (mean rank difference = 6.857, p > 0.999) nor in the fear recall group (mean rank difference = 8.000, p = 0.864) was Fos expression higher than in the home group. cell (Fig. 7A). mvHPC 中Fos 表达的良好消退、不良消退、恐惧回忆和家庭笼组之间存在显着差异（X2 (3) = 8.056, p = 0.045），因此良好消退（平均秩差= 13.29） ，p = 0.031），但无论是弱消退（平均秩差= 6.857，p > 0.999）还是恐惧回忆（平均秩差= 8.000，p = 0.864）组都显示出比家庭笼组更多的Fos 表达（图2）。 mvhpc 中 fos 表达 的 消退 、 不良 消退 恐惧 回忆 和 家庭 笼组 之间 存在 显着 差异 （（x2 (3) = 8.056, p = 0.045） ， 良好 （（平均 秩差 秩差 = 13.29） p = 0.031 ） ， 但 无论是 弱消退 （平均 秩差 秩差 = 6.857 ， p> 0.999） 还是 恐惧 （平均 秩差 秩差 = 8.000 ， p = 0.864） 组都 出 家庭 笼组 多 的 fos 表达 图 图。。。。））））））））））））））））））））））））））））））））））） Имелась значительная разница между группами «хорошо», «плохо», «припоминание страха» и «домашняя клетка» для экспрессии Fos в mvHPC (X2(3) = 8,056, p = 0,045) и, следовательно, хорошая регрессия (средняя разница рангов = 13,29), p = 0,031), но группы со слабым угасанием (средняя разница рангов = 6,857, p > 0,999) и воспоминания о страхе (средняя разница рангов = 8,000, p = 0,864) показали более высокую экспрессию Fos, чем группа в домашней клетке (рис. 2). There was a significant difference between the good, bad, fear recall, and home cell groups for Fos expression in mvHPC (X2(3) = 8.056, p = 0.045) and therefore a good regression (mean rank difference = 13.29), p = 0.031), but groups with weak extinction (mean rank difference = 6.857, p > 0.999) and fear memories (mean rank difference = 8.000, p = 0.864) showed higher Fos expression than the group in home cage (Fig. 2). 7A). However, there was no significant difference (X2(3) = 4.893, p = 0.180) (Figure 7C). In addition, there was no significant correlation between global Fos expression in mvHPC (rs = -0.233, p = 0.309) (Figure 7B) and Fos expression in mvHPC afferent IL cells (rs = 0.056, p = 0.810) (Figure 7D) . and a report on the disappearance. Further, there was no significant difference between good extinction, poor extinction, fear recall, and home cell groups in Fos expression in pvHPC (X2(3) = 3.623, p = 0.353) (Figure 7E), and there were no significant differences. difference in good regression of Fos expression in pvHPC afferent IL cells, poor regression, fear memory, and home cell groups (X2(3)=3.871, p=0.276) (Fig. 7G). Finally, there was no significant correlation between pvHPC global Fos expression (rs = −0.127, p = 0.584) (Figure 7F) and Fos expression in IL-afferent pvHPC cells (rs = 0.176, p = 0.447) and extinction recall (Figure 7F). 7H).
Fos expression is elevated in the ventral hippocampus of rats, indicating good memory extinction. (A) The well extinct group, but not the other groups, showed increased expression of Fos in mvHPC compared to the home cell group. (B) There was no significant correlation between Fos expression and extinction recall in mvPHC. (C) Fos expression in mvHPC afferent IL cells did not differ significantly between groups. (D) There was no significant correlation between Fos expression in IL afferents and extinction recall in mvHPC. (E) There were no significant differences between groups in Fos expression in pvHPC. (F) There was no significant correlation between Fos expression and extinction recall in pvHPC. (G) Fos expression in pVHPC afferent IL cells did not differ significantly between groups. (H) There was no significant correlation between Fos expression in IL afferents and extinction recall in pvHPC. Error bars represent the standard deviation of the mean. *p < 0.05. *p < 0.05. *р < 0,05. *p < 0.05. *p < 0.05。 *p < 0.05。 *р < 0,05. *p < 0.05. Our primary analysis for all regions showed comparison at three levels along the anteroposterior axis, although we also analyzed each region that collapsed along the anteroposterior axis. The results of these analyzes are presented in table 1.
Here we tested whether individual differences in extinction recall would be reflected in different patterns of afferent activity in the lower limbic cortex. To this end, we assessed Fos activity in IL projections from the paraventricular thalamus, claustrum, basolateral tonsil, and ventral hippocampus after extinction replay. In IL-projecting cells, we found higher activity in the posterior region of PVT in rats that exhibited good extinction recall compared to rats with poor extinction. There were no differences in IL afferents from the clavicular nucleus, ventral hippocampus, or basolateral tonsil. In addition to IL-projecting cells, increased neural activity was observed in selected areas of the rat claustrum and ventral hippocampus with good resolution. Our results indicate that successful extinction memory is organized by specific PVT projections to IL and non-IL-targeting cells in the claustrum and ventral hippocampus.
We found that PVT IL prediction was active in rats that showed good extinction recall, which is consistent with a recent study showing that PVT is required for extinction recall. This study did not use subregion-specific manipulations, but showed that both PVT projections on the lateral central amygdala and IL projections on PVT were needed to reproduce extinction. Our results indicate that, in addition to the IL-PVT-CeL chain, a post-PVT entry into IL may also be required for quench recall. Thus, it turns out that both efferent and afferent IL connections are involved in the reproduction of extinction. An important next step is to determine what causes pPVT to signal extinction reproduction at the neural circuit level. In addition to the relationship with IL, previous duct-tracking studies31,32 have shown that pPVT receives input from the ventral periaqueductal gray (vPAG), which is associated with extinction learning33,34,35,36. Although the role of vPAG in extinction recall has not been established, the prediction of pPVT by vPAG is an attractive candidate due to their density and the involvement of both regions in generating previous evidence of fear extinction.
Another important aspect of our PVT results is that they are specialized along their anterior-posterior axis. Strikingly, neuronal activity of the PVT projection in IL correlates with the opposite behavioral state, such that pre-PVT projection activity in IL is associated with fear recall, while pPVT projection is active after successful recall extinction (i.e., fear). This functional heterogeneity within the PVT is not surprising given previous work [discussed in 37]. A striking example of functional distribution in PVT has recently emerged in a study that has characterized the properties of specific cell types in PVT. This study shows that DRD2-expressing dopamine cells are predominantly expressed in pPVT, innervate the forelimb cortex, and respond to aversive stimuli. The second cell population is predominantly expressed in aPVT and marks the transition to a state of low physiological arousal and innervates the cortex of the lower limbs. Our results hardly fit into this pattern, as IL-projecting aPVT cells are active during fear recall, while pPVT projections are active and animals show low levels of fear. There are at least two possible explanations for the apparent discrepancy. First, the identified cell types are not located exclusively in one anterior-posterior site of the TVV. Thus, active IL-projecting pPVT cells in rats with good extinction memory may belong to a class of cells that are more likely to be detected in aPVT and signal a transition to a low arousal state. The same may be true for IL-projecting cells in aPVT activated after fear memory. Second, previous tracing studies have identified the presence of IL3-projecting pPVTs, although some appear to be derived from DRD2-containing cells, other cell types may project to IL and become activated upon successful reproduction of quenching.
Although the aim of this study was to identify differences between rats exhibiting different extinction phenotypes, these experiments also revealed new data related to fear memory mechanisms. Interestingly, we found increased Fos activity at the anterior CLA in rats with fear memory.
The clavicle is positioned as the center of cortical communication and is involved in processes ranging from sensory integration to attention and sleep40,41,42,43. There is limited evidence on how the claustrum is involved in fear conditioning or fear expression, however, earlier research has shown that contextual fear expression is involved in Fos activity in the claustrum. It has recently been reported that inhibition of atresia projections to the entorhinal cortex during contextual fear conditioning impairs long-term memory formation, although their need for fear expression has not been tested. In the same study, increased Fos activation was observed when animals were exposed to a new environment compared to mice exposed to a familiar environment. With this in mind, the CLA activation we report here may be due to exposure to the new camera during testing, rather than fear of recall itself. To more accurately characterize the function of locks in fear and situational processing, future studies should use targeted lock manipulation.
Although previous work has shown that PVT is associated with fear memory expression,45,46,47 we did not observe any change in total Fos expression in rats when they recalled fear 48 hours after conditioning. This difference can be explained by several factors, including previous work testing fear of discrete cues in the same context in which the conditioning took place, whereas in our experiment, testing was done in a new room. In addition, we euthanized our animals 60 minutes after testing, while the previous work used a time point of 90 minutes. Finally, in previous studies, the test was carried out in a room where the animals could respond with an appetite, whereas in our work, rats were tested without an appetite response. While this allows for some degree of conditioned inhibition, there is evidence that allowing animals to inhibit pressure to obtain food while testing whether they fear cues creates motivational conflict (i.e., fear versus reward), which is a key stimulating factor. participation PVT48, 49. .
The basolateral amygdala is known to be involved in the acquisition of fear extinction50,51 and there is evidence that BLA projections to IL are also involved in this process23. However, it is not clear if the BLA and its connections are involved in the extinction return. Imaging studies23,28 have shown increased BLA activity in animals recalling faded memories. While our previous work showed no difference in BLA activation between good and bad extinction rats, our results here suggest that extinction recall generally does not affect BLA or activation in predicting BLA IL. Consistent with our findings, although circuit manipulation studies suggest that IL inputs to the BLA are important for extinction learning, they are not necessary for extinction recall. However, the role of the BLA cannot be completely ignored as recent evidence suggests that certain cell types in the BLA are required to reproduce extinction.
Notably, fear recall did not result in Fos activation in the BLA, as previous lesion, drug, and imaging studies have implicated this area in fear expression and/or fear reconsolidation after retrieval54,55,56,57. The data presented here combine basal and lateral subnucleus of the amygdala, and previous data suggest that fear expression drives Fos activity on the dorsal side of the lateral nucleus. We analyzed the underlying and lateral data separately, but there was no difference in either case (data not shown) and both areas collapsed in the data we present here. We did not analyze subregions of the lateral amygdala, so specific changes in this area may be masked. Another possibility of no change in Fos activity in BLA is due to timing of fear memories compared to conditioning. Some previous work has shown that the contribution of BLA to fear expression decreases with time after conditioning, such that expression is BLA dependent at 24 hours post conditioning but independent at 7 days (ref. 45 but see 58). occurred 48 hours after training, making the lack of change in Fos activity at this time point likely reflect time-dependent changes in BLA participation in fear expression.
Finally, we find evidence that successful extinction memory is associated with the ventral hippocampus. This is characteristic of “intermediate” vHPCs as the same pattern was not seen in the posterior region. Consistent with previous work, we found no change in Fos activation in afferent vHPC ILs. There is substantial evidence that vHPC28,60,61 is required for fear actualization when CS occurs outside the context in which fading occurs, and that this depends at least in part on vHPC entry into IL13. Based on these previous results, we would expect poor fading to be associated with increased predicted IL activity of vHPC. However, this was not the case as there was no difference in Fos activity in IL-projected retrograde-labeled vHPCs or unlabeled cells in vHPCs. This suggests that the inability to remember fading in the context of fading may trigger a different mechanism than the fear of renewal.
It is important to note some of the inherent design and analysis limitations and how they affect our conclusions. First, we divided animals into upper and lower thirds, and mice into “good” and “bad” based on extinction recall scores. This is done in order to avoid group schemes that divide animals into separate groups from the middle of the distribution, or group schemes that exclude animals from the middle of the distribution, such as separation by median or comparison of the upper and lower thirds of rats. we want to avoid this situation because the median division does not reflect the variability of human responses to trauma that we are trying to model. In addition, while comparing the upper and lower thirds of rats allows us to compare groups of similar size, this approach ignores animals at the center of distribution and does not accurately reflect variability in reactions to injury. While our method may suffer from problems with heterogeneous variance and comparing groups with unequal sample sizes, it captures what we are trying to mimic better than alternative methods.
The results presented here help us better understand how individual differences in extinction recall are reflected in differences in neural circuit activity. Our findings may be relevant to post-traumatic stress disorder, which is known to be associated with excessive fear and an inability to eliminate fear responses. We show that differences in extinction recall are associated with differences in intrinsic and extrinsic neural activity projected onto IL. These differences were distributed across distinct regions along the anteroposterior axis, further highlighting the importance of assessing brain function at the subregional level. The disadvantages of the current method include the relevance of the study and the focus on male rodents. Future research should determine the neurobiological mechanisms underlying extinction learning in female rodents and use methods to draw causal inferences.
The datasets used and/or analyzed in the current study are available from the respective authors upon reasonable request.
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