Supplemental Digital Content is available in the text To determine prognostic factors of clinically relevant radiographic progression (CRRP) in patients with rheumatoid arthritis (RA) in clinical practice.We performed a multicenter prospective study in Japan of biological disease-modifying antirheumatic drug (bDMARD)-naive RA patients with moderate to high disease activity treated with conventional synthetic DMARDs (csDMARDs) at study entry. We longitudinally observed 408 patients for 1 year and assessed disease activity every 3 months. CRRP was defined as yearly progression of modified total Sharp score (mTSS) > 3.0 U. We also divided the cohort into 2 groups based on disease duration (<3 vs ≥3 years) and performed a subgroup analysis.CRRP was found in 10.3% of the patients. A multiple logistic regression analysis revealed that the independent variables to predict the development of CRRP were: CRP at baseline , time-integrated Disease Activity Score in 28 joints-erythrocyte sedimentation rate (DAS28-ESR) during the 1 year postbaseline , RA typical erosion at baseline (95%CI 1.56–21.1), and the introduction of bDMARDs (95%CI 0.06–0.38). The subgroup analysis revealed that time-integrated DAS28-ESR is not a predictor whereas the introduction of bDMARDs is a significant protective factor for CRRP in RA patients with disease duration <3 years.We identified factors that could be used to predict the development of CRRP in RA patients treated with DMARDs. These variables appear to be different based on the RA patients’ disease durations. Magnetic resonance imaging (MRI) and ultrasonography (US) are sensitive enough to detect active synovitis and erosions in early RA.14 Accordingly, the identification of individual RA patients at high risk of CRRP is critical for achieving the goal of RA control. Various clinical and biological markers including C-reactive protein (CRP), the erythrocyte sedimentation rate (ESR), and the presence of autoantibodies have been identified as risk factors for CRRP in patients with RA, especially those enrolled in clinical trials treated with bDMARDs.18The primary goal of RA treatment is to control disease activity and prevent structural damage, but some patients develop clinically relevant radiographic progression (CRRP) despite conventional treatment with DMARDs. In these patients, a treatment strategy providing strict control of the progression of RA should be considered in order to alter the course of radiographic progression.20 but to the best of our knowledge there has been no large-scale clinical study investigating the prevention of CRRP by using RA patients treated in accord with the EULAR recommendations in daily clinical practice.These cohort studies adopted tender joint counts and swollen joint counts as clinical indices. The matrix models based on these variables did not include a commonly used composite measure such as the Disease Activity Score in 28 joints (DAS28). Some of the studies of RA cohorts in clinical practice have investigated a CRRP model,To assess the relevance of the updated EULAR recommendations and to determine prognostic factors of CRRP in Japanese RA patients in clinical practice, we conducted a large-scale prospective study and evaluated the associations between clinical variables and the risk of developing CRRP among csDMARD-refractory RA patients.http://www.umin.ac.jp/ctr/] (#UMIN000014791), conducted in the daily clinical practice for RA in Japan. The inclusion criteria were as follows: RA patients who met the American College of Rheumatology (ACR) 1987 criteria or the 2010 RA classification criteria;21 the patient's clinical disease activity determined by the DAS28-ESR is moderate to high or, obvious plain radiographic erosion is confirmed at enrollment; and RA patients taking csDMARDs but not bDMARDs at enrollment.This was a prospective, observational cohort study registered with the University Hospital Medical Information Network Clinical Trials Registry [22Overall, 887 patients with csDMARDs-refractory RA from 26 related centers of Nagasaki University and Tohoku University in Japan were recruited in our cohort between May 2009 and March 2012. All of the patients were examined and treated by Japan College of Rheumatology-certified rheumatologists. Although this was a prospective, observational cohort study, we recommended that all of the participating rheumatologists treat the patients using a T2T strategy. We did not recommend the choice of DMARDs. We observed the patients for 1 year after enrollment and assessed the RA disease activity every 3 months, including use of the DAS28-ESR and the Health Assessment Questionnaire.According to the T2T strategy, physicians were allowed to introduce bDMARDs including infliximab, adalimumab (ADA), golimumab, certolizumab pegol, etanercept, tocilizumab (TCZ), and abatacepet (ABT) if the patient's disease activity was not controlled by csDMARDs alone. All of the above bDMARDs were available in Japan during the observational period. To determine the efficacy of bDMARDs for the prevention of CRRP in the present investigation, we selected the patients for whom a bDMARD was introduced within 3 months after their study enrollment. All patients gave their signed informed consent to be subjected to the protocol, which was approved by the Institutional Review Board of Nagasaki University, Tohoku University, and the related centers.24 The images were scored by 2 independent rheumatologists, trained and certified by Prof van der Heijde , and blinded to the clinical evaluation as described.26 The interobserver reliability (as determined by the interclass correlation coefficient) was 0.97. The smallest detectable change of mTSS in the present study was calculated as 2.96 as described.27 We therefore defined an annual increase of the mTSS > 3.0 units as the development of CRRP, according to a previous report.28 In addition, we considered the patients as having typical RA erosion if erosion score is ≥3 according to the EULAR definition of erosive disease.29Radiographs of each patient's hands and feet were taken at baseline and at 1 year, and we evaluated the radiographic progression by determining the changes over the year by obtaining each patient's modified total Sharp score (mTSS), joint erosion, and joint space narrowing at 1 year.31 Since all of the patients at entry had already been treated by csDMARDs in the present study, the titer of autoantibodies may not represent the exact serologic characteristics. Therefore, we adopted the presence of ACPA/RF, instead of the titer, in the present study. To determine the independent predictive factors toward the development of CRRP at 1 year, we subsequently performed multiple logistic regression analysis. We selected variables with P-values <0.3 by univariate analyses as model 1. We then set model 2 by including variables with P-values <0.05 in model 1.Baseline demographic, clinical, and radiographic characteristics of RA patients with or without CRRP were compared with Fisher exact tests for discrete variables and Wilcoxon test for continuous variables. Previous studies have shown that the titer of autoantibodies such as anticitrullinated peptide antibodies (ACPA) or rheumatoid factor (RF), especially the latter, is influenced by DMARDs.To test the interaction variables, we created model 3 by adding interaction terms between treatment (early introduction of bDMARDs) and baseline disease parameters (CRP at baseline and disease duration) as independent variables. We finally determined model 2 as the final model.P-value <0.05 was considered significant.We also performed subgroup analysis based on disease duration (<3 vs ≥3 years). Each subgroup was analyzed with a logistic regression analysis by using the final model. In order to adjust for nonrandom assignment to bDMARDs treatment, we further analyzed using propensity scores. Variables used in constructing the propensity score were age, gender, disease duration, CRP at baseline, erosion score ≥3 at baseline, methotrexate (MTX) use at baseline, and Prednisolone use. Statistical analyses were performed using SAS 9.4 software . A Figure The patients’ mean age was 60.7 years, and the mean disease duration was 5.5 years. During the 1-year observation period, the treatment of 249 patients was strengthened with 1 or more csDMARDs or bDMARDs according to the EULAR recommendation, as decided by the participating rheumatologists. Among these 249 patients, the dosage of csDMARDs for 124 patients was increased. For 43 patients, the csDMARD(s) were switched or 1 or more other csDMARD was added. For 85 patients, a bDMARD was initiated after study entry. The average period before the introduction of a bDMARD was 2.5 months. bDMARDs  were initiated within 3 months after the enrollment for 76 patients. During the study, 12 patients dropped out; the retention rate of csDMARDs or bDMARDs was >90%. The therapeutic course during the 1 year following the baseline is shown in Supplementary Figure S1.CRRP was observed in 42 of the 408 patients (10.3%). Cumulative probability plots during the 1 year postbaseline as assessed by mTSS are shown in Figure P-values <0.3 by univariate analyses and a performed logistic regression analysis, which revealed 5 independent variables that could be used to predict the development of CRRP, as follows: disease duration, time-integrated DAS28-ESR during the 1 year postbaseline, CRP at baseline, erosion score ≥3 at baseline, and the introduction of bDMARDs.To determine which variables are associated with the development of CRRP at 1 year, we evaluated 14 variables as shown in Table P-values <0.05 in the 1st model and found 4 prognostic factors of CRRP, as follows: CRP at baseline , time-integrated DAS28-ESR during the 1 year postbaseline , RA typical erosion (erosion score ≥3) at baseline , and the introduction of bDMARDs  , we considered that these variables had their interaction. Accordingly, we included the interaction terms (disease duration × the introduction of bDMARDs and CRP at baseline × the introduction of bDMARDs) with the final model in a logistic analysis and found there is no significant interaction between these variables .Since the mean disease duration was significantly shorter and the mean serum CRP level at baseline was significantly higher in the early bDMARDs introduction group than in the other patients .33 We therefore speculated that the variables that could be used to predict CRRP might be different between early-stage RA and established RA. Accordingly, we divided the cohort into 2 groups based on disease duration  and conducted a subgroup analysis. The patients’ characteristics according to disease duration are shown in Table In our cohort, we found that the patients’ disease duration at baseline was associated with CRRP. There have been some reports showing the rate of radiographic progression is more rapid in the first 2 to 3 years of RA.P = 0.0256), the rate of remission at 1 year was also significantly higher in the former group . The estimated yearly progression calculated by dividing mTSS at baseline by disease duration at baseline was significantly higher in the <3 years group compared to the ≥3years group , whereas no significant difference was observed in the percentage of CRRP between the 2 groups .Although the DAS28-ESR values at baseline were significantly higher in the subgroup with disease duration <3 years  compared to the ≥3 years group  , and the introduction of bDMARDs  are independent variables to predict the development of CRRP  was the only variable for predicting CRRP in the group with ≥3 years’ disease duration  whereas the rate of CRRP was comparable between these groups . In addition, time-integrated DAS28-ESR did not contribute to the development of CRRP in the RA patients with <3 years’ disease duration.In line with this observation, the DE019 clinical trial of ADA revealed that the efficacy of ADA toward ΔmTSS was greater among patients with RA of <3 years’ duration compared to those with ≥3 years’ duration.7 We consider that this statement is also applied in cases of CRRP.These data suggest that radiographic progression in RA  may not be suppressed in early-stage RA patients only through the control of clinical disease activity, that is, DAS28-ESR. Thus, an evaluation by MRI or US is thought to be useful to predict the subgroup of patients who will develop CRRP among early-stage RA patients. The EULAR recommendations for the use of imaging of the joints in the clinical management of RA state, in recommendation #5, that MRI bone edema as well as the synovitis detected by MRI and US are strong independent predictors of subsequent radiographic progression in early RA and should be considered for use as a prognostic indicator.42 Thus, taken together, the past and present findings indicate that physicians should pay close attention to the timing of bDMARD introduction – especially among early-stage RA patients – since bDMARDs can inhibit radiographic progression regardless of clinical disease activity.44In the present study, the estimated yearly progression of mTSS from baseline was significantly high in the RA subgroup with disease duration <3 years. These results are consistent with the well-known observation that radiographic progression occurs early (within the first 2–3 years) in patients with RA.45The time-integrated DAS28-ESR seems to be a more valuable predictive factor in established RA, since our multivariate analysis revealed that in our cohort, the time-integrated DAS28-ESR was not an independent predictive factor in the patients with <3 years’ disease duration but that it was an independent predictive factor in the patients with ≥3 years’ duration. We speculate that the speed of radiographic progression starts to decline in cases of established RA, and thus the control of clinical disease activities  is enough and essential in patients with ≥3 years’ RA duration. Nevertheless, long-term observation is needed to confirm these results, since a previous study showed that the time-integrated DAS28-ESR over 3 years is associated with CRRP in early RA patients.46 Furthermore, it has also been proposed that Japanese patients with RA showed high concentration of active form of MTX-polyglutamate in red blood cells with very low-dose MTX.47 Accordingly, it is assumed that patients in this cohort were treated according to the EULAR recommendations.The dose of MTX in our Japanese cohort was lower than the other cohort. Previous clinical study has shown that the optimum dosage of MTX for a Japanese individual is 6 to 8 mg/week, and that higher dosages will increase the incidence of adverse effects.38 Thus, long-term verification studies are required to confirm the present results. Second, the mean DAS28-ESR at baseline in our study was 4.54, which seems to be low compared with the previous research providing the pilot risk model of rapid radiographic progression.19 Verification studies targeting RA patients with high clinical disease activity are thus also needed.This study has some limitations. First, as stated above, a relatively short period of observation was used. The EULAR T2T recommendations state in recommendation #5 that the desired treatment target should be maintained throughout the remaining course of the disease.In conclusion, the present study is the first prospective observation to examine predictive variables of CRRP among Japanese RA patients being treated with DMARDs in daily practice. Our findings indicate that CRRP in our cohort was closely associated with the persistent disease activity, CRP at baseline, and the early introduction of bDMARDs. It is of note that some of these risk variables vary according to the disease duration. Our results provide useful information in relation to strict disease control by T2T strategies in accord with the EULAR recommendations 2013 update.


Poor adherence to inhaled medications and incorrect inhaler technique are known to adversely affect outcomes in asthma, contributing to the continuing failure for many patients to achieve control despite the availability of effective therapies.2 (ELLIPTA is a trademark of the GlaxoSmithKline group of companies). The actuated dose is subsequently inhaled from the mouthpiece.2 The ELLIPTA DPI is used to deliver fluticasone furoate (FF), a new inhaled corticosteroid licensed in Europe in combination with vilanterol, a new long-acting β2-agonist, for asthma and chronic obstructive pulmonary disease and in development as a monotherapy for asthma. The aim of this analysis was to investigate patient perception of the ease of use, and investigator-reported competence in use, of the ELLIPTA DPI.The ELLIPTA dry powder inhaler (DPI) is a handheld inhaler with single-step activation, featuring a cover that is opened by the patient to uncover the mouthpiece and activate a dose2 The primary clinical trial data are reported separately.3–5We describe a sub-analysis of the ease of use and inhaler competence data in patients with asthma from three randomised, multicentre clinical trials of FF/vilanterol combination therapy (HZA106827 (100/25 μg) and/or FF monotherapy ; FFA115283 (50 μg); HZA106827 (100 μg)), in which the ELLIPTA DPI was used to deliver study medication (including placebo where applicable). Preliminary results have been published in abstract form.Patients completed a questionnaire at week 4 of each trial, rating the ease of use of the inhaler and how easy it was to tell how many doses of medication were left in the inhaler. For both questions, patients selected their response from the following ordinal scale: very easy, easy, neutral, difficult, and very difficult. Investigators assessed, by observation, patients’ competence in using the ELLIPTA DPI following one demonstration of correct usage at randomisation, at week 2 and at week 4. Data were analysed and interpreted descriptively; no statistical inference was planned.A total of 1,050 asthma patients  participOverall, 65% of questionnaire respondents reported that the inhaler was very easy to use, and 94% reported that it was easy or very easy to use. Only 1% of patients reported that the inhaler was difficult or very difficult to use. Similarly, 74% reported that they found it very easy to tell how many doses of medication were left in the inhaler using the in-built numerical dose counter, and 96% found it easy or very easy. Less than 1% of patients found it difficult or very difficult to tell how many doses were left in the inhaler.At randomisation, investigators reported that 95% of patients used the inhaler correctly after the initial demonstration of correct usage at randomisation (week 0), and did not require additional instruction. A further 4% of patients were able to use the inhaler correctly at randomisation after one additional instruction. The most common error made at randomisation  was to open the cover incorrectly (20 (1.9%) of all patients), followed by inhaling the dose incorrectly (15 (1.4%)), unspecified reason (12 (1.1%)), and closing the cover incorrectly (3 (0.3%)). At week 2 and week 4, >99% of patients used the inhaler correctly; four (0.4%) patients made errors at week 2 and week 4, respectively .6 Patient preference data obtained from a separate interview-based study7 are consistent with our findings, suggesting that patients with asthma and chronic obstructive pulmonary disease generally perceive the ELLIPTA DPI positively and find it easy to use.In all three studies, the majority of participants found the inhaler to be easy to use, and were observed to use the inhaler correctly following a single demonstration. The design of the inhaler and appropriateness of the delivery mechanism to the patient may boost patient satisfaction with the medication regimen and competence in device use.9 Similarly, our single-device study was conducted against the background of randomised, controlled clinical trials in which all patients were given clear instruction in correct use of the inhaler at randomisation. Such thorough instruction is unlikely to be replicated in real-world clinical practice;10 this could therefore be considered a limitation in interpretation of this study.A similar questionnaire has previously been used to assess comparative ease of use in asthma patients participating in randomised controlled trials, whose responses indicated that the DISKUS DPI is easier to use than DiskHaler.The perceived and observed ease of use findings reported in this analysis suggest that the ELLIPTA DPI may have the potential to reduce inhaler-related handling errors and improve adherence; however, further studies are required to specifically assess these possibilities.


Orienting attention to a new target requires prior disengagement of attention from the current focus. Previous studies indicate that the superior colliculus (SC) plays an important role in attention. However, recordings of responses of SC neurons during attentional disengagement have not yet been reported. Here, we analyzed rat SC neuronal activity during performance of an attention-shift task with and without disengagement. In this task, conditioned stimuli  were sequentially presented. To obtain an intracranial self-stimulation reward, rats were required to lick a spout when an infrequent conditioned stimulus appeared . In the disengagement reward trials, configural stimuli consisting of an infrequent stimulus and frequent stimulus in the former trials were presented; in the non-disengagement reward trials, only an infrequent stimulus was presented. Of the 186 SC neurons responding to the CSs, 41 showed stronger responses to the CSs in the disengagement reward trials than in the non-disengagement reward trials (disengagement-related neurons). Furthermore, lick latencies in the disengagement reward trials were negatively correlated with response magnitudes to the CSs in half of the disengagement-related neurons. These disengagement-related neurons were located mainly in the deep layers of the SC. Another 70 SC neurons responded to the CSs in both disengagement and non-disengagement reward trials, suggesting that these neurons were involved in attention engagement. Our results suggest complementary mechanisms of attentional shift based on two subpopulations of neurons in the SC. The superior colliculus (SC) constitutes part of the brain network involved in visual attention , and conOrienting attention to a new target requires three sequential mental operations: (1) disengagement of attention from its current focus; (2) moving attention to the new target; and (3) engagement of the new target . Thus, t∘C) and light (07:00–19:00) were automatically controlled. Food and water were available ad libitum. Treatment of all rats was in strict compliance with the United States Public Health Service Policy on Human Care and Use of Laboratory Animals, National Institutes of Health Guide for the Care and Use of Laboratory Animals, and Guidelines for the Care and Use of Laboratory Animals at the University of Toyama. All experimental procedures were approved by our institutional committee for experimental animal ethics.Eleven male Wistar rats, weighing 270–320 g at the time of surgery , were used. The rats were individually housed in a room where temperature  and after training , rats were again anesthetized  and mounted with the artificial earbars. A small hole  was drilled through the cranioplastic acrylic and the underlying skull for chronic, repeated recordings. The exposed dura was excised, and the hole was covered with hydrocortisone ointment ; alternatively, one or two drops of chloramphenicol  solution (0.1 g/mL) were dropped into the hole. The hole was covered with a sterile Teflon sheet and sealed with epoxy glue.Before surgery, the rats were acclimated by handling and were accustomed to being placed into a small, plastic restraining cage for brief periods. After recovery from the first stage of surgery, the threshold level for ICSS  was determined, and any rat for which the threshold exceeded 300 μA was excluded. In these ICSS parameters, rats produced stable 40–70 lever presses/min for ICSS in an operant chamber . Then, tFigure 1A). The attention-shift task included five sessions, and each session consisted of 36 trials including 12 reward trials  and 24 non-reward trials . In each trial, a CS (light flash or sound) appeared for 1 s, followed by spout protrusion close to the mouth for 2 s. In the reward trials , rats could obtain ICSS reward if they licked the spout . A touch sensor detected individual spout licks.During task training, the rat was placed in the restraining cage with its head fixed rigidly and painlessly in the stereotaxic device by the artificial ear bars. A midrange speaker, located 1 m in front of the rat, delivered the auditory stimuli, and each white light, 3 cm in front of each eye, delivered the visual stimuli  were sequentially presented, and when rats detected infrequent CSs and licked the spout, rats could acquire ICSS reward . Trial sequence was set sub-randomly by a computer in that at least one non-reward trial always preceded each reward trial. Thus, the task required shift of attention to infrequent CSs. In session 1, right light associated with non-reward was sequentially presented , and when the left light, an infrequent stimulus, appeared, the rat could acquire ICSS reward if it licked the spout . In session 2, left light associated with non-reward was sequentially presented , and when right light, an infrequent stimulus, appeared, the rat could acquire ICSS reward if it licked the spout . In sessions 3 and 4, right, or left light associated with non-reward was similarly sequentially presented , and when both right and left lights were simultaneously presented, the rat could acquire ICSS reward if it licked the spout . The CS in the reward trials included not only the infrequent stimuli but also frequent stimuli of the former non-reward trials. Therefore, in sessions 3 and 4, the rat must disengage attention from the frequent CSs to detect the infrequent stimuli. In session 5, a tone (frequent stimulus) was sequentially presented, and when both right and left lights (infrequent stimuli) were simultaneously presented, the rat could acquire reward if it licked the spout. Session 5 was used as a control session for sessions 3 and 4; the CSs in the reward trials in session 5 were same as those in sessions 3 and 4, although attentional disengagement from the CSs in the former trials was not required in session 5. SC neurons related to attention disengagement are supposed to respond stronger to the CSs in sessions 3 and/or 4 than in session 5 . Sequence of the sessions was run pseudo-randomly. The rats were trained to lick the spout only in the reward trials.The sequence of the CSs in each session are shown in Z = 0.5–1.5 MΩat 1 kHz) was stereotaxically and vertically inserted into the SC in a stepwise fashion by a pulse motor-driven manipulator . Extracellular neuronal activity was passed through a multi-channel differential amplifier with a preamplifier , monitored on an oscilloscope, and recorded on a data recorder . Only neuronal activity with a signal-to-noise ratio >3:1 was recorded. The analog signals of amplified neuronal activity, triggers for CSs, ICSS reward, and spout licking were digitized at a 40-kHz sampling rate and stored on a computer via a multichannel acquisition processor system . The digitized neuronal activities were isolated into single units by their waveform components using the Oﬄine Sorter program (Plexon). Spike sorting was performed with the oﬄine sorter program for cluster analysis (Oﬄine Sorter). Each cluster was checked manually to ensure that the cluster boundaries were well separated and the waveform shapes were consistent with action potentials. For each isolated cluster, an autocorrelogram was constructed and only units with refractory periods >1.2 ms were used for further analyses. Finally, superimposed waveforms of the isolated units were drawn to check the consistency of the waveforms. These units were transferred to the NeuroExplorer program  for further analyses.After the rats had learned the conditioned licking task to discriminate the CSs in the reward and non-reward trials, SC neurons were recorded during performance of the task. Neuronal activity of an individual rat was usually recorded every other day. After being placed in the enclosure, the ointment was removed, and a glass-insulated tungsten microelectrode (P < 0.05) of neuronal activity between the 500-ms pretrial control period and the 1,000-ms CS periods. Neuronal response magnitude was defined as follows: the mean firing rate during the 1,000-ms CS period minus the mean firing rate during the 500-ms pretrial period. For each neuron, neuronal response magnitudes to all CSs were compared by the one-way analysis of variance (ANOVA) and Tukey post hoc tests (P < 0.05). Based on response patterns to the CSs, we then classified SC responsive neurons (see Results).Since the rats had to adapt to new rules in the beginning of the sessions, initial trials in each session were discarded and only data after the third reward trial were analyzed. Both neuronal and behavioral data on each trial were counted from the peristimulus histograms in successive 50-ms bins for three periods: a pretrial control period (500 ms), a CS stimulation period , and rewarding stimulation (reinforcement) period . Significant excitatory or inhibitory responses to each CS were defined by a Wilcoxon signed rank (WSR) test  was constructed using the data of the whole reward and non-reward trials across five sessions in each neuron. Neuronal response latency was defined as the interval from the onset of stimulus presentation to the time at which neuronal firing rate exceeded the mean ± 2.0 SD of the baseline firing rate. Mean response latencies to the CSs were compared among SC neuronal types by one-way ANOVAs at a significance level of P < 0.05.For some SC neurons , not only response magnitudes, but also response latencies and durations were analyzed in each CS. For this purpose, peri-event histograms of individual CSs were constructed. Neuronal response duration was defined as the duration during which the neuronal firing rate exceeded the mean ± 2.0 SD of the baseline firing rate. Because these neurons showed no responses in the non-reward trials, we analyzed latencies and durations of neuronal responses to each stimulus only in the reward trials. Mean latencies and duration of responses to the CSs were similarly compared among the CSs by one-way repeated-measure ANOVAs at a significance level of post hoc comparisons were performed using the Bonferroni-correction method with a significance level of P < 0.05. Correlations between individual lick latencies in the reward trials and neuronal activity were analyzed using simple linear regression.Lick latency was defined as the interval from spout protrusion to the moment when the rat licked the spout. Mean lick latencies in reward trials were compared among the five sessions by one-way repeated-measure ANOVAs. The After the last recording session, rats were anesthetized with pentobarbital , and several small electrolytic lesions (20 μA for 20 s) were made stereotaxically around the recording sites with a glass-insulated tungsten microelectrode. Rats were then given an additional overdose of anesthetic and perfused transcardially with heparinized 0.9% saline followed by 10% buffered formalin. The brain was removed and cut into 50-μm frontal sections with a freezing microtome. Sections were Nissl-stained with cresyl violet. The sites of electrical lesions were carefully determined microscopically. The location of each recording site was then calculated by comparing the stereotaxic coordinates of the recording sites with those of the lesions. Positions of neurons were stereotaxically located on the tissue sections and plotted on the corresponding sections on the atlas of P < 0.05).Based on the locations of SC neurons, the ratio of SC neurons in the superficial layers, the intermediate layers and the deep layers were calculated for each type of SC neuron. The ratio (percent) of each layer for each type of SC neuron was defined as follows: (the number of a given type of SC neuron × 100)/the number of SC neurons recorded in that layer. The ratio of SC neurons among the different SC layers was compared with a Chi-squared test . Statistical comparison indicated that there was no significant main effect of session , nor significant interaction between session and trial type  = 0.221, P > 0.05; two-way ANOVA). The results indicated that the rats similarly performed the five sessions.During the task, rats almost always licked the spout in the reward trials, while they seldom licked the spout in the non-reward trials. We analyzed the behavioral data that were collected during recordings of 156 responsive SC neurons. Figure 2B shows the mean lick latencies in the reward trials of the five sessions (R1–R5). Statistical comparison indicated that there was a significant main effect of session  (one-way repeated-measures ANOVA). Post hoc tests indicated that mean lick latencies in sessions 3 and 4 requiring disengagement  were significantly longer than those in the other sessions requiring no disengagement .Lick latencies were analyzed in the reward trials. We analyzed the behavioral data with lick latencies less than 300 ms, which were collected during recordings of 156 responsive SC neurons. Table 1 summarizes the response patterns of these 583 neurons. One hundred and eighty-six (31.9%) neurons responded to the CSs. These 186 responsive neurons were classified into four types: disengagement-related neurons , reward and attention shift-related neurons , visually responsive neurons , and inhibitory-responsive neurons .Over a period of 1–3 months for each rat, recordings were made from 611 neurons located in and around the SC during the attention-shift task. Of these neurons, 583 were located in the SC. P < 0.05), and if they also showed significantly higher response magnitudes during presentation of these CSs than other CSs associated with and without reward . A typical example of this type of neuron in the right SC is shown in Figure 3A. The neuron responded during presentation of all infrequent CSs associated with reward in sessions 1–5 , but not during presentation of frequent CSs associated with non-reward . Comparisons of the response magnitudes to the CSs are shown in Figure 3B. The response magnitudes during presentation of the infrequent CSs contralateral to the recording site requiring attention disengagement in session 3 (R3) were significantly stronger than those to the other CSs with and without disengagement . It is noted that this SC neuron was recorded from the right SC, and the left light was the infrequent CS in session 3. To detect the left light associated with reward, the rat must disengage attention from the right light in session 3. On the other hand, the rat had to detect the left light in session 1 and the left and right lights in session 5, which required no disengagement. The above results indicate that the response magnitude to the CS requiring disengagement in session 3 was significantly larger than to the same CSs requiring no disengagement in sessions 1 and 5.Attention disengagement-related neurons were defined as such if the neurons satisfied the following two comparisons; if neurons showed excitatory responses during presentation of the infrequent CSs requiring attentional disengagement  contralateral to the recording sites , mean response latencies (b), and the mean response durations (c) of the disengagement-related neurons recorded from the left SC (A), right SC (B), and both sides of the SC (C). In the left SC neurons , the mean response magnitude was significantly stronger (a), the mean response latency was significantly shorter (b) and the mean response duration was significantly longer in session 3 requiring disengagement than those in other sessions . In the right SC neurons , the mean response magnitude was significantly stronger (a), the mean response latency was significantly shorter (b) and the mean response duration was significantly longer in session 4 requiring disengagement than those in other sessions . In SC neurons bilaterally (n = 41) (C), the mean response magnitude was significantly stronger (a), the mean response latency was significantly shorter (b), and the mean response duration was significantly longer in sessions 3 and 4 requiring disengagement than those in other sessions . These results indicate that the disengagement-related neurons responded stronger and faster to the CSs in the contralateral visual field requiring attentional disengagement.Figure 5 shows the relationships between neuronal response magnitudes in R3 shown in Figure 3 and lick latencies in individual trials. Statistical analysis by simple linear regression indicated that there was a significant negative correlation between response magnitudes and lick latencies . Thus, stronger neuronal responses were accompanied by the shorter licking latencies. Of the 41 disengagement-related neurons, 17   showed similar significant negative correlations between neuronal response magnitudes in session 3 or 4 with disengagement and lick latencies . Furthermore, another five neurons tended to show similar significant negative correlations . These results indicate that the attention disengagement-related neurons guide behaviors in the trials requiring attentional disengagement.The above results suggest that activity of this type neurons might correlate with lick behaviors in the sessions requiring disengagement. P < 0.05), regardless of attentional disengagement, and if response magnitudes to these CSs were larger than those to the frequent CSs without reward . In 46 of the 70 reward and attention shift-related neurons, responses to the infrequent CSs contralateral to the recording sites were larger than those to the infrequent CSs ipsilateral to the recording sites . A typical example of this type of neuron recorded from the right SC is shown in Figure 6. This neuron showed excitatory responses during presentation of all infrequent CSs including those contralateral to the recording site  that were associated with reward , and not to the frequent CSs associated with non-reward  (A). Comparison of the response magnitudes to the CSs are shown in Figure 6B. The response magnitudes to the infrequent CSs contralateral to the recording site  were significantly stronger than that to the CSs associated with non-reward CSs , and also larger than those to the infrequent CSs ipsilateral to the recording site . We found that the response magnitudes to the configural CSs in session 4, in which left light was the frequent stimuli , was significantly smaller than those to the same configural CSs in session 3, in which left light was the infrequent stimuli . These results indicate that this neuron responded stronger to the same contralateral CS (left light) when the rat attended to it than when the rat did not attend to it, suggesting that activity of this neuron reflects visual attention.Reward and attention shift-related neurons were defined as neurons that showed excitatory responses during presentation of the infrequent CSs in reward trials contralateral to the recording sites  to the visual CSs (light flash) contralateral to the recording sites regardless of reward association. A typical example of this type of neuron recorded from the left SC is shown in Figure 7. The neuron responded to the CSs that included the right light  regardless of reward association . Comparison of the response magnitudes to the CSs are shown in Figure 7B. The response magnitudes to the CSs that included the right light were significantly stronger compared to those to the other CSs .Visually responsive neurons were defined as neurons that showed excitatory responses . This type of SC neuron sometimes showed transient excitatory responses to those CSs in short latencies. Of the 12 inhibitory-responsive neurons, four neurons showed inhibitory responses to the CSs associated with reward if the CSs included the light in the contralateral visual field. These neurons showed responses similar to reward and attention shift-related neurons. A typical example of this type neuron recorded from the right SC is shown in Figure 8. This neuron showed inhibitory responses to the infrequent CSs contralateral to the recording site that were associated with reward regardless of attention disengagement , and not to the frequent CSs associated with non-reward . Comparison of the response magnitudes to the CSs are shown in Figure 8B. The absolute values of the response magnitudes to the infrequent CSs contralateral to the recording site were significantly stronger than responses to the other CSs . The remaining eight inhibitory-responsive neurons showed inhibitory responses to the contralateral light regardless of reward.Inhibitory-responsive neurons were defined as neurons that showed inhibitory responses to some of the CSs .The mean response latency of the visually responsive neurons was short (26.5 ± 0.7 ms). However, attention disengagement-related neurons (157.2 ± 21.9 ms), reward and attention shift-related neurons (130.4 ± 13.3 ms), and inhibitory-responsive neurons (123.0 ± 20.2 ms) showed significantly longer mean response latencies , 181 in the intermediate layers , and 190 in the deep layers . The ratios of neurons in each layer of each neuronal type are shown in Figure 10. For the disengagement related-neurons, the ratios of the deep layers were significantly higher than those of the intermediate and the superficial layers , and the ratios of the intermediate layers were significantly higher than those of the superficial layers  in the left SC (A), right SC (B), and both sides of the SC (C). These results indicate that the disengagement-related neurons were located mainly in the deep layers. For the reward and attention shift-related neurons, the ratios of the deep layers as well as the intermediate layers were significantly higher than those of the superficial layers  in the left SC (A), right SC (B), and both sides of the SC (C). There were no significant differences in the ratios between the deep and intermediate layers . These results indicate that the reward and attention shift-related neurons were located mainly in both the deep and intermediate layers. For the visually responsive neurons, the ratios of the superficial layers were significantly higher compared to the intermediate layers , and the ratios of the intermediate layers were significantly higher compared to that of the deep layers . Visually responsive neurons were mainly located in the superficial layers.The recording sites of all SC neurons are shown in In the present study, to induce attention to a specific side, the frequent stimulus was repeatedly presented to that given side in sessions 1–4. Then, the infrequent stimulus was presented in the opposite side simultaneously with the frequent stimulus in sessions 3 and 4, while the infrequent stimulus was presented alone in the opposite side in sessions 1 and 2. Therefore, to engage attention to the infrequent stimulus in the opposite target side in sessions 3 and 4, the animals were required to disengage attention from the frequent stimulus in the previous side in sessions 3 and 4. Consistent with the idea of Figure 6 to the single stimulus (left light) in R1 and the double stimuli (left and right lights) in R3 were similar, suggesting that the divided attention effect was not evident in R3. However, further studies are required to confirm this idea.However, the delayed lick latencies in sessions 3 and 4 could be explained by ambiguousness of reward availability due to the distractor effect instead of attentional disengagement. This possibility seems to be not likely based on the following results. First, there was no difference in correct ratios among the five sessions. This indicates that the animals performed similarly sessions 3 and 4 with simultaneous presentation of the infrequent and frequent stimuli as well as sessions 1 and 2 with the infrequent stimulus only, suggesting that ambiguousness was not evident in sessions 3 and 4. The only difference was lick latencies. A human psychological study reported that presentation of a stimulus in a specific position induces attention to that location , and project directly to the deeper layers of the SC and the pulvinar . ConsistThe reward and attention shift-related neurons, which responded to the contralateral CSs in the reward trials, were located mainly in both the intermediate and deep layers of the SC. The intermediate and deep layers of the SC have intimate connections with various cortical and subcortical structures and relatively few connections with the retina . One of Although the mean response latency of these neurons was longer than that of the visually responsive neurons, some of these neurons had short latencies comparable to visually responsive neurons, which are shorter than cortical neurons . These nThe attention disengagement-related neurons responded more strongly to the CSs requiring attention disengagement in sessions 3 and 4 than to other rewarding CSs in sessions 1 and 2, requiring no attention disengagement, although behavioral requirement was the same. The only difference between the sessions 1 and 2 vs. sessions 3 and 4 was the lights; only one of the two lights was turned on in sessions 1 and 2 while two lights were simultaneously turned on in sessions 3 and 4. Therefore, differences in response magnitudes between sessions 1 and 2 vs. sessions 3 and 4 might be ascribed to differences in total luminance of the stimuli. However, this is unlikely since response magnitudes to the two lights in sessions 3 and 4 were significantly larger than response to the same stimuli in session 5, in which attentional disengagement was not required. Furthermore, we discovered that response latencies of attention disengagement-related neurons were faster and response magnitudes stronger specifically in the sessions 3 and 4 requiring attentional disengagement when compared to those in sessions 1, 2, and 5, in which lick latencies were faster than in sessions 3 and 4. These results strongly suggest that activity of attention disengagement-related neurons does not reflect simple motor-preparatory activity. In addition, activity of some attention disengagement-related neurons was negatively correlated with lick latencies in sessions requiring attention disengagement. Together, these results suggest that attention disengagement-related neurons specifically play a role in attentional disengagement processes to guide licking. These characteristics are contrasted with those of the reward and attention shift-related neurons that responded to the contralateral rewarding CSs requiring no attention disengagement.Another possibility is that the present results could be ascribed to a distractor effect since the animals must detect a target stimulus from the two stimuli in R3 and R4. However, it is not likely based on the following reasons. First, previous studies reported that the neuronal responses to target stimuli with distractors were smaller than those to target stimuli without distractors (see the above section). Therefore, it is noted that the present results in R3 and R4 were opposite to those to the target stimuli with distractors in the previous studies; the neuronal responses of attention disengagement-related neurons to the infrequent stimuli in R3 and R4 were rather larger than those to the infrequent stimuli in R1 and R2. Second, behavioral latencies were negatively correlated with response magnitudes of these attention disengagement-related neurons in R3 and R4. These results suggest that the distractor argument is less likely.A previous human case study reported that a patient with lesions including the right SC showed deficits in saccades to the contralateral (left) target in an overlap condition requiring disengagement . These hSeveral studies suggest deficits in disengagement of visual attention as a unique feature of autism in young children , 2013. Tgap effect; In monkey studies, when animals were required to saccade to an eccentric saccadic target from an initially fixated target, introduction of a temporal gap between the disappearance of the initially fixated target and the appearance of the eccentric saccadic target reduces saccadic reaction time . These results can be interpreted as follows. When an ipsilateral target without disengagement is presented, contralateral reward and attention shift-related neurons are activated to engage attention to the ipsilateral target, and at the same time activity of the attention-disengagement neurons in the ipsilateral SC is suppressed so that these attention disengagement-related neurons do not inhibit the reward and attention shift-related neurons in the contralateral SC. Further studies are required to prove or disprove this idea.The present study demonstrated the existence of rodent SC neurons that are comparable to those in monkeys , as well as a new type of SC neuron that has not been reported in previous studies (attention disengagement-related neurons). The visual attention system can remain either engaged or disengaged  and baseOn the other hand, in the present study we required a licking response from the rats and not a saccade. Previous studies reported SC involvement not only in eye movements (saccades), but also in hand control  and locoThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anxiety disorders often emerge during childhood. Rodent models using classical fear conditioning have shown that different types of fear depend upon different neural structures and may emerge at different stages of development. For example, some work has suggested that contextual fear conditioning generally emerges later in development  than explicitly cued fear conditioning  in rats. This has been attributed to an inability of younger subjects to form a representation of the context due to an immature hippocampus. However, evidence that contextual fear can be observed in postnatal day 17 subjects and that cued fear conditioning continues to emerge past this age raises questions about the nature of this deficit. The current studies examine this question using both the context pre-exposure facilitation effect for immediate single-shock contextual fear conditioning and traditional cued fear conditioning using Sprague-Dawley rats. The data suggest that both cued and contextual fear conditioning are continuing to develop between PD 17 and 24, consistent with development occurring the in essential fear conditioning circuit. The time course of limbic-system development and the ontogeny of fear and anxiety are critical for our understanding of anxiety disorders. Indeed, there is now growing appreciation that anxiety may be a developmental disorder, or at least a disorder with a large developmental component Several studies suggest that the ability of rats to exhibit conditioned freezing to an explicit cue (such as a tone) emerges over a different time course during development than the ability to exhibit freezing to a context, such as the conditioning apparatus Other research suggests that contextual freezing may emerge earlier and can be observed under a variety of circumstances closer to PD 17 In addition to evidence that contextual conditioning may emerge prior to weaning, there is also evidence that auditory conditioning may be continuing to develop later than previously believed. For example, when examining extinction of fear conditioning, Rick Richardson and colleagues report giving twice as many CS-US pairings to PD 16 subjects than to PD 23 subjects in order to achieve equivalent initial freezing, suggesting that PD 23 subjects otherwise demonstrate greater conditioning To address this, our experiments will examine the emergence of contextual and auditory fear conditioning in the periweaning period using two behavioral approaches. Our first set of studies will examine the emergence of contextual conditioning using a contextual pre-exposure task. This capitalizes upon a pair of observations Our second set of experiments will assess the specificity of the deficit in pre-weaning subjects by examining the effects of age on a cued conditioning paradigm. A deficit in conditioning to the auditory cue in younger subjects would suggest that development is occurring in the basic fear conditioning circuit and may not be limited to the contextual learning system. Furthermore, during cued conditioning fear develops not only to the explicit cue, but also to the background context. Indeed, some studies have suggested that background conditioning is stronger than explicit contextual conditioning in pre-weaning subjects These studies capitalized upon the pre-exposure reversal of the immediate shock deficit in contextual conditioning to examine the ontogeny of contextual fear conditioning.These studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of New England . All efforts were made to minimize suffering.3 studies were conducted using rats at different ages. 100, 96 or 163 offspring from 11, 11, or 18 Timed-Pregnant Sprague-Dawley (Charles River) rats were used for Experiments 1–1, 1–2 and 1–3 respectively. Timed-Pregnant Sprague-Dawley rats arrived on either gestational day (GD) 12 or 18 and were checked daily for birth. All subjects were born on GD 21 or 22 and GD 22 was considered the day of birth  for all litters. Litters were housed in 43 cm×44 cm×20 cm transparent PET cages  in the University of New England rat vivarium. On postnatal day (PD) 3, litters were culled to 10 pups per litter  whenever possible. All subjects lived with littermates and mother prior to weaning, which occurred on PD 21. Post-weaning subjects were housed with their same sex littermates (approximately 5 per cage). All subjects were maintained on a 12∶12 hour light/dark cycle and had free access to food and water at all times in the vivarium. At the end of the experiment, animals were euthanized by CO2 overdose.Four Startfear chambers (Harvard Apparatus/Panlab model #58722) were used to collect all behavioral data In order to compare to our previous work On the second day (training), subjects were again transported into the lab in their home cages and stored in a holding room. Subjects were then individually removed from their home cage, weighed, and placed in a transport cage. They were then transported into the conditioning room in the same groups as the day before. All subjects were individually placed into the same context as the previous day where they received a 1.5 mA foot shock that was either immediate or had a 5, 10, 20, or 30 second delay  between the time that they were placed in the context and the time that they received the shock. This relatively high shock level is required to observe the pre-exposure facilitation effect when using freezing as the dependent measure On the third day (testing), subjects were again transported into the lab in their home cages and stored in a holding room. Subjects were individually removed from their home cage, weighed, and placed into a transport cage. They were then transported into the conditioning room in the same groups as the previous two days. All subjects were then placed into the same context as the previous day for five minutes during which their activity was digitized and recorded. Latency to first freezing bout, average movement amplitude during the initial movement bout, and percent freezing were recorded. Following this session, subjects were removed from the conditioning apparatus, placed back into their transport cage, and then back into their home cage.Each experiment was run over 3–5 separate replications, the data of which was pooled to create the final dataset. The various behavioral groups were conducted in pseudo-random order (human designed and precluding repetition) and under the condition that each condition was run equally often in any given cage. For Experiment 1–1: 17/18/19, pre-exposure occurred on PD 17, conditioning on PD 18 and testing on PD 19. For Experiment 1–2: 24/25/26, pre-exposure occurred on PD 24, conditioning on PD 25 and testing on PD 26. For Experiment 1–3: 17/25/26, pre-exposure occurred on PD 17, conditioning on PD 25 and testing on PD 26.The data was collected using Startfear “Freezing” computer software. The subjects in Experiment 1 were either pre-exposed, or not, to the context on Day 1 and given a single shock on Day 2 following various placement-to-shock delays. Subjects were randomly assigned to groups. In general, no more than 1 same-sex littermate was assigned to an experimental condition. In cases where this rule was accidentally deviated from, data from the subjects were averaged and treated statistically as a single observation. Subjects in each experiment were distributed across the groups as shown in We expected that our prepubescent subjects would not show any differences as a function of sex and that the different contexts would not affect conditioning. Indeed, initial omnibus ANOVAs showed no main effects or interactions with sex or cage , therefore two-way ANOVAs (pre-exposed or not) X placement-to-shock interval  were conducted using JMP10 for Macintosh to examine the effect of pre-exposure and placement-to-shock interval on initial movement, latency to freeze and percent freezing during the day of testing.Experiment 1–1: 17/18/19. Subjects were pre-exposed to a context or not on PD 17, given a shock on PD 18, and tested on PD 19. We expected that pre-exposure would fail to enhance conditioning in subjects conditioned at this age. The results supported this hypothesis . A 2 . Instead, we saw a general facilitation of conditioning due to pre-exposure regardless of placement-to-shock interval . A 2  we expected that subjects run in this paradigm would show enhanced fear conditioning based on pre-exposure, but only at moderate intervals. The results of Experiment 3 do not support this hypothesis . A 2  from 18 litters of Timed-Pregnant Sprague-Dawley rats were used (conditioning with either a tone paired or unpaired 0.3 mA or 1.0 mA footshock). Husbandry conditions were exactly as above.The conditioning apparatus was exactly as above.Rats were randomly assigned to one of two conditioning groups (paired or unpaired tone-shock) and each experiment had no more than one male and one female littermate per condition. Each cohort was run on 3 consecutive days and began on either PD 17 or PD 24. The same experimenter performed the three-day behavioral protocol between 8∶30 a.m. and 1∶00 p.m. each day with any given cohort. Start times for each day were within one hour of the start time of the first day. On each day, the home cage was brought into the lab. Rats were weighed, labeled (only on the first day), and placed into individual transport cages. The transport cages were 24 cm×18 cm×13 cm and made of clear plastic. All ambient sound was muted and the rats were transported to the testing room and placed into individual Startfear conditioning chambers. After each test, all rats were returned to their original cages. The experimental chambers were then cleaned thoroughly using Sparkleen and water, followed by either ethanol (Context A) or ammonia (Context B).On the first day, (PD 17 or 24), animals in the “paired training” group were habituated to the experimental chamber for 5 minutes at the start of training. This habituation period was followed by 10 conditioning trials in which a 10-s 70-dB 4-Khz tone coterminated with an aversive stimulus (a 2-s 0.3 mA or 1.0-mA footshock). Trials were separated by randomly chosen intervals . Animals in the “unpaired training” were also habituated to the experimental chamber for 5 minutes. However, the subsequent 10 auditory tones and 10 aversive stimuli were presented in random order. Any two stimuli were separated by a randomly chosen interval between 45 and 105 s (mean  = 75 s). Although our software continuously records movement, due to the confounding influence of footshock on freezing to the tone, we only report freezing for the 5 minutes of context exposure prior to the onset of the aversive stimulus.The following day, rats underwent one of two testing conditions: “Context Testing: or “Novel Context and Tone Testing”. In “Context Testing,” the rats were returned to the same experimental chamber with the same contextual configuration for 5 minutes. Freezing behavior was recorded. In “Novel Context and Tone Testing,” rats were placed into a novel chamber, which was in the opposite contextual configuration, for 5 minutes during which freezing was recorded. This was followed by 10 exposures to the 10-s 4-Khz tone, separated by 20-s intervals. The software reports freezing behavior to the tone as percent freezing across all 10 trials. On the third day, rats were returned to the experiment and subjected to the alternate test. However these data are not included in the current report due to the difficulty in interpreting the data due to the potential effects of the first test session.Rats were divided into 16 groups see . To reduTo use freezing as an index of fear requires observing an increase in the levels of freezing in the presence of a putative fear-inducing CS. This can be complicated when there are differences in baseline freezing levels. In these studies, initial freezing during habituation differed as a function of age F  = 35.5, p<.05  and shock level F  = 4.11, p<.05 . The difference in baseline freezing by age was expected We conducted a 2  X 2  X 2 (shock intensity: 0.3 mA or 1.0 mA) ANOVA to look for effects and interactions of age, shock intensity, and condition on auditory conditioning see . ConsistWe also conducted a 2  by 2  X 2 (shock intensity: 0.3 mA or 1.0 mA) ANOVA to look for effects and interactions of age, shock intensity, and condition on contextual conditioning see . ConsistThese experiments further investigate the ontogeny of fear conditioning in rats. Our data suggest that both auditory and contextual fear conditioning continue to emerge between PD 17 and 24, consistent with development occurring in the essential fear circuit. This is in contrast to some previous work suggesting that behavioral changes during this period were largely limited to contextual learning The current data suggest pre-exposure at PD 17 generally fails to enhance aversive contextual conditioning occurring at PD 18 regardless of the placement-to-shock interval, although there is evidence of context-induced freezing in these subjects at this age . This is consistent with previous work demonstrating that freezing to a conditioning context can be observed in pre-weaning rats, although conditioning tends to be weaker than in post-weanlings Previous work has suggested that contextual learning around PD 17 can be more strongly elicited by enriching the context or conducting simultaneous auditory conditioning The findings at PD 17 stand in contrast to the effects of contextual exposure after weaning. Pre-exposure at PD 24 generally facilitated conditioning at PD 25, as shown by the significant effect of pre-exposure on all three dependent measures of fear (Experiment 1–2). Elevated levels of context-induced freezing were also observed following auditory fear conditioning at this age (Experiment 2). This is consistent with the large body of evidence that post-weaning rats demonstrate elevated levels of context-elicited freezing. However, we found little interaction between contextual pre-exposure and placement-to-shock interval. This is in contrast to work reported by Fanselow (1990), which demonstrated that pre-exposure only enhanced single-shock conditioning at intermediate delays (9 or 27 s) in two strains of adult rats. Conditioning with longer and shorter placement-to-shock intervals was unaffected. One explanation for Fanselow's findings is that at longer placement-to-shock intervals, the shock is no longer “immediate” and all subjects have time to learn about the context and reach asymptotic fear. At shorter placement-to-shock intervals, the benefits of the pre-exposure are also lost, perhaps because the subjects do not have time to recognize the context. Subsequent studies using adult Finally, in contrast to the conclusions of Foster & Burman (2010), pre-exposure at PD 17 generally fails to facilitate contextual freezing following conditioning at PD 25. Foster & Burman (2010) made the observation that contextual exposure at PD 17 could facilitate fear conditioning at PD 24–25 even though it failed to do so at PD 18. This suggested that the developmental deficit at PD 17/18 was not in the ability to learn about a context, but rather in the ability to use contextual information for fear conditioning. Yap & Richardson  make a similar point regarding latent inhibition and renewal of fear, suggesting that the ability to acquire, but not express, contextual learning exists prior to weaning. Although more recent evidence suggests that preweaning subjects do show contextual renewal in an odor-enhanced context Our confidence in the current results is increased because three measures of fear, all based on movement, were consistent in assessing the effects of pre-exposure on contextual fear conditioning. Duration of freezing behavior is perhaps the most common measure of fear conditioning in rodents and has been used for over 45 years Overall, the current data clearly demonstrate that context-induced freezing is impaired in rats at PD 17 compared to rats at PD 24.Although the current studies were initially designed to examine the development of contextual fear conditioning, Experiment 2 also demonstrated a relative impairment in auditory fear conditioning at PD 17 relative to PD 24. Although this initially appears to be a novel finding, in conflict with previous literature The current findings suggest that the development of contextual learning during this period may be secondary to development occurring in the fear conditioning circuit more generally. Although the current data cannot rule out development of sensory or motor systems as contributing to the behaviors we observe, the increase in conditioned freezing between PD 17 and 24 is consistent with the known development of the relevant limbic circuitry. Indeed, a variety of amygdalar nuclei continue to develop well past this period in terms of volume, cell number and especially the number of oligodendrocytes rd and 4th weeks of life can be shown to be equivalent, there is now sufficient evidence that there exist other situations in which fear conditioning differs at these ages. Once differences in fear conditioning as a function of age have been shown, that other parameters exist in which conditioning is equivalent does not affect our overall conclusion that age (perhaps interacting with other variables) determines the emergence of fear conditioning during this developmental period. The current data support the overall conclusion that critical development is occurring in the fear conditioning circuit between PD 17 and 24.Even though there are clearly situations in which rats at the 3Checklist S1NC3R Guidelines Checklist.(PDF)Click here for additional data file.