Reduced adult neurogenesis alters behavioural and endocrine discriminative fear conditioning

Overview: The purpose of these experiments was to investigate the role of adult hippocampal neurogenesis in behavioural pattern separation, the ability to perceive, learn and remember fine details of sensory experience. And avoid interference between related experiences. Here, we have used discriminative context fear conditioning to probe this putative function for adult-born neurons. In these paradigms mice or rats are tested in their ability to discriminate (based on their freezing behaviour) a context paired with footshock from a different context that was not paired with footshock.
In the first experiment (“circles vs stripes”, Fig 1) we chose a difficult discrimination test, where contexts were very similar except for the pattern on the walls. Wild type mice and neurogenesis-deficient GFAP-TK mice did not “learn” this discrimination. However, WT mice did show discrimination on a test 1 week later, supporting a role for adult neurogenesis in context discrimination/pattern separation and perhaps also in memory consolidation.
In the second experiment (“mo diff”, Fig 2) we made the contexts more distinct from each other (more different à “mo diff”) to enable WT mice to discriminate during training, and determine whether TK mice showed deficits during the learning phase. They did not (if anything TK mice may have been better discriminators). All mice performed rather poorly in the 1 week tests, however.
In the third experiment (“stress + mo diff”, Fig 3) we repeated the mo diff discrimination but subjected mice to chronic stress prior to training, since chronic stress can enhance fear conditioning (and therefore may enhance 1w memory relative to mo diff) and since neurogenesis-deficient mice have an altered stress response and may learn differently following stress. Here, WT and TK mice both showed strong, similar levels of behavioural discrimination.
Notables:
All WT and TK mice were tested 1 week after training in both contexts in a counterbalanced fashion (i.e. ½ tested in shock context then in safe context, other ½ in safe context then in shock context). The testing order had significant effects on behaviour that were obscured when the tests were pooled. For example, on test 1 in the “circles vs stripes” experiment, WT mice discriminated but TK mice did not. In test 2, WT mice again discriminated but in the “wrong” direction (i.e. they froze more in the safe context, suggesting a carryover effect from test 1; note this effect on test 2 did not reach p < 0.05).
In all experiments, in addition to performing behavioural analyses, we also took blood samples after tests 1 and 2 to measure the stress hormone, corticosterone. In some cases corticosterone levels matched behavioural performance, in some cases it didn’t. TK mice had much higher levels of corticosterone in the “mo diff’ experiment (Fig. 2E).
Figure 1: Circles vs Stripes
A) Methods overview: These experiments tested the ability of wild type (N = 12) and neurogenesis-deficient mice (GFAP-TK mice, N = 19) to discriminate 2 contexts in a fear conditioning paradigm. Both contexts were Med Associates fear conditioning chambers that had their 4 walls lined with black and white circle or stripe patterns (mouse always in same chamber, but panels changed). Both patterns were 50% black and 50% white and the 2 contexts did not differ in any other way. Mice were treated for ~12 weeks (beginning at 8 weeks of age) with valganciclovir as described (Snyder, Nature 2011), which completely inhibited adult neurogenesis. Mice were handled daily for 1 week and then given 10 min habituation sessions to each of the 2 contexts (circle in the AM, stripe in the PM). The day following habituation mice were trained for 5 days on the fear conditioning paradigm. Mice were placed in the safe context in the AM for 3 min and in the shock context in the PM for 3 min. In the PM session, mice received a footshock at 2:28 (2s, 0.75mA). Contexts were counterbalanced such that for half of the mice the stripe context was paired with footshock. One week after training half of the mice (i.e. 6 WT, 9 TK) were given a 3min test in the shock context and, the next day, a 3min test in the safe context. The other half of the mice were given a 3min test in the safe context and, the next day, a 3min test in the shock context.
B) Habituation: No significant genotype or context differences in freezing (2 way RM ANOVA)
C) Training: Both WT and TK mice showed an increase in freezing over days but neither genotype showed increased freezing in the shock context (1st 2:28 of each training session shown, i.e. prior to shock). A discrimination index was used to compare genotypes but no significant differences were found. Index = (shock – safe) / (shock + safe); positive values indicate discriminative fear memory.
D) 1 week behaviour tests: When the 1w tests were pooled, there was no context fear discrimination in either genotype (bar graph). However, analyzing by day & context, WT mice froze significantly more when first tested in the shock context (an effect that carried over to the next day’s test, when they were tested in the safe context). The unemoticons indicate a significant group effect and significant difference on day 1 (2 way ANOVA with Bonferroni post hoc, both P < 0.05). TK mice showed no behavioural discrimination.
E) Corticosterone was measured 30min after the 2 tests (submandibular blood samples). WT mice showed discriminative stress hormone levels on the day 2 test (genotype x context interaction P = 0.05, Bonferroni post hoc P < 0.05)
Figure 2: “Mo Diff”
A) Methods Overview: Since mice failed to show behavioural context discrimination during training in the circles vs stripes experiment, contexts were adjusted to make them more different, hence “mo diff”. The paradigm is the same as the circles vs stripes experiment except that the shock context was a standard fear conditioning chamber with house and cue lights on, no fan, and was cleaned with dilute Simple Green. The safe context had a solid floor, curved blue walls, houselight only, and was cleaned with vanilla-scented ethanol. For obvious reasons, context-footshock pairing was not counterbalanced. N = 11 WT, 13 TK.
B) Habituation: Mice froze significantly more in the safe context during habituation (context x genotype RM ANOVA, effect of context indicated by the number 2 plastics recycling symbol, P < 0.05).
C) Training: WT and TK mice showed increased freezing over days and a significant context x day interaction (2 way RM ANOVA both P < 0.001). Mice froze significantly more in the shock context during the latter phases of training, as indicated by the crossed swords (Fisher’s LSD, P < 0.05). Context discrimination indices were not different between WT and TK mice, but TK mice showed significantly greater discrimination indices than chance on days 3, 4 and 5, as indicated by the word FAX (one sample t-test).
D) 1 week behaviour tests revealed significantly greater freezing in the shock context than in the safe context (context x genotype RM ANOVA, effect of context P < 0.05, indicated by the snowman). There was a group x day interaction (P < 0.05) such that mice that were first tested in the safe context froze significantly more in the shock context on day 2 (Fisher’s LSD P < 0.05, both WT and TK mice, indicated by the anchors).
E) Pooled 1w corticosterone levels were significantly greater in TK mice than in WT mice (context x genotype RM ANOVA, effect of genotype P < 0.01, indicated by the Israeli new Shekel symbol). Context x genotype ANOVAs on each testing day reveal that, in the shock context on day 1 and in the safe context on day 2, TKs had significantly greater corticosterone levels than WT mice (data aren’t really graphed to illustrate this).
Figure 3: chronic stress + Mo Diff
A) Methods overview: Methods identical to “mo diff” with the exception that mice were subjected to 3 weeks of daily 2hr restraint stress prior to fear conditioning (same mice as in Snyder, Nature 2011 Fig. 2A,B). N = 34 WT, 36 TK.
B) Habituation: Mice froze significantly more in the safe context, in the PM/evening (genotype x context RM ANOVA, main effect of context P < 0.0001, indicated by the number 5 plastics recycling symbol).
C) Training: For both WT and TK mice, 2 way RM ANOVAs revealed significant effects of context (P < 0.05), day (P < 0.0001) and significant interactions (P < 0.0001). Both genotypes froze more in the shock context on days 2-5 (Fisher’s LSD P < 0.001 for all; indicated by yield signs). Comparing freezing discrimination indices, there was no difference between genotypes.
D) 1 week behaviour tests: Pooling the 1w tests, mice froze significantly more in the shock context than in the safe context (P < 0.001, indicated by the “drug free zone” sign) and there was no difference between genotypes. Broken down by context testing order, WT mice showed a significant group x day interaction (RM ANOVA, P < 0.0001). WT mice successfully discriminated on test day 1 but not day 2 (see figure for post hoc details). TK mice also showed a group x day interaction (P < 0.0001), discriminating on both day 1 and day 2.
E) Corticosterone levels: Blood samples were obtained 30min after the 1w tests for corticosterone analyses but we never got there. Hence, no panel E.

概述：本实验旨在探究成年海马神经发生对行为模式分离的作用，即感知、学习与记忆感官体验细微细节的能力，以及避免相关经验间的相互干扰。本研究采用辨别性情境恐惧条件反射来检验成年神经元所具有的潜在功能。在这些范式下，对小鼠或大鼠进行测试，以评估它们在辨别（基于其冻结行为）与足部电击配对的情境与未配对足部电击的不同情境的能力。在第一项实验（“圆形与条纹”，图1）中，我们选择了一项困难的辨别测试，其中情境非常相似，唯一的区别在于墙壁上的图案。野生型小鼠和神经发生缺陷型GFAP-TK小鼠并未“学习”这种辨别。然而，野生型小鼠在1周后的测试中表现出了辨别能力，这支持了成年神经发生在对情境辨别/模式分离以及可能也是对记忆巩固过程中的作用。在第二项实验（“mo diff”，图2）中，我们将情境之间的差异进一步扩大（更不同，即“mo diff”），以便野生型小鼠在训练期间进行辨别，并确定TK小鼠在学习阶段是否表现出缺陷。它们没有（如果有的话，TK小鼠可能成为了更好的辨别者）。然而，在1周后的测试中，所有小鼠的表现都相当差。在第三项实验（“压力+mo diff”，图3）中，我们重复了mo diff辨别测试，但在训练前对小鼠施加了慢性压力，因为慢性压力可以增强恐惧条件反射（因此可能增强相对于mo diff的1周记忆），并且由于神经发生缺陷型小鼠的应激反应改变，它们在压力后的学习可能有所不同。野生型小鼠和TK小鼠在行为辨别方面均表现出强烈且相似的水平。值得注意：所有野生型和TK小鼠在训练后一周内在两种情境中以平衡的方式进行了测试（即一半在电击情境后测试安全情境，另一半在安全情境后测试电击情境）。测试顺序对行为有显著影响，当测试合并时，这种影响被掩盖。例如，在“圆形与条纹”实验的第1次测试中，野生型小鼠进行了辨别，但TK小鼠没有。在第2次测试中，野生型小鼠再次进行了辨别，但方向“错误”（即在安全情境中冻结更多，这表明第1次测试的延续效应；请注意，这种效应在第2次测试中没有达到p < 0.05）。在所有实验中，除了进行行为分析外，我们还在测试1和2后采集血液样本以测量应激激素皮质酮。在某些情况下，皮质酮水平与行为表现相匹配，在另一些情况下则不匹配。在“mo diff”实验中（图2E），TK小鼠的皮质酮水平显著高于野生型小鼠。图1：圆形与条纹A）方法概述：这些实验测试了野生型小鼠（N = 12）和神经发生缺陷型小鼠（GFAP-TK小鼠，N = 19）在恐惧条件反射范式中辨别两种情境的能力。两种情境均为Med Associates恐惧条件反射室，其四壁均用黑白圆形或条纹图案装饰（小鼠始终处于同一室内，但面板会改变）。两种图案均为50%黑色和50%白色，两种情境在其他方面没有区别。小鼠在12周内（从8周龄开始）接受valganciclovir治疗，如所述（Snyder，Nature 2011），这完全抑制了成年神经发生。小鼠每天处理1周，然后对两种情境各进行10分钟的习惯化训练（上午为圆形，下午为条纹）。习惯化后的一天，小鼠在恐惧条件反射范式中进行了5天的训练。小鼠在上午将安全情境放置3分钟，下午将电击情境放置3分钟。在下午的会话中，小鼠在2:28时接受了2秒、0.75毫安的足部电击。情境以平衡的方式配对，即一半的小鼠将条纹情境与足部电击配对。在训练后一周，一半的小鼠（即6只野生型小鼠，9只TK小鼠）在电击情境中进行了3分钟的测试，第二天在安全情境中进行了3分钟的测试。另一半的小鼠在安全情境中进行了3分钟的测试，第二天在电击情境中进行了3分钟的测试。B）习惯化：无显著的基因型或情境差异在冻结（2向重复测量方差分析）C）训练：野生型和TK小鼠在几天内都表现出冻结的增加，但两种基因型在电击情境中都没有表现出增加的冻结（每个训练会话的前2:28显示，即在电击之前）。使用辨别指数来比较基因型，但没有发现显著差异。指数 =（电击-安全）/（电击+安全）；正值表示辨别性恐惧记忆。D）1周行为测试：当将1周测试合并时，两种基因型都没有情境恐惧辨别（条形图）。然而，按天数和情境分析，当首次在电击情境中测试时，野生型小鼠的冻结显著增加（这种效应延续到第二天测试，当时他们在安全情境中测试），未表情符号表示有显著的小组效应和第1天的显著差异（2向重复测量方差分析，Bonferroni事后检验，P < 0.05）。TK小鼠没有表现出行为辨别。E）皮质酮的测量：在2次测试后30分钟测量皮质酮（下颌下血液样本）。野生型小鼠在第二天测试中表现出辨别性应激激素水平（基因型×情境交互作用P = 0.05，Bonferroni事后检验P < 0.05）。图2：“Mo Diff”A）方法概述：由于在“圆形与条纹”实验中，小鼠在训练期间未能表现出行为情境辨别，因此调整情境以使它们之间更加不同，即“mo diff”。范式与“圆形与条纹”实验相同，但电击情境为一个标准的恐惧条件反射室，室内有房屋和提示灯光开启，无风扇，并用稀释的Simple Green清洁。安全情境有一个实心地板，蓝色曲线墙壁，只有房屋灯光，并用香草味的乙醇清洁。由于显而易见的原因，情境-足部电击配对没有进行平衡。N = 11 WT，13 TK。B）习惯化：在习惯化期间，小鼠在安全情境中的冻结显著增加（情境×基因型重复测量方差分析，情境的主要效应由数字2的塑料回收标志表示，P < 0.05）。C）训练：野生型和TK小鼠在几天内都表现出冻结的增加，并且情境×天数重复测量方差分析显示有显著效应（P < 0.001）。小鼠在训练的后阶段在电击情境中表现出显著的冻结增加，如交叉剑符号所示（Fisher的LSD，P < 0.05）。野生型和TK小鼠之间的情境辨别指数没有差异，但TK小鼠在第3、4和5天显示出比随机更高的辨别指数，如FAX一词所示（单样本t检验）。D）1周行为测试显示，在电击情境中的冻结显著高于在安全情境中的冻结（情境×基因型重复测量方差分析，情境的效应P < 0.05，由雪人符号表示）。存在组×天数交互作用（P < 0.05），即在第一天测试时，首先在安全情境中测试的小鼠在第二天在电击情境中的冻结显著增加（Fisher的LSD P < 0.05，野生型和TK小鼠，由锚符号表示）。E）1周皮质酮水平汇总：TK小鼠的皮质酮水平显著高于野生型小鼠（情境×基因型重复测量方差分析，基因型的效应P < 0.01，由以色列新谢克尔符号表示）。每个测试日的情境×基因型方差分析显示，在第1天的电击情境和第2天的安全情境中，TK小鼠的皮质酮水平显著高于野生型小鼠（数据未真正绘图以说明这一点）。图3：慢性压力+Mo DiffA）方法概述：方法与“mo diff”相同，但小鼠在恐惧条件反射之前每天接受3周的2小时束缚压力（与Snyder，Nature 2011图2A,B中的小鼠相同）。N = 34 WT，36 TK。B）习惯化：小鼠在安全情境中冻结显著增加，在下午/晚上（基因型×情境重复测量方差分析，情境的主要效应P < 0.0001，由数字5的塑料回收标志表示）。C）训练：对于野生型和TK小鼠，2向重复测量方差分析显示情境（P < 0.05）、天数（P < 0.0001）和显著交互作用（P < 0.0001）的显著效应。两种基因型在2-5天时在电击情境中冻结更多（Fisher的LSD P < 0.001对所有；由指示标志表示）。比较冻结辨别指数，两种基因型之间没有差异。D）1周行为测试汇总：小鼠在电击情境中的冻结显著高于在安全情境中的冻结（P < 0.001，由“无药物区”标志表示），两种基因型之间没有差异。按情境测试顺序分解，野生型小鼠在测试日1表现出显著的组×天数交互作用（RM ANOVA，P < 0.0001）。野生型小鼠在测试日1成功进行了辨别，但在测试日2没有（请参阅图以获取事后细节）。TK小鼠也表现出组×天数交互作用（P < 0.0001），在测试日1和测试日2都进行了辨别。E）皮质酮水平：在1周测试后30分钟采集血液样本以进行皮质酮分析，但我们从未到达那里。因此，没有E图。