paw withdrawal thresholds ( pwts ) is the most commonly used method to assess pain - related behaviors in rodents [ 1 , 2 ] . due to the absence of verbal communication with animals , pain can not be monitored directly in preclinical research but can be estimated by examining behavioral responses to nociceptive stimuli . hence , a nociceptive withdrawal reflex response is used to explore pain mechanisms in rats . however , pain is a subjective and multidimensional experience , including sensory - discriminative , affective , and cognitive components , which requires cortical involvement and modulation [ 57 ] . previous studies have shown that spinal dorsal horn neurons receive nociceptive information , convey it to the ventral horn , and contribute to spinally - mediated nocifensive reflexes . if the mechanical stimulus - induced withdrawal behavior is only a simple reflex that does not activate cortical areas , the pwts can not be used as a behavioral standard for evaluating pain . thus , it is crucial to establish whether a synchronous cortical response accompanies the withdrawal behavior used in preclinical pain research . emerging evidence suggests that cortical areas are adaptive processors , subject to top - down expectations , in which neural activity reflects internal goals and states in addition to external stimuli . the rostral anterior cingulate cortex ( racc , brodmann s areas 24 and 32 ) , a major area implicated in the descending pain modulatory system , is also involved in prefrontal top - down processing . previous research , using positron emission tomography ( pet ) , documented that racc served to prepare for upcoming pain by regulating anticipatory threat responses , consistent with activity in the racc during visual stimuli predicting disagreeable noises observed in an fmri study . expectation is highly subjective and subject to the intrinsic dynamics of oscillations and synchrony in cognitive processing . neural oscillations observed in local field potentials ( lfps ) reflect coherent neuronal population activity . the mechanisms of oscillation generation are computationally interesting and may serve as a means of explaining various aspects of perception and cognition . therefore , our key interest in this study was to simultaneously record the lfps of the racc in quietly resting , unanesthetized rats under multiple mechanical stimulus conditions to elucidate pain expectation modulation coincident with the withdrawal behaviors . male adult sprague - dawley rats , 250300 g body weight , were obtained from the experimental animal center of zhejiang chinese medical university . the animals were housed in groups of 5 in plastic cages with soft bedding at the university animal care facility with an artificial 12/12 h light - dark cycle ( lights on at 8 a.m. ) . animals received food and water ad libitum with a constant room temperature of 2325c and a relative humidity of 4070% . before experimental manipulations , rats were given a period of 1 week to adjust to the new surroundings . the whole experiment was performed in accordance with the guidelines of the international association for the study of pain and the institutional animal ethics committee ( iaec ) . the paw withdrawal threshold was measured automatically using a dynamic plantar aesthesiometer ( model 37450 ; ugo basile , comerio , italy ) . animals were habituated to the testing surroundings daily for 2 consecutive days ( between 9 a.m. and 12 a.m. ) before testing . each rat moved freely in a transparent plastic compartment of a 6-compartment box with a wire mesh floor and were acclimatized for 20 min before the test session . a paw - flick response was elicited by applying an increasing vertical force ( increased steadily from 0 to 50 grams in 20 s ) using a stainless - steel probe ( a straight 0.5-mm diameter ) placed underneath the mesh floor and focused on the middle of the plantar surface of the ipsilateral hind paw . the ipsilateral paw of each rat was measured 5 times at intervals of 1-min or more and the value of the applied force was determined and used for statistical analysis . 1 recording microwire array was surgically implanted under urethane anesthesia ( 1.2 g / kg i.p . surgical procedures were the same as in previous studies , with the exception of the location of the microwires arrays . rats were placed in a stereotaxic frame on a heated surgical platform maintained at a constant temperature of 37c , and a midline scalp incision was made to expose the skull to allow the implantation of 4 screws and an array . the array was driven into the right side racc at a 20-degree angle using a hydraulic microdrive ( model 51421 , stoelting , wood dale , usa ) ; the array was advanced while recording the neural ensemble activity to achieve the correct placement . each microwire array consisted of 8 filaments of nickel chromium wire ( 35-m diameter , stablohm 675 ; california fine wire company , grover beach , ca , usa ) . the array was constructed in 42 architecture , with 200 m between recording wires . the following coordinates ( relative to the bregma ) were used to center the array : racc ( + 2.7 mm rostrocaudal , + 0.8 mm mediolateral , 2.0 mm dorsoventral ) . after a 7-day recovery period , rats were allowed to adapt to the recording environments ( nociceptive behavioral testing surroundings ) for 2 days . with the application of cone - beam computed tomography ( cbct ) , we could evaluate the location of microwire array more accurately ( figure 1 ) , since cbct image data are acquired in digital format from a single 360 rotational scan and through a kavo ct 3d examination ( kavo , germany ) with 0.2-mm voxel size . we performed 3d modeling and panoramic reconstructions , as well as cross - sections obtained using reconstruction software ( examvision , version 1.9.3.13 , kavo dental , germany ) . extracellular local field potentials ( lfps ) were recorded from the implanted microwire array by a cerebus neural signal processing system ( blackrock microsystems , salt lake city , ut , usa ) . lfps signals were preamplified ( 300 ) , bandpass filtered ( 0.3250 hz ) , and sampled at 1 khz . a plantar video camera and video tracking system ( any - maze , stoelting , co , usa ) was used to provide real - time imaging of the rats plantar and the stimulus probe in the dynamic plantar aesthesiometer , permitting synchronization of the probe s stimulus process with the acquired neuronal data . no animals were removed from the study due to poor placement of the recording wires . paw withdrawal thresholds to 5 sequential mechanical stimuli were expressed as a scatter plot and violin plot ( figure 2 ) . the scatter plot was an important tool for assessing the trend shown by the data ; it represents each observation as a point , with paw withdrawal threshold plotted on the y - axis . the violin plot was a combination of a box plot and a kernel density plot , which retained more information about the shape of the distribution . the main advantage of the violin plot was that it presented the density ; a wider violin plot shows higher density . levene s test was used to assess the homogeneity of variances across samples . a small p value of levene s test ( typically 0.05 ) suggests that differences in sample variances were unlikely to have resulted from random sampling from populations with equal variances , leading to rejection of the hypothesis that the variances were homogenous . data were processed and validated by offline analysis using neuroexplorer 5.021 ( nex , plexon inc . ) and exported to matlab 2014a ( mathworks , natick , ma ) for complementary analysis . spectrogram analysis was used to visualize lfp power at different frequency bands as a function of time for each condition . the raw racc lfps were bandpass filtered at 245 hz using a non - causal zero - phase - shift filter ( fourth - order butterworth ) . next , the power spectral densities ( psd ) were calculated by hanning window , 2 frequency bins over the 245 hz range , and 50% overlapping windows . the power was normalized by the logarithm of the psd ( in decibels ) and a smoothing was applied ( gaussian filter , width=3 ) . five frequency band intervals were considered : 24 hz ( delta ) , 49 hz ( theta ) , 915 hz ( alpha ) , 1530 hz ( beta ) , and 3045 hz ( gamma ) . all data for parametric analysis was originally assessed using levene s test for homogeneity of variance . one - way or two - way repeated measures analysis of variance ( rmanova ) with bonferroni post hoc analysis was used when the variances were equal . when the variance was not homogeneous , dunnett s t3 post hoc test was used to perform pairwise comparisons . in all cases , male adult sprague - dawley rats , 250300 g body weight , were obtained from the experimental animal center of zhejiang chinese medical university . the animals were housed in groups of 5 in plastic cages with soft bedding at the university animal care facility with an artificial 12/12 h light - dark cycle ( lights on at 8 a.m. ) . animals received food and water ad libitum with a constant room temperature of 2325c and a relative humidity of 4070% . before experimental manipulations , rats were given a period of 1 week to adjust to the new surroundings . the whole experiment was performed in accordance with the guidelines of the international association for the study of pain and the institutional animal ethics committee ( iaec ) . the paw withdrawal threshold was measured automatically using a dynamic plantar aesthesiometer ( model 37450 ; ugo basile , comerio , italy ) . animals were habituated to the testing surroundings daily for 2 consecutive days ( between 9 a.m. and 12 a.m. ) before testing . each rat moved freely in a transparent plastic compartment of a 6-compartment box with a wire mesh floor and were acclimatized for 20 min before the test session . a paw - flick response was elicited by applying an increasing vertical force ( increased steadily from 0 to 50 grams in 20 s ) using a stainless - steel probe ( a straight 0.5-mm diameter ) placed underneath the mesh floor and focused on the middle of the plantar surface of the ipsilateral hind paw . the ipsilateral paw of each rat was measured 5 times at intervals of 1-min or more and the value of the applied force was determined and used for statistical analysis . to study electrophysiological properties , 1 recording microwire array was surgically implanted under urethane anesthesia ( 1.2 g / kg i.p . surgical procedures were the same as in previous studies , with the exception of the location of the microwires arrays . rats were placed in a stereotaxic frame on a heated surgical platform maintained at a constant temperature of 37c , and a midline scalp incision was made to expose the skull to allow the implantation of 4 screws and an array . the array was driven into the right side racc at a 20-degree angle using a hydraulic microdrive ( model 51421 , stoelting , wood dale , usa ) ; the array was advanced while recording the neural ensemble activity to achieve the correct placement . each microwire array consisted of 8 filaments of nickel chromium wire ( 35-m diameter , stablohm 675 ; california fine wire company , grover beach , ca , usa ) . the array was constructed in 42 architecture , with 200 m between recording wires . the following coordinates ( relative to the bregma ) were used to center the array : racc ( + 2.7 mm rostrocaudal , + 0.8 mm mediolateral , 2.0 mm dorsoventral ) . animals were placed in a new cage with warm bedding until awakening from anesthesia . after a 7-day recovery period , rats were allowed to adapt to the recording environments ( nociceptive behavioral testing surroundings ) for 2 days . with the application of cone - beam computed tomography ( cbct ) , we could evaluate the location of microwire array more accurately ( figure 1 ) , since cbct image data are acquired in digital format from a single 360 rotational scan and through a kavo ct 3d examination ( kavo , germany ) with 0.2-mm voxel size . we performed 3d modeling and panoramic reconstructions , as well as cross - sections obtained using reconstruction software ( examvision , version 1.9.3.13 , kavo dental , germany ) . extracellular local field potentials ( lfps ) were recorded from the implanted microwire array by a cerebus neural signal processing system ( blackrock microsystems , salt lake city , ut , usa ) . lfps signals were preamplified ( 300 ) , bandpass filtered ( 0.3250 hz ) , and sampled at 1 khz . a plantar video camera and video tracking system ( any - maze , stoelting , co , usa ) was used to provide real - time imaging of the rats plantar and the stimulus probe in the dynamic plantar aesthesiometer , permitting synchronization of the probe s stimulus process with the acquired neuronal data . no animals were removed from the study due to poor placement of the recording wires . paw withdrawal thresholds to 5 sequential mechanical stimuli were expressed as a scatter plot and violin plot ( figure 2 ) . the scatter plot was an important tool for assessing the trend shown by the data ; it represents each observation as a point , with paw withdrawal threshold plotted on the y - axis . the violin plot was a combination of a box plot and a kernel density plot , which retained more information about the shape of the distribution . the main advantage of the violin plot was that it presented the density ; a wider violin plot shows higher density . a small p value of levene s test ( typically 0.05 ) suggests that differences in sample variances were unlikely to have resulted from random sampling from populations with equal variances , leading to rejection of the hypothesis that the variances were homogenous . data were processed and validated by offline analysis using neuroexplorer 5.021 ( nex , plexon inc . ) and exported to matlab 2014a ( mathworks , natick , ma ) for complementary analysis . spectrogram analysis was used to visualize lfp power at different frequency bands as a function of time for each condition . the raw racc lfps were bandpass filtered at 245 hz using a non - causal zero - phase - shift filter ( fourth - order butterworth ) . next , the power spectral densities ( psd ) were calculated by hanning window , 2 frequency bins over the 245 hz range , and 50% overlapping windows . the power was normalized by the logarithm of the psd ( in decibels ) and a smoothing was applied ( gaussian filter , width=3 ) . five frequency band intervals were considered : 24 hz ( delta ) , 49 hz ( theta ) , 915 hz ( alpha ) , 1530 hz ( beta ) , and 3045 hz ( gamma ) . paw withdrawal thresholds to 5 sequential mechanical stimuli were expressed as a scatter plot and violin plot ( figure 2 ) . the scatter plot was an important tool for assessing the trend shown by the data ; it represents each observation as a point , with paw withdrawal threshold plotted on the y - axis . the violin plot was a combination of a box plot and a kernel density plot , which retained more information about the shape of the distribution . the main advantage of the violin plot was that it presented the density ; a wider violin plot shows higher density . a small p value of levene s test ( typically 0.05 ) suggests that differences in sample variances were unlikely to have resulted from random sampling from populations with equal variances , leading to rejection of the hypothesis that the variances were homogenous . data were processed and validated by offline analysis using neuroexplorer 5.021 ( nex , plexon inc . ) and exported to matlab 2014a ( mathworks , natick , ma ) for complementary analysis . spectrogram analysis was used to visualize lfp power at different frequency bands as a function of time for each condition . the raw racc lfps were bandpass filtered at 245 hz using a non - causal zero - phase - shift filter ( fourth - order butterworth ) . next , the power spectral densities ( psd ) were calculated by hanning window , 2 frequency bins over the 245 hz range , and 50% overlapping windows . the power was normalized by the logarithm of the psd ( in decibels ) and a smoothing was applied ( gaussian filter , width=3 ) . five frequency band intervals were considered : 24 hz ( delta ) , 49 hz ( theta ) , 915 hz ( alpha ) , 1530 hz ( beta ) , and 3045 hz ( gamma ) . all data for parametric analysis was originally assessed using levene s test for homogeneity of variance . one - way or two - way repeated measures analysis of variance ( rmanova ) with bonferroni post hoc analysis was used when the variances were equal . when the variance was not homogeneous , dunnett s t3 post hoc test was used to perform pairwise comparisons . in all cases , we reanalyzed 1220 previous mechanosensitivity measurements on the ipsilateral paws of 244 rats measured 5 times in succession . variances were not equal across all 5 successive measurements ( levene statistic=14.136 , df1=4 , df2=1215 , p<0.001 ) . however , this was due entirely to the first measurement ; the variances of the subsequent 4 measurements were homogeneous ( levene statistic=1.769 , df1=3 , df2=972 , p=0.151 ) . pairwise comparisons using dunnett s t3 post hoc test found no significant differences among the 5 sequential measurements . in figure 2 , the scatter and violin plots illustrate the shape and density of the distributions . these results suggest that during 4 later measurements , the distributions of pwts were more robust and volatility was smaller than in the first one . compared to the first mechanical stimulus , higher - amplitude lfp signals were observed during the pre - stimulus period of the subsequent 4 stimuli ( figure 3a ) , yielding a generally higher psd in the longitudinal heat maps ( figure 3b ) . the pre - stimulus psd of lfp signals is further shown in figure 4a and 4b . the statistical comparison of the total psd of each pre - stimulus period across the 5 sequential stimuli revealed significant differences ( one - way rmanova ; f= 246.6 , p<0.001 ) . the pre - stimulus total psd of the last 4 stimuli was significantly higher than that of the first stimulus ( p<0.001 , p<0.001 , p<0.001 , p<0.001 ) ( figure 4a ) . furthermore , during the pre - stimulus period , significant differences were encountered across frequency bands ( two - way rmanova ; f=4739.22 , p<0.001 ) and the number of stimuli ( two - way rmanova ; f=102.50 , p<0.001 ; figure 4b ) . compared to the first pre - stimulus period , post hoc analysis revealed an increase in psd in the delta frequency band ( p<0.001 for the subsequent 4 times ) , and a decrease in the theta band ( p<0.001 for the third time ) , as well as in the alpha , beta , and gamma bands ( p<0.001 , p<0.001 , and p<0.001 for the subsequent 4 times ) . together , these data suggest that paw withdrawal behavior increased the total power and delta band power of the pre - stimulus lfps in the racc during subsequent stimuli . we reanalyzed 1220 previous mechanosensitivity measurements on the ipsilateral paws of 244 rats measured 5 times in succession . variances were not equal across all 5 successive measurements ( levene statistic=14.136 , df1=4 , df2=1215 , p<0.001 ) . however , this was due entirely to the first measurement ; the variances of the subsequent 4 measurements were homogeneous ( levene statistic=1.769 , df1=3 , df2=972 , p=0.151 ) . pairwise comparisons using dunnett s t3 post hoc test found no significant differences among the 5 sequential measurements . in figure 2 , the scatter and violin plots illustrate the shape and density of the distributions . these results suggest that during 4 later measurements , the distributions of pwts were more robust and volatility was smaller than in the first one . compared to the first mechanical stimulus , higher - amplitude lfp signals were observed during the pre - stimulus period of the subsequent 4 stimuli ( figure 3a ) , yielding a generally higher psd in the longitudinal heat maps ( figure 3b ) . the pre - stimulus psd of lfp signals is further shown in figure 4a and 4b . the statistical comparison of the total psd of each pre - stimulus period across the 5 sequential stimuli revealed significant differences ( one - way rmanova ; f= 246.6 , p<0.001 ) . the pre - stimulus total psd of the last 4 stimuli was significantly higher than that of the first stimulus ( p<0.001 , p<0.001 , p<0.001 , p<0.001 ) ( figure 4a ) . furthermore , during the pre - stimulus period , significant differences were encountered across frequency bands ( two - way rmanova ; f=4739.22 , p<0.001 ) and the number of stimuli ( two - way rmanova ; f=102.50 , p<0.001 ; figure 4b ) . compared to the first pre - stimulus period , post hoc analysis revealed an increase in psd in the delta frequency band ( p<0.001 for the subsequent 4 times ) , and a decrease in the theta band ( p<0.001 for the third time ) , as well as in the alpha , beta , and gamma bands ( p<0.001 , p<0.001 , and p<0.001 for the subsequent 4 times ) . together , these data suggest that paw withdrawal behavior increased the total power and delta band power of the pre - stimulus lfps in the racc during subsequent stimuli . in previously published studies , pwts were generally measured multiple times , and the arithmetical average was used to assess mechanical hyperalgesia in rats . this view is built on the assumption that the paw withdrawal behavior is nothing more than a spinal reflex ; therefore , the method of averaging multiple measures in physics was deemed valid in living animals with perceptual ability . our results indicate that a top - down expectation is manifested by changes in pre - stimulus lfp activity in the racc over the course of multiple withdrawal behaviors . in the dataset of withdrawal behaviors , the distributions of pwts in the subsequent 4 measurements were denser and more stable than in the first one . taken together , our measurements during paw withdrawal are indicative of cognitive processing of pain , as exemplified by changes in the racc lfp activity in the pre - stimulus period , and expectations increase the stability of later withdrawal behaviors . the lfp , measured with very fine microwires , reflects the synaptic activity of only tens to perhaps thousands of nearby neurons . power increase is usually interpreted as increased synchrony in the neuronal assembly surrounding each microwire , implying the connectivity between these neurons . our results are in agreement with the recent characterization of the brain as a  dynome    or chronectome  , highlighting the role of dynamic communication within or between networks in shaping cognition and behavior . as aaron and karen reported , the processing of pain is encoded by a  pain connectome  , the spatiotemporal signature of brain network communication that represents the integration of all cognitive , affective , and sensorial aspects of pain . additionally , a strong delta band component was encountered during pre - stimulus periods of the last 4 withdrawal behaviors . in fact , it has been shown that the pre - stimulus modulation of delta oscillations serves as a neural mechanism underlying the faster processing of expected events . our results recorded during multiple withdrawal stimuli also confirm an increase of the delta band power , which is correlated with an increase in top - down expectations , inducing more stable withdrawal behaviors . another important point is that the method of averaging multiple measures in physics can not be directly used in the detection of pwts . previous research of pain in rodents has usually treated pwts as a key index of pain behavior in the study of pain - related molecular targets , while only a few of these targets play an important role in humans . the reason for this may be that the pwts is not equivalent to pain threshold , defined as  the minimal intensity of a stimulus that is perceived as painful  . our results show that the pre - stimulus period of the last 4 withdrawal responses exhibited a change in cortical activity and brain state . taken together , these results suggest that the average pwt , under conditions of cortical engagement , might be regarded as a behavioral indicator of pain in rats . therefore , the results may be conducive to the translation from basic research of pain - related biomarkers to potential clinical applications . this study provides evidence that the first withdrawal behavior causes changes of pre - stimulus lfp activity in the racc during subsequent stimuli , and this preparation in advance could affect subsequent stimuli - induced withdrawal behaviors , which contribute to the dynamics of bottom - up input and top - down expectation .