为了达到这一目的，我们采用了行为，药理，电生理方法探讨了疼痛对时距知觉的影响及其潜在神经机制。研究一在大鼠身上建立了时间二分动物模型（Temporal bisection task），探讨了三种常见疼痛的动物模型下时距知觉行为的变化，包括（1）脊神经结扎（Spinal nerve ligation，SNL）建立的神经病理痛模型；（2）足底注射完全弗氏佐剂（Complete freund's adjuvant，CFA）建立的慢性炎症痛模型；（3）福尔马林足底注射建立急性炎症痛模型。这一研究观察到福尔马林注射建立的急性炎症痛模型引起了时距知觉(1.2 – 2.4s范围)的延长，与前人研究结果一致。疼痛过程中所伴随的高唤醒可能导致了时距知觉的延长。
对较短的时长容易受到唤醒的影响，对较长的时长则需要持续性注意的参与。研究二比较了福尔马林疼痛及疼痛后效对秒下到秒上（0.6 -2.4s）和秒上（2 – 8s）时长时距知觉的影响。我们观察到福尔马林痛延长秒下到秒上时长知觉，并且在疼痛后的三天也出现时距知觉延长的效果，提示了疼痛及疼痛后效也能引起时距知觉延长。
前人研究观察到内侧前额叶皮层(Medial prefrontal cortex, mPFC)编码了时距知觉信息，但mPFC亚区在其中的角色尚不清楚。研究三采用了双侧微量注射MK801(N-甲基-D-天冬氨酸受体拮抗剂，6μg每侧)以及蝇蕈醇(γ-氨基丁酸A型受体兴奋剂，0.1 μg每侧)的方式干预了mPFC三个亚区：前扣带回皮层(Anterior cingulate cortex，ACC）, 前边缘皮层(Prelimbic cortex，PrL)和基底边缘皮层(Infralimbic cortex，IL)，同时观察了大鼠在时间二分任务中的表现。结果显示，药理学干预IL区域对时间二分任务的影响最大。
研究四采用了在体多通道电生理记录的方式，在清醒大鼠进行时间二分任务时同步记录4个脑区，基底边缘皮层(IL)，背侧纹状体(Dorsal striatum, DS)，初级感觉皮层(Primary sensory cortex, SI)，背内侧丘脑核(medial dorsal thalamus，MD)中神经元活动。结果观察到多个脑区存在对时间反应的“Timing neurons”，其中IL中发现“Timing neurons”的概率较高。在福尔马林痛及疼痛后第二天的测试中，动物表现出了时距知觉延长，伴随“Timingneuron”活动模式发生了变化：所知觉时间超过1s时，疼痛及疼痛后效降低了“Timingneurons”的Firingrate。
失匹配负波(mismatch negativity, MMN)常被用来检验对刺激之间差异的觉察程度，也被作为前注意的一个指标。研究五以MMN为指标，探讨了福尔马林疼痛对不同刺激间隔（ISI）的Oddball范式诱发MMN的影响。这项研究观察到疼痛能够降低1.5s ISI，响度偏差刺激引起MMN的幅度，但不影响0.5s ISI时，响度偏差刺激引起的MMN幅度。这项研究提示了疼痛能够影响ISI间隔较长时神经元的活动，与研究四结论对应。
依据以上结果，可以得出以下结论：（1）动物处于急性疼痛状态下或者处于有过疼痛经历的环境下，时距知觉倾向于延长；（2）疼痛及疼痛引起的条件化所伴随的高唤醒可能中介了时距知觉的延长；（3）编码时距知觉信息的“Timing neurons”分布在了多个脑区。（4）疼痛及疼痛引起的条件化对时距知觉的影响由所知觉时间长短决定：疼痛状态下，当所知觉时长低于1s时，“Timing neurons”活动变化不大；当所知觉时长超过1s时，“Timing neurons”的神经元显著降低。这一系列研究为疼痛对时距知觉的影响提供了行为及神经机制上的证据。|
|Other Abstract||Interval timing refers to the time perception of hundred milliseconds to several minutes, which related to cognitive activities such as movement control, musical rhythm, behavioural prediction andlearning etc. Time perception wasaffected by a lot of factors, such as arousal and attention. Pain is a multi-dimensional psychological and physiological phenomenon, which consist of sensory discrimination, emotional motivationand cognitive evaluation. Previous studies have found that pain affects the interval timing in human:1) Healthy subjects usually judge the duration of painful faces longer than netural faces; 2) Healthy subjects also prolonged the subjective duration when in state of laboratory pain or after pain conditioning;3) Interval timing behaviors in chronic pain patients was abnormality changed too, and the depressive moodcombiditywith chronic pain may mediate this process. So far, there has been a lack of systematic research to explore how pain affects time perception and the potiential neural mechanisms behind it. Interval timing may provide a new window for study in pain. On the one hand, it is worthy to discuss whether interval timing could be a biomarker of chronic pain.On the other hand, misleadinglyshorten the interval timing on pain, healthy subjects’intensity of pain reduced, suggestingstudying on interaction of pain and interval timing would develop a new method to cope with pain.
For this purpose, we used behavioral, pharmacological and electrophysiological methods to explore how pain affects interval timing and its potiential mechanisms. In Study 1, we established the temporal bisection task in rats and investigated the interval timing behaviors (1.2 – 2.4s) on three widely-used pain models, including 1) Spinal nerve ligation (SNL) induced neuropathic pain model; 2) Plantar injection of Complete Freund's Adjuvant (CFA) induced chronic inflammatory pain model; 3) Plantar injection of formalin-induced acute inflammatory pain. Our study found that formalin pain prolonged interval timing, which is consistents with previous human studies.
Generally, time perception within short duration was mainly affected by arousal, while time perception within long duration was mainly affected by the sustained attention. In Study 2, we compared the effects of formalin pain on the interval timing behavior withinsub-second to supra- seconds (0.6- 2.4s) and supra-seconds (2-8s) range. We observed that formalin pain prolonged the time perception within 0.6-2.4s range, this effect could sustain until three days after pain.
Previous studies suggested medial prefrontal cortex (mPFC) plays a key role in interval timing behaviors. In Study 3, ahead of rats performing temporal bisection task, we bilateralmicroinfusion of MK801 (N-methyl-D-aspartic acid receptor antagonist, 6 μg per side) or Muscimol (γ-aminobutyric acid type A receptor agonist, 0.1 μg per side) into three subregions of mPFC [Anterior cingulate cortex (ACC), Prelimbic cortex (PrL) and Infralimbic cortex (IL)] respectively in rats. Results showed that pharmacological disruption in the IL region had the greatest influence on the interval timing behaviors.
In Study 4, we adopted multiple single-unit recording technique to synchronously recording the neuronal activity of four brain regions, IL, Dorsal striatum (DS), the Primary sensory cortex (SI), and the medial dorsal thalamus (MD), during rats’ performing temporal bisection tasks. Results showed all the recording brain areas contained the neurons which neural activity changes over time (“Timing neuron”). The probability of “Timing neuron” found in IL was higher than SI and MD. Rats exhibited a prolongation of time perception in the temporal bisection task performing immediately after formalin injection and one day post-formalin injection. The neural activity of “Timing neuron” were changed by pain and post-effect of pain: pain and its post-effects lower the firing rate of 'Timing Neurons' when the to-be-timed tone more than 1 second.
Mismatch negativity (MMN) was usually interpreted to reflect the detection of difference in a comparsion process. In study 5, we used amplitude of MMN as the index, and explored the effect of formalin pain on Oddball paradigm induced MMN with 1.5s and 0.5s interstimulus-interval (ISI). Results shown formalin pain reduced the amplitude of MMN with 1.5s ISI, but not effect on the MMN with 0.5s ISI. This study suggests that attentional resources are reduced in the presence of pain, but this effect was only observed in the short ISI.
Base on above results, we concluded that: 1) Time perceptionwas prolonged when animals in the state of acute pain or post-effects of pain;2) The high arousal associated with pain and post-effects of pain may contribute to prolongation of interval timing behaviors; 3) The neurons that encoding the temporal information are widely distributed inbrain;4) Effects of pain and post-effects of pain on interval timing behaviorswere determined by the length of to-be-timed duration:Pain had no significant effect on interval timing when the to-be-timed duration was less than 1s, while pain significantly changed the activity pattern of neurons encoding time information when the to-be-timed duration was longer than 1s. This series of studies providedbehavioral and neural evidences for how pain modulatesinterval timing.|