We aimed to 1) determine if subcortical volume deficits are common to mesial temporal lobe epilepsy (MTLE) patients and their unaffected siblings 2) assess the suitability of subcortical volumetric traits as endophenotypes for MTLE.MRI-based volume measurements of the hippocampus, amygdala, thalamus, caudate, putamen and pallidium were generated using an automated brain reconstruction method (FreeSurfer) for 101 unrelated ‘sporadic’ MTLE patients , 83 unaffected full siblings of patients and 86 healthy control subjects. Changes in the volume of subcortical structures in patients and their unaffected siblings were determined by comparison with healthy controls. Narrow sense heritability was estimated ipsilateral and contralateral to the side of seizure activity.MTLE+HS patients displayed significant volume deficits across the hippocampus, amygdala and thalamus ipsilaterally. In addition, volume loss was detected in the putamen bilaterally. These volume deficits were not present in the unaffected siblings of MTLE+HS patients. Ipsilaterally, the heritability estimates were dramatically reduced for the volume of the hippocampus, thalamus and putamen but remained in the expected range for the amygdala. MRI-negative TLE patients and their unaffected siblings showed no significant volume changes across the same structures and heritability estimates were comparable with calculations from a healthy population.The findings indicate that volume deficits for many subcortical structures in ‘sporadic’ MTLE+HS are not heritable and likely related to acquired factors. Therefore, they do not represent suitable endophenotypes for MTLE+HS. The findings also support the view that, at a neuroanatomical level, MTLE+HS and MRI-negative TLE represent two distinct forms of MTLE. Temporal lobe epilepsy (TLE), the most prevalent form of partial epilepsy in adults, has long been considered an acquired condition. However, epidemiological evidence has indicated a significant genetic predisposition Based on seizure semiology, TLE can be classified into mesial and lateral subtypes. The pathologic hallmark of the more common mesial TLE (MTLE) is hippocampal sclerosis (HS), which is identified in approximately 65–70% of patients Quantitative MRI (QMRI) studies of MTLE+HS patients have detected a variety of extra-hippocampal neuroanatomical abnormalities, predominantly ipsilateral to the side of seizure focus, including volume deficits in other subcortical structures  In sporadic forms of MTLE, if subcortical volume deficits  are under the direct control of genetic variation, then volume changes can be expected in unaffected first-degree relatives of patients and the heritability of the impacted structures should be high. Accordingly, subcortical volumetric traits may represent endophenotypes reflecting particular genetic risk factors highly relevant to the condition. In the present study, we examined the changes and heritability of the volume of subcortical structures in 101 unrelated patients with ‘sporadic’ MTLE and 83 unaffected full siblings of patients. We aimed to:Determine if volumetric changes in subcortical structures are common to both ‘sporadic’ MTLE cases and their unaffected siblings.Assess the suitability of volume measures of subcortical structures as endophenotypes for MTLE, the identification of which could aid in the mapping of genetic factors influencing the development of this condition.The following research ethics committees independently approved this study:Beaumont Hospital Ethics  Committee.St. James’s Hospital/AMNCH Research Ethics Committee.Clinical Research Ethics Committee of the Cork Teaching Hospitals.Written informed consent was provided by all study participants.One hundred and one unrelated patients with mesial temporal lobe epilepsy (MTLE) were recruited from three tertiary epilepsy centres in Ireland: Beaumont Hospital, St. James’s Hospital, and Cork University Hospital. Patients underwent a comprehensive evaluation that confirmed the clinical features of MTLE and the side of seizure activity based on the International League Against Epilepsy (ILAE) guidelines In total, 83 unrelated, asymptomatic (for seizures), same-gender full siblings of MTLE patients were also recruited. Of those, 50 were siblings of MTLE+HS patients  and 33 were siblings of MRI-negative TLE patients . The mean age difference between patients and their unaffected siblings was 5.1 years (range: 1–13). Siblings with known neurological or psychiatric illness were excluded. Further, those with a positive history of childhood febrile seizures, intracranial infection or significant head trauma were also excluded. See Our control population consisted of 86 healthy individuals with no known neurological or psychiatric illness . Controls with a history of childhood febrile seizures, intracranial infection or significant head trauma were excluded. Control subjects with a family history of epilepsy or febrile seizures were also excluded.Participants were divided into two groups for the purpose of MR image acquisition.1-weighted spoiled gradient recalled sequence  with 124 sagittal slices (slice thickness = 1.5 mm) was used to acquire the images.MTLE+HS patients (n = 70), their unaffected siblings (n = 50), and healthy control subjects (n = 58) were scanned using a 1.5 T MRI scanner  at Beaumont Hospital. A three-dimensional (3D) T1-weighted turbo field echo sequence  with 160 slices and an isotropic spatial resolution of 1.0 mm3 was used to acquire the images.MRI-negative TLE patients (n = 31), their unaffected siblings (n = 33), and healthy control subjects (n = 28) were scanned using a 3.0 T MRI scanner  at the Centre for Advanced Medical Imaging, St. James’s Hospital. A three-dimensional (3D) TMR images were processed using FreeSurfer, a fully automated image analysis software (version 4.50). The FreeSurfer process has been described in detail previously For each participant, MRI-based volume measurements were generated for the following subcortical structures in each hemisphere: the hippocampus, thalamus, amygdala, putamen, pallidum, and caudate. Data from group 1 and 2  were analysed independently and accordingly no formal comparisons were made across the two MRI-platforms. Multivariate ANCOVA’s  were employed to compare volume measurements of the subcortical structures between MTLE patients and their unaffected siblings to the healthy controls. Unpaired t-tests were used to test for significant mean differences.MTLE+HS patients were divided into left (n = 35) and right (n = 35) MTLE+HS based on the side of seizure activity/HS and were compared separately to the healthy controls scanned on the 1.5T scanner (n = 58). Similarly, their unaffected siblings were divided into siblings of left MTLE+HS patients (n = 27) and siblings of right MTLE+HS patients (n = 23) and each group was compared to the same healthy controls.MRI-negative TLE patients with left (n = 12) and right (n = 12) seizure activity focus were compared separately to the healthy control group (n = 28) scanned on the 3T scanner. MRI-negative TLE patients with bilateral (n = 2) or undetermined (n = 5) side of seizure activity were excluded from the analysis. Siblings of left and (n = 12) right (n = 12) MRI-negative TLE patients were also treated as separate groups and were compared independently to the same healthy controls (n = 28).p = 0.05 Correction for multiple comparisons were made using a false discovery rate (FDR), setting the level of significance at ) of the volume of each subcortical structure was estimated in MTLE patients and their unaffected siblings using the maximum-likelihood variance components model implemented in the Sequential Oligogenic Linkage Analysis Routines (SOLAR) version 2.1.4 software package p2, for a quantitative trait was decomposed into a genetic component, σg2, and a residual component that includes all effects not accounted for by the genetic component, σe2, while accounting for the population and pedigree structure using the following equation a2 is the additive genetic variance, Δ is the probability that the two relatives share both alleles identical by decent, σd2 is the dominant genetic variance, δ is the identity matrix, and σe2 is the variance accounted by non-genetic variation, including random environmental effects and experimental errors. For any pair of relatives, the value for Φ and Δ can be estimated. For example, for full sibling pairs, the values are ½ and ¼ respectively.The heritability  which is estimated as the ratio of σg2to σp2 where the genetic component (σg2) of the phenotypic variance is defined by an additive genetic component (σa2).The term heritability is commonly associated with the narrow sense heritability (2h) of the volume of each subcortical structure was estimated ipsilateral and contralateral to the side of seizure activity. For this analysis, regional volumetric measurements for the patients and their discordant siblings were re-assembled into ipsilateral and contralateral measurements according to the side of seizure focus in patients. A separate univariate model was set up to calculate the heritability for each trait  with ICV, gender, and age included as covariates. Heritability estimates were compared to those reported for healthy middle-aged male twins (n = 474) by Kremen et al In order to determine the influence of the disease, the narrow-sense heritability (p<0.0001), amygdala , and thalamus (left and right MTLE+HS: p<0.0001)  and right MTLE+HS patient groups . Patients with left MTLE+HS also displayed volume reduction in contralateral hippocampus (p = 0.012). No significant volume differences across the same structures were detected between the unaffected siblings of MTLE+HS patients and the healthy controls. However, a non-significant trend of volume reduction in ipsilateral amygdala was observed ; see p-values survived correction for multiple comparisons (FDR correction threshold p = 0.05).Comparing patients to controls, left and right MTLE+HS patient groups displayed significant volume deficits in ipsilateral hippocampus (left and right MTLE+HS: 001) see , 2, 3. ANo significant volume change was observed in MRI-negative TLE patients when compared to healthy controls. Similarly, their unaffected siblings showed no significant volume changes when compared to the healthy controls. Results are shown in 2h = 0) and that of ipsilateral thalamus showed very small evidence of heritability (2h = 0.04). Further, reduced heritability was observed for the volume of ipsilateral caudate (2h = 0.28), putamen (2h = 0.35) and pallidum (2h = 0.40). In contrast, high heritability was noted for the volume of ipsilateral amygdala (2h = 0.81). The heritability estimates for the volume of contralateral subcortical structures were higher than those estimated ipsilaterally (ranged from 0.43–0.83) and were relatively comparable to those reported in healthy individuals The results of the heritability estimates for the volume of subcortical structures calculated in MTLE patients and their unaffected siblings ipsilateral and contralateral to the side of seizure focus are shown in In MRI-negative TLE, the heritability estimates for the volume of all ipsilateral and contralateral subcortical structures ranged from 0.42–0.85 and were comparable to those reported by Kremen et al In the present study, the volume and heritability of brain subcortical structures were examined in a large sample of unrelated patients with ‘sporadic’ MTLE and their unaffected siblings. The results indicate that subcortical volume deficits are significant in MTLE+HS but apparently absent from MRI-negative TLE patients. Subcortical volume deficits were not present in the unaffected siblings of patients. These findings suggest that, in sporadic MTLE+HS, most of the significant volume deficits in subcortical structures are largely determined by non-genetic factors.Previous QMRI-based studies that investigated subcortical structures in patients with MTLE+HS have consistently identified volume loss in ipsilateral hippocampus, amygdala and thalamus Several studies have reported significant correlations between duration of epilepsy and MTLE+HS-related volume deficits in ipislateral hippocampus, amygdala and thalamus, indicating progressive volume loss In the current study, MRI-negative TLE patients and their unaffected siblings displayed no evidence of volume deficits across any of the subcortical structures. The lateralization effect of the seizure activity, which was very apparent in the MTLE+HS group, was absent in the MRI-negative TLE patients group. This was reflected by the heritability estimates where no difference was noted between the ipsilateral and contralateral subcortical structures. These observations may reflect the subtlety of subcortical structural abnormalities in MRI-negative TLE or indicate the involvement of distinct epileptic networks. Subtle atrophy affecting particular hippocampal subfields was previously reported in a small number of MRI-negative TLE patients in a pattern that was different from that usually seen in MTLE+HS patients In the present study, we focused on volumetric measures of subcortical structures. Future work is required to assess MTLE-related cortical grey matter alterations, including neocortical thinning Figure S1p<0.01; *p<0.05 (corrected for multiple comparisons).The volume of left (top panel) and right putamen (bottom panel) in MTLE patients and their unaffected siblings relative to the healthy controls. Volume measurements are reported in z-scores which were derived from the mean of the controls data. Error bands represent 95% confidence intervals (CI). **Mean is significantly different from the controls at (TIF)Click here for additional data file.Figure S2The volume of left (top panel) and right (bottom panel) of caudate in MTLE patients and their unaffected siblings relative to the healthy controls. Volume measurements are reported in z-scores which were derived from the mean of the controls data. Error bands represent 95% confidence intervals (CI).(TIF)Click here for additional data file.Figure S3The volume of left (top panel) and right (bottom panel) pallidum in MTLE patients and their unaffected siblings relative to the healthy controls. Volume measurements are reported in z-scores which were derived from the mean of the controls data. Error bands represent 95% confidence intervals (CI).(TIF)Click here for additional data file.


Numerous studies have reported subliminal repetition and semantic priming in the visual modality. We transferred this paradigm to the auditory modality. Prime awareness was manipulated by a reduction of sound intensity level. Uncategorized prime words (according to a post-test) were followed by semantically related, unrelated, or repeated target words (presented without intensity reduction) and participants performed a lexical decision task (LDT). Participants with slower reaction times in the LDT showed semantic priming  and negative repetition priming . This is the first report of semantic priming in the auditory modality without conscious categorization of the prime. The extent to which words can be processed unconsciously has been a topic of considerable debate. Unlike studies of implicit word processing In the auditory modality, subliminal perception has been considerably less investigated. Similarly to the visual modality, masking techniques  have been used to reduce prime awareness. However, most of these studies reported contradictory results Studying auditory subliminal priming requires adaptations of the experimental design because of the sequential nature of speech presentation and because of longer processing times in the auditory compared to the visual domain. Unlike visual words, which can be fully presented in a short time window (without distortion), spoken words require time for presentation/pronunciation. In addition, processing is lengthened in the auditory domain as compared to the visual domain Here, we investigated auditory subliminal repetition and semantic priming by using primes presented at low intensity, i.e. in competition with the internal noise  was presented to check that participants were unable to categorize the low-intensity primes. The prime categorization was used as a measure of prime awareness, as in previous studies Forty-five volunteers were tested: 16 in a pre-experiment  and 29 others in the main experiment. All participants were right-handed according to the Edinburgh Handedness Inventory One hundred and sixty words were selected from a French database  or moderate digital attenuation (35 dB for primes of the main experiment) was combined with an analog fixed attenuation. This attenuation was analog rather than digital to prevent acoustic distortion at low levels of presentation. All stimuli were binaurally presented to participants through headphones.To check that repetition priming and semantic priming were elicited with our experimental material, a first group of participants heard a (prime) word, followed by a (target) word or pseudo-word. Participants were asked to decide whether the target was a word or a pseudo-word (i.e. performed a LDT). Primes and targets were presented at a comfortable hearing level (60 dB-A) to sixteen participants. They performed a LDT on the target as fast and accurately as possible by pressing one of two buttons. One hundred and twenty prime-target pairs were presented in random order: sixty with a word target , 20 semantically unrelated to the prime, and 20 repeated) and sixty with a pseudo-word target. Across participants, words were used as either semantically related, semantically unrelated, or repeated. For each participant, none of the words was repeated, except inside the pairs of the repeated condition. A fixation cross was displayed in the center of a monitor screen while the prime word was presented. The target was presented 50 ms after the end of the prime (and of the fixation cross).A second group of participants performed first the LDT in a subliminal priming paradigm and then two post-tests: a prime detection task and a prime categorization task.Submitted).The priming phase was the same as in the pre-experiment, except for the sound level  and the instructions: participants were not told about the presence of the prime. The 10 dB-A intensity level was chosen because previous experiments by our team suggested an absence of conscious categorization at this level , d′D) of 2.7±0.1. Five participants were excluded from the analysis of the main experiment because (1) their performance at the LDT in the priming phase of the main experiment were below the group's mean accuracy minus two SD, or (2) their performance at the prime categorization task were two SD above the group mean d′ sensitivity of categorization (d′C) , or (3) their detection of the prime was two SD below the group mean d′D. Priming is assumed to be subliminal when the performance on the target is above zero, but the performance on the prime is null (here at d′C = 0) d′C = 0 (corresponding to the y-intercept of the regression) is significantly different from zero. d′C was computed for each participant. Thus, the regression was based on a sample size equal to the participant sample size.Since our aim was to study subliminal auditory perception in a homogenous participant group, participants had to be (1) able to perform the LDT in the priming phase of the main experiment, (2) unable to categorize the prime, but nevertheless (3) able to detect it. To test for prime detection, participants performed a detection task (present/absent) in which a word (n = 60) or a silence (n = 60) was randomly presented at the same time as a fixation cross. To avoid participants searching for words, this prime detection task was performed before the prime categorization task. Analysis of the prime detection task showed an average accuracy of 87.4±1.7% and a p-values were adjusted with Greenhouse–Geisser correction for nonsphericity when appropriate. Fisher's Least Significant Difference test was applied for post-hoc comparisons as the Relatedness factor had only three levels Accuracy and correct RTs were analyzed with ANOVAs using Relatedness  as within-subject factor and Rapidity (slow responders/fast responders) as between-subjects factor. Participants were separated into slow and fast responders with a median split. All reported F = 6.90; p = .004; ηp2 = .330] with better accuracy for semantically related (98.7±0.6%) and repeated word pairs (97.5±0.6%) than for unrelated pairs (92.5±2.2%) (ps<.01). There was no main effect of Rapidity (slow/fast) [p = .22] and no interaction with Relatedness [p = .12]. For RTs  = 21.8; p<.001; ηp2 = .609]: RTs were faster for repeated targets (921±38 ms) than for semantically related (1005±38 ms) (p = .003) and unrelated targets (1092±32 ms) (p<.001); RTs were also faster for semantically related versus unrelated targets (p = .002). A main effect of Rapidity  confirmed that slow and fast responders' RTs were significantly different. No interaction between Rapidity and Relatedness was observed [p = .67]. These results indicated that our material, when both primes and targets were presented at a comfortable hearing level (60 dB-A), elicited the expected semantic and repetition priming effects.Average LDT accuracy was 96.2±0.7%. There was a main effect of Relatedness . For slow responders only, RTs were smaller for semantically related targets (1105±29 ms) than for unrelated (1153±28 ms) (p = .034) and repeated targets (1166±28 ms) (p = .008). RTs did not differ between unrelated and repeated targets (p = .549). For the fast responders, no significant differences were observed (ps>.470). In addition, a main effect of Rapidity  confirmed that slow and fast responders' RTs were significantly different.In the priming phase, the overall LDT accuracy was high (93.9±0.7%) and there was no significant effect of Relatedness nor an interaction between Relatedness and Rapidity . The d′C was not significantly greater than zero for the slow group  and the fast group  and did not differ significantly between the fast and slow groups , thereby suggesting that participants were unable to categorize the prime.In the post-test prime categorization task, the average accuracy was 52.0±1.2% (chance level: 50%) with d′C = .10±04, t(22) = 2.54, p = .02), priming at d′C = 0 was estimated through regression analyzes y = −120x+29 with a y-intercept that was significantly above zero  . The regression line of priming effects between related and repeated pairs was y = −80.2x+31.0 with a y-intercept that was significantly above zero  . As these estimated priming effects at d′C = 0 (corresponding to the y-intercepts) differed from zero, these regression suggested semantic and negative repetition priming when participants were not able to categorize the stimuli.As categorization sensitivity for the entire group was slightly greater than zero . Participants were faster to discriminate target words that followed semantically related words than target words that followed semantically unrelated words. Moreover, they showed faster responses for semantically related words than for repeated words (a negative repetition priming). These two priming effects were particularly well observed for slow responders.Our study is the first study to report subliminal semantic priming with auditory primes. In the visual domain, only a few studies that properly controlled the level of prime awareness have reported unconscious semantic priming effects Interestingly, the size of the auditory semantic priming effect we report here (48 ms) is larger than what has been observed in these studies. This difference might be due to the modality. At least under conscious perception, larger semantic priming can be found in the auditory modality than in the visual modality In the auditory modality, subliminal semantic priming has never been reported up to now. The absence of semantic priming in Kouider and Dupoux Participants responded faster to target words that followed semantically related prime words than to target words that repeated the prime, an effect referred to as negative priming One explanation of negative repetition priming is that inhibitory mechanisms might affect RTs. Eimer and Schlaghecken The lack of RTs difference between unrelated and repeated targets see  would thIn contrast to the negative repetition priming observed here, Kouider and Dupoux Even though regression analyzes suggest subliminal priming in all our participants, the RT data showed these effects only in the group of slow responders. This could be a confirmation of our prediction that a differential facilitation between fast- and slow-reacting participants would occur because the processing time of the subliminally presented primes would be lengthy and difficult. Slow responders took more time than fast responders to achieve matching processes between the prime and the target In contrast, when participants were aware of the prime, priming did not differ between slow and fast responders. These results suggest that participants need more time and cognitive load to process the prime when it is presented subliminally as compared to when it is presented supraliminally.As done by Kouider and Dupoux


A fully automated method for the detection and quantification of bird migration was developed for operational C-band weather radar, measuring bird density, speed and direction as a function of altitude. These weather radar bird observations have been validated with data from a high-accuracy dedicated bird radar, which was stationed in the measurement volume of weather radar sites in The Netherlands, Belgium and France for a full migration season during autumn 2007 and spring 2008. We show that weather radar can extract near real-time bird density altitude profiles that closely correspond to the density profiles measured by dedicated bird radar. Doppler weather radar can thus be used as a reliable sensor for quantifying bird densities aloft in an operational setting, which—when extended to multiple radars—enables the mapping and continuous monitoring of bird migration flyways. By applying the automated method to a network of weather radars, we observed how mesoscale variability in weather conditions structured the timing and altitude profile of bird migration within single nights. Bird density altitude profiles were observed that consisted of multiple layers, which could be explained from the distinct wind conditions at different take-off sites. Consistently lower bird densities are recorded in The Netherlands compared with sites in France and eastern Belgium, which reveals some of the spatial extent of the dominant Scandinavian flyway over continental Europe. Comprehensive monitoring of bird migration at continental scales can provide fundamental insight into migration patterns, the impact on migratory flight of synoptic scale factors like weather and orography and the selection of stop-over areas by migratory birds –3. StronRadar has had an immense impact on ornithology and the study of bird migration, because of its unique ability to monitor bird movements up to high altitudes and distances during both night and day ,4–7. Weapera . Althourmation; –15. If wAlthough weather radars have been used in ornithological research for several decades , to our 2.λ = 3 cm) radar of the type ‘Superfledermaus’ [To obtain reference data for validating weather radar bird observations, we organized extensive field campaigns with a high precision dedicated bird radar. An ex-military pencil-beam X-band . It has been suggested that spectrum width can be useful in target identification of various biological scatterers , but in The developed bird detection algorithm produces a vertical profile of bird density, speed and direction every 5–15 min at 200 m altitude resolution, based on data at the 5–25 km range. At these close distances, the radar beam is sufficiently narrow to probe distinct altitudes, and range-dependent biases are largely avoided, which otherwise need to be compensated for . Each ex3.1.et al. [σr is calculated  occurred at altitudes, where simultaneously bird migration was detected by an independent bird radar, which demonstrated that σr is also a good indicator for the presence of birds. Reliable bird density quantification could not be demonstrated in the study of van Gasteren [σr as a discriminator for bird presence, where we will also discuss possible events unrelated to bird migration that can cause high values of σr.The algorithm is based on the existing wind-profiling algorithms for Doppler weather radars, using the volume velocity profiling (VVP) technique ,34. Thiset al. . The VVPequation ). Recentprofiles ,30,31. v3.2.σr criterion, we developed a target identification scheme to filter out non-bird echoes from the radar volume data. All reflectivity factors above 20 dBZe are masked as non-bird echoes, since such high values occurred rarely during bird migration in our study period and exceed reflectivity factors expected for broad-fronted passerine migration (20 dBZe corresponds to approx. 3500 passerine-sized birds per cubic kilometre at C-band (see equation (In addition to the equation  and figuequation  legend. a (i)) and precipitation (figure 2b (ii)). To identify selected cells in bird migration areas and discard them from the precipitation map, for each selected cell an average nearest neighbour variance σcell is computed for the radial velocities  than for birds performing active flight. This difference is illustrated in figure 2b, where σcell is plotted as a function of the average cell reflectivity factor for a number of cells detected during intense bird migration events (green bullets) and cells detected during events with convective showers (blue bullets). A combined criterion using reflectivity and radial velocity information is used to assign cells to the precipitation map: cells with either a low variance or a high reflectivity factor (σcell < 5 m s−1 or Zcell > 15 dBZe) are removed from the data as non-bird scattering. From the remaining areas, an average reflectivity is calculated for each 200 m altitude interval.When selecting contiguous reflectivity areas, spurious precipitation cells may be detected in areas of intense bird migration as well. Examples of selected cells are given in figure 2equation ). Analogσcell, the identification of outliers is less straightforward and has not been implemented. As a result, values of σcell for bird migration are typically higher than for σr, and therefore also the optimal threshold in σcell is higher (5 m s−1) than for σr (2 m s−1).We find that bird migration gives rise to a relatively high fraction of radial velocity dealiasing outliers (up to 15%) compared with precipitation (1%) and daytime clear air echoes  using thρHV > 0.9 . By taking advantage of the additional information, especially the removal of precipitation from the data is improved. We exclude contiguous areas with a high correlation coefficient, meteors; ) or high insect; –40). For3.3.λ, the reflectivity η, equivalent reflectivity factor Ze and bird density ρbird are related according to [η in cm2 km−3, Ze in mm6 m−3, Km = (m2 − 1)/(m2 + 2) with m the complex refractive index of the scatterers, λ (in cm) the radar wavelength, ρbird in birds per km−3 and σbird the bird radar cross section at C-band in square centimetres. The bar over the ‘ρbirdσbird’ product denotes that this quantity is strictly an average over the different bird types or species i according to ∑i ρibird,σibird, and over the scanned viewing angles. With |Km|2 = 0.93 for water [η is proportional to Ze by a factor of 28.0 at S-band (λ = 10 cm)  and 3.51 × 103 at X-band (λ = 3.0 cm). Bird radar cross sections and therefore reflectivities η are of the same order of magnitude at these radar bands. However, because of the λ−4 proportionality in equation (Ze in S-band compared with C-band (and much lower reflectivity factors in X-band).Bird density information can be obtained from reflectivity measurements, analogous to quantitative precipitation estimation. For S-band weather radars, empirical relationships have been found between reflectivity and the volumetric density of migrating birds ,41, but rding to ,41,423.1or water , we findη in equation  during this period. While on 4 October, migration does not exceed 1.5 km, on 6 and 7 October, the bird radar records migration extending above 3 km height. These altitude profiles are closely reproduced by the weather radar algorithm (figure 3b). Also, a remarkable correspondence is observed between both sensors for the recorded absolute numbers of birds, as illustrated by the closely overlaying height-integrated bird densities (figure 3c).In figure 3For the quantitative verification, measurements were used from all three campaigns in De Bilt, Wideumont and Trappes, comprising 118 days of continuous data. We limit the quantitative verification of the weather radar algorithm to night-time hours only. During daytime bird flocks are common , which s4.1.1.σr  and as a function of bird reflectivity . Bird densities as determined by the bird radar are indicated by colours. The large majority of non-zero bird densities are observed for time–height layers with σr > 2 m s−1, which confirms that a radial velocity standard deviation σr = 2 m s−1 successfully discards non-bird echoes. In figure 4a, a large number of points cluster around σr = 1 m s−1 at high raw reflectivity values, which can be attributed to precipitation events. A considerable number of time–height layers with σr > 2 m s−1 have obtained a high raw reflectivity from precipitation contaminations. The effect of removing these non-bird echoes on the reflectivity is shown in figure 4b. Precipitation-contaminated scatter points shift horizontally to lower reflectivity values, and high reflectivity gets always associated to high bird densities. Some residual precipitation contaminations remain in the region σr < 2 m s−1, while time–height layers for which all resolution volumes get assigned to the precipitation map are fully removed.equation ) as a fuρbird determined by bird radar exceeds 1 bird per km−3 and define a criterion for bird presence in weather radar data by σr > 2 m s−1. By collocating the bird radar and weather radar measurements, we can thus group all time–height layers into categories for the number of correct detections H, missed detections M, false detections F and correct non-bird detections Z. For the combined set of campaigns, we find that the weather radar system has a high probability of detection (POD) to be H/(H + M) (POD = 97%). The weather radar algorithm detects birds even at very low densities and bird migration events are unlikely to be missed. The false alarm ratio (FAR), F/(H + F), is however rather high (FAR = 42%), but the majority of these false alarms occur in a regime of very low bird reflectivity. Precipitation contaminations with reflectivity factors of −30 to −10 dBZe are frequent, but, as will be discussed below, such reflectivity factors correspond to very low bird densities below 1 bird per km−3 only. The rate of false detection steeply decreases with increasing bird reflectivity. Calculating the POD and FAR statistics for the subset of time–height layers with a reflectivity factor greater than −5 dBZe, we find POD = 100% and FAR = 3% only. This regime of bird reflectivity factors is of most interest, as it corresponds to events with moderate to high bird densities greater than 10 birds per km−3.We define true bird presence when the bird density σr, can also occur when the actual velocity field is not uniform  or a low value of σcell. Since σcell is a locally calculated quantity over the nearest neighbours of each resolution volume, its value increases only owing to velocity variations over scales of up to a few kilometres and not by large-scale non-uniformities. Our bird radar measurements verify that most daytime weather radar echoes in clear air were caused by other scatterers than birds, which were correctly removed by the weather radar algorithm based on a low value of σr. This confirms the suggestion by Caya & Zawadzki [Large radial velocity standard deviations, d shear; ,44) or wd shear; ). Such nZawadzki  that in −3, a radar resolution volume contains only one up to a few individual birds . The radar resolution is therefore sufficient to resolve part of the speed and directional variation between individual birds. In contrast, insect aerial densities tend to be much higher than bird aerial densities [σr slightly decreases when the bird density increases. A second cause of the high radial velocity variance observed for birds is their relatively fast active flight  up to bird densities of a few hundred birds per kmensities . Each ra5 m s−1; ), which 5 m s−1; .4.1.2.η (measured by weather radar) and bird density ρbird (measured by bird radar), as illustrated in σbird = η/ρbird . From mid-September to mid-October (a), the average daily cross section is significantly increasing  from 7 to 15 cm2 by 0.3 ± 0.1 cm2 d−1. A reverse temporal trend is observed in spring when the cross section significantly decreases  from 16 to 10 cm2 by 0.1 ± 0.04 cm2 d−1 in the period mid-March to early May (b).In agreement with equation , we findequation ). We fin-October a, the avarly May b.σbird must thus be explained from changes in the average body size of the migrating passerines. We can exclude insect scattering as a contribution to the observed cross-section changes, as the number of identified insect echoes by the bird radar decreased in autumn and increased over the course of spring, which are in fact the reverse trends that would explain the observed cross-section variations . All large passerines  in westvulgaris ,50, it iσbird of 11 cm2 when converting weather radar reflectivity into bird density by equation . These cross sections are too large for the predominantly small passerines that migrate during this time of the season [σbird. During nocturnal spring migration, insect contamination is minor up to early June. Weather radar-extracted nocturnal bird densities drop below 5 birds per km−3 by mid-May at the end of the migration season. In spring, insect contaminations thus result in a bias of at most 5 birds per km−3; however, the contamination is likely less since by mid-May birds (e.g. Swifts) are still aloft and insect densities are much lower in March and April.During some nights in early September, larger effective e season . We may e season ,51; howeAlthough the aerial mass density of insects can be larger than for birds , scatter2 km3 volume, while for the same time–height layer the fixed pencil beam of the bird radar surveys only a 0.01–0.1 km3 volume, depending on the altitude (see appendix A for the exact scanning strategies). Only in the case of ideal intense broad front migration [Differences in the representativity of the bird radar reference data and the weather radar data will contribute to the spread in the correlation between bird radar and weather radar in igration  will the4.2.irlam numerical weather prediction model [irlam wind profile shows that on the night of 3–4 October wind conditions at low altitude were more favourable than at high altitude, where migrants would have encountered strong south-westerly head and side winds. On this occasion, migration did not extend above 1 km. On the other hand, on the night of 5–6 October, tail winds were present above 1 km and a large fraction of migration took place at high altitude.The effect of the environmental wind on flight altitudes becomes evident by comparing the bird density profile with the wind profiles calculated by the Hon model   to south (bottom). Arrows indicating flight directions are overlaid with the bird densities, while barbs in the pink boxed insets show the Hirlam wind profile at 00.00 UTC. Large differences are visible between the sites, both in total density, timing and altitudinal profile of the migration.Figure 7Migration in Trappes is characterized by strong departure and ascent to high altitudes above 2 km, where birds experience favourable tail winds along the predominant migratory direction  as revealed by the wind profile. Low-altitude migration is avoided because of unfavourable easterly low-level winds. The migration intensity quickly drops after 00.00 UTC and the arrival of birds from southern latitudes is limited. The drop in bird density is related to the weather conditions south of Trappes, which, due to rain and easterly winds, were unsuitable for migration.−1 to birds having travelled over a distance of about 280 km. These birds must therefore have departed in the vicinity of Trappes in northern France, where birds chose a flight altitude of 2500 m. Note that Wideumont is located 585 m mean sea level (MSL), therefore the 1900 m altitude band closely matches the cruising altitude after departure near Trappes. The double-layered density profile is not explained by the local wind profile in Wideumont. Rather, it is consistent with the migrants maintaining a constant flight altitude (above sea level) once they had completed the ascent phase of their migratory flight.Migration in Wideumont (280 km north east of Trappes) starts out with strong departure at lower altitudes. Below 2 km, altitude tail winds are more favourable than above 2 km, where initially strong westerly winds prevail (wind data not shown). After 22.00 UTC, a remarkable double-layered bird density height profile is observed, with one band centred around 800 m AGL and a second band centred around 1900 m AGL. Since we started observing bird migration by weather radar in autumn 2007, such double-layered altitude profiles have been observed regularly during spring migration. The top altitude flight band appears after 4 h of flight time, which corresponds with a measured bird ground speed of 70 km hIn De Bilt north of Wideumont, no birds depart in the early night. This is explained by the weak occlusion front slightly south of de Bilt, which blocks most migration. With this front weakening in activity, migration conditions become more favourable over the course of the night, and after 00.00 UTC, we do observe the passage and arrival of migrating birds. No migration is observed on the most northerly located weather radar in Den Helder.pera weather radar network become available, it will be possible to map for the first time the extent and dimensions of entire migratory flyways over Europe.Consistently lower bird densities are recorded in De Bilt and even more so in Den Helder compared with the sites in Trappes and Wideumont. We calculated migration traffic rates  defined 5.−3 are discarded) and weather radar reflectivity can be quantitatively correlated to the bird densities determined by the bird radar.We have developed an automated method for bird detection and bird density quantification by weather radar, extracting bird density, speed and direction as a function of altitude. The algorithm can run in near real-time with a time and altitude resolution of 5 min and 200 m, respectively, and was applied to four different Doppler weather radars in The Netherlands, Belgium and France. Generated altitude profiles are based on data up to 25 km range and thus provide a local characterization of the migration profile. The extracted weather radar bird densities have been validated with data from a high-accuracy dedicated bird radar, which was stationed in the measurement volume of different weather radars during the peak migration season of autumn 2007 and spring 2008. We find that Doppler weather radar is highly successful in detecting migrating birds and quantifying bird densities as a function of altitude. The probability of detection is (POD) is very high (99%), the false alarm ratio (FAR) is low (3%, provided that bird densities less than 10 birds kmBy applying our bird quantification method to a network of weather radars, we observed how mesoscale weather structured the intensity and timing of bird migration. In particular, the altitude profiles at individual sites were shown to be affected by the weather conditions at other locations. While the local wind field is an important variable for understanding local bird densities aloft, weather conditions in the vicinity of take-off sites can also strongly determine the observed flight altitude profiles.pera data centre for radar data within the coming 2 years [The current study shows that automated bird migration monitoring by operational weather radar networks is feasible. The developed methods for bird detection and quantification can be easily extended to full operational weather radar networks. With the development of the O 2 years , the est 2 years .Currently, polarimetric weather radar is rapidly becoming the new operational standard. Dual-polarization techniques will likely contribute to future improvements of the current bird migration quantification algorithm. We expect that automated bird migration quantification can also be implemented for weather radars operating at different radar wavelengths like S-band and X-band; however additional research and validation is required, because of the very different relative cross section of hydrometeors, insects and birds at these wavelengths. Weather radars are capable of providing spatial information on bird migration by using data from their full surveillance area ,3,33. A For early-warning systems of bird migration, operational bird density forecasts are essential, which could be made in combination with spatially explicit bird migration models ,58. Such


This increase in single spike efficiency is, however, counterbalanced by a temperature-dependent decrease in the amplitude and duration of the spike afterhyperpolarization, resulting in a nonlinear increase in the spike firing rate, particularly at temperatures above approximately 35°C. Interestingly, the total energy cost, as measured by the multiplication of total Na+ entry per spike and average firing rate in response to a constant input, reaches a global minimum between 37–42°C. Our results indicate that increases in temperature result in an unexpected increase in energy efficiency, especially near normal body temperature, thus allowing the brain to utilize an energy efficient neural code.The energy efficiency of neural signal transmission is important not only as a limiting factor in brain architecture, but it also influences the interpretation of functional brain imaging signals. Action potential generation in mammalian, versus invertebrate, axons is remarkably energy efficient. Here we demonstrate that this increase in energy efficiency is due largely to a warmer body temperature. Increases in temperature result in an exponential increase in energy efficiency for single action potentials by increasing the rate of Na Conserving energy is essential to life. The brain, while only 2% of the body mass, uses an astounding 20% of its energy. It has long been assumed that this large energy consumption was due to the need to generate the electrical signals through which brain cells communicate: the action potentials. However, recent results reveal that the wires of the mammalian brain – the axons – are remarkably energy efficient. How is this energy efficiency obtained? Here we addressed this question by performing recordings and computational models of mammalian brain cells. We found that the increase in body temperature associated with the evolution of warm-blooded animals had an energetic benefit. The action potentials of warm-blooded animals became remarkably energy efficient, owing simply to the increase in body temperature. These results indicate that mammalian brains, although requiring a great deal of energy to operate, are actually more efficient than expected. These influxes of Na+ into neurons occur in addition to a background leak of Na+ ions through the neuronal membrane. Thus, to understand the energy costs of neuronal signaling in the cortex, it is essential to understand the entry of Na+ into neurons and neuronal processes during neuronal activity.Brain signaling is metabolically expensive. Energy expenditure not only constrains the size and architecture of the brain, which limits its computational power, but is critical to the interpretation of functional brain imaging signals through related metabolic mechanisms (e.g. oxygen consumption and blood flow) + current that generates the upstroke and the outward K+ current that facilitates the downstroke. Any overlap of these two opposing currents would merely result in an electrically neutral exchange of positive ions. Classic investigations by Hodgkin of squid giant axon revealed an excess entry of approximately 4 times as much Na+ as minimally required to generate the action potential + entry has figured prominently in estimates of the distribution of the sources of energy consumption in the mammalian brain + entry needed is obtained [for action potential generation] by quadrupling [the minimal Na+ entry] to take account of simultaneous activation of Na+ and K+ channels” + entry (and K+ exit) during action potentials A maximally energy efficient action potential would entail no overlap of the inward Na+ and outward K+ currents occurring during action potential generation in mammalian cortical axons revealed, in contrast to the results predicted, an excess ratio of Na+ entry of only 1.3 + channel inactivation prior to substantial activation of the outward K+ current Reconstruction of the inward Na+ and K+ currents underlying action potential generation Loligo) lives in an ocean environment varying in temperature from approximately 10–23°C, which is far colder than naturally occurring in most mammals. Since temperature is a strong determinant of ion channel kinetics, which in turn can dramatically change action potential efficiency, we explored here the possibility that mammalian neurons may generate action potentials with maximal energy efficiency at normal body temperatures.Prior recording and modeling studies have demonstrated that the efficiency of action potential generation is highly sensitive to the kinetics of underlying ionic currents + entry between these two species by performing simple Hodgkin-Huxley style simulations of action potentials in either the traditional HH single compartment model or in a simple uniform cylindrical model of a cortical axon Na, IK, and ILeak so as to clearly demonstrate the principles of the effects of temperature on excess Na+ entry during action potentials. Simulations with more complete computational models of layer 5 pyramidal neurons, including back propagating action potentials from the axon 10 for both Na+ and K+ currents of 2.3 10 used from 1.5 to 3 yielded qualitatively similar results as those shown in + entry occurring during action potential generation, such that at 18°C a value of approximately 4X excess is obtained, while at 37°C, a value of 1.41 is observed for our model of cortical axons  while injecting the fixed 37°C action potential waveform, resulted in a marked decrease of the total Na+ current and overlap of Na+ and K+ currents . Thus, increasing temperature does not result in a decrease in total Na+ current or a decrease in Na+/K+ current overlap by decreasing the action potential duration. Rather, the increase in temperature results in both a decrease in spike duration as well as a decrease in excess Na+ entry owing largely to the increased rate of Na+ channel inactivation (see below).Examining the relationship between action potential duration and excess Nainset in  may leadres e.g. . To testres e.g. . We then to 37°C . When ficurrents  and redu18°C cf. . As with18°C cf. , making currents . Our ana+ and outward K+ currents results in systematic changes in dV/dt of the action potential, and the ratio of the maximal falling to rising dV/dt values, with temperature (min)/(IdV/dtImax). This variable is strongly influenced by the level of separation of the inward Na+ and outward K+ currents, and by other factors such as the peak amplitude of INa and IK. We include it here because it is a readily measureable variable in real neurons, and therefore useful for comparison with results of our model. + entry ratio as a function of γ revealed that as γ increases (representing in part decreased overlap of INa and IK), the excess Na+ entry ratio decreases  and IK (τn) and inactivation (τh) of INa, revealed an exponential and strong decrease in all three with increases in temperature (Na activation (m) and inactivation (h) and IK activation (n) variables versus membrane potential during generation of an action potential at 18 and 36°C revealed significant and important effects of temperature  constant while allowing τm and τn to vary  or IK activation rate (τn) constant individually did not abolish the strong decrease in Na+ entry/spike with temperature  was invariant . Interestingly, at higher temperatures (approximately 38–40°C), the firing rate in our models increases rapidly with increases in temperature  to a constant square pulse input decreases with temperature to a minimum at approximately 38–40°C. At temperatures above this minimum, the rapid increase in firing rate results in an increasing Na+ load on the neuronal process , IK became especially large even 25–60 msec after the spike. These changes resulted in a larger, more prolonged spike AHP  of IK constant, while allowing the activation (τm) and inactivate rates (τh) of INa to vary with temperature, nearly abolished the ability of changes in temperature to cause non-linear changes in discharge rate (m) or inactivation rate (τh) of INa constant, while allowing the other kinetic time constants to vary with temperature, did not change the presence of this non-linear relationship, although it did alter the range of temperatures over which it occurred (h) of INa invariant inverted the relationship between the total Na+/spike and temperature, while keeping τm or τn invariant does not fundamentally alter this relationship  is strongly affected by keeping INa inactivate rate (τh) invariant (Na (τm) constant has relatively little effect, while keeping the activation rate of IK (τn) constant exaggerates the decrease in total Na+/current pulse  The firing rate of the neuronal process to a constant increases will increase non-linearly with changes in temperature, particularly at temperatures above approximately 37°C. Next we tested whether or not these predictions would be confirmed in somatosensory layer 5 and entorhinal layer 2/3 cortical pyramidal cells recorded in vitro in response to constant current pulse injection  action potentials will become shorter in duration and smaller in amplitude, with a marked decrease in overlap of the inward Nanjection ,9, or dunjection .2+ signaling As predicted by HH simulations, increases in temperature resulted in a significant decrease in spike duration , spike h+ entry per current pulse  decreased exponentially with increases in temperature , 9F, actOne possible confounding factor in our in vitro recordings is that increasing temperature indirectly increased neuronal excitability through decreasing the oxygen content of the bathing solution. To examine this possibility, we measured the oxygen content of the ACSF at the upper interface of layer 2/3 of the entorhinal cortical slice and the bath solution while varying temperature . We then+ channel inactivation and subsequent decreased spike amplitude, duration and overlap between inward Na+ and outward K+ currents. Thus, increases in temperature naturally result in a marked decrease in excess Na+ entry during spike generation, reducing the need for activation of the Na+/K+ ion pump (ATPase), and thus reducing energy expenditure. This result suggests that the higher body temperature of endotherms such as mammals (versus ectotherms such as squid) has the advantage of resulting in a marked increase in energy efficiency of single action potential generation. However, increases in temperature also resulted in an increase in spike discharge rate to a constant amplitude input or during spontaneous network activity, owing in part to decreases in the amplitude and duration of K+ currents initiated by action potentials Increases in temperature have marked and strong effects on action potential generation, and these in turn impact upon the energy efficiency of neuronal activity. Here we demonstrate that increases in temperature result in a large increase in the energy efficiency of single action potential generation owing to the increased rate of Na+ entry during spike generation in the squid giant axon + entry during action potential generation(excess ratio of around 1.3)  . Recent  at 37°C –3.+ currents activated by action potentials. These decreases in K+ current amplitude and duration may result from decreases in activation owing to the shortened and smaller action potential , changes in the kinetics of second messenger events within the neuron following the action potential, or changes in the kinetics of the K+ channels themselves 2+ activated K+ currents, in real neurons that were not included in our simple simulation Normal mammalian brain temperature varies from approximately 36–39°C, depending upon state of the animal  and location within the brain, although some mammals (and some species of birds) can exhibit brain temperatures as high as 45°C + and K+ currents + currents responsible for spike repolarization + channel inactivation, significantly increases the energy demands on the neuron, owing to significant overlap of the inward Na+ and outward K+ currents + and K+ currents, resulting in high energy efficiency + channel properties between the soma and axon, with Kv1.1/1.2 channels being prevalent in the axon initial segment + channels in the axon initial segment are approximately 2X faster than those in the soma + channels may result from the presence of 1–3 auxiliary subunits in axonal locations Different neuronal subtypes in the brain vary in their energy efficiency of action potential generation owing in part to differences in overlap of action potential-related NaSparse neuronal discharge, in which the impact of each action potential is maximized in the task of the network in which it is embedded, is a powerful means to reduce overall energy expenditure + load (spike rate×Na+/spike) on a neuron reached its minimum at about 37°C suggests that neurons may have optimized their energy use for this temperature range, as has been observed for a number of cellular and organ functions such as enzymatic function and the balance of heat production/loss in an ambient external environment of 25°C Our observation that total Na4 2, CaCl2, 2, NaHCO3 26, NaH2PO4 1.25, dextrose 25 . For maintaining the slow oscillation in vitro, the extracellular [Ca2+] and [Mg2+] were reduced to 1.0 mM Experiments were carried out in accordance with all relevant NIH and institutional guidelines on the proper care and use of animals in research. Experiments were performed on slices of mouse somatosensory (20–45 days old) or entorhinal (12–21 days old) cortex maintained in vitro in a submerged style recording chamber. The ACSF contained (in mM): NaCl 126, KCl 2.5, MgSO2 2, Na2ATP 2, HEPES 10, EGTA 0.2 mM, pH 7.2 with KOH (288 mOsm). All statistical comparisons were performed using a two-tailed t-test. All results are reported as mean +/− standard deviation, and illustrated as mean +/− standard error of the mean.Whole-cell recordings were achieved from the soma using a Multiclamp 700B amplifier  as described previously Original Hodgkin-Huxley Model. The classical Hodgkin-Huxley equation describing the membrane potential as a function of all the currents that flow across the axon membrane, is2 is the specific membrane capacitance, Istim is the input stimulus, gL = 0.3 mS/cm2, gNamax = 120 mS/cm2, gKmax = 36 mS/cm2, are the leak, maximal sodium and maximal potassium conductance per unit membrane area, respectively. VL = −54.4 mV, VNa = 50 mV, and VK = −77 mV are reversal potentials of leak, sodium and potassium channels, respectively. The gate variables m, h, and n are dimensionless activation and inactivation variables, which describe the activation and inactivation processes of the sodium and potassium channels, each of which is governed by the following differential equations:Φ regulating the temperature dependence, known as the Law of Arrhenius, with Q10 = 3. The sensitivity of the Na+ current kinetics to temperature has been studied in a variety of preparations, where the Q10 for inactivation is, on average, 2.95 and the Q10 for activation is 2.68  within the cortical slice at temperatures varying from 30–42°C. Following this, we then independently manipulated oxygen content, while keeping temperature constant at 30°C and examined the effect of changes in oxygen concentration on neuronal responsiveness. The oxygen concentration was measured at 1 Hz in acute slices of mouse entorhinal cortex (12–18 days) using an OxyLite system . The oxygen probe (Model BF/OT/E), which has a tip diameter of 350 µm, was placed 100 µm beneath the surface of the tissue in layer 2/3 of entorhinal cortex. Each temperature change was brought to steady state for at least two minutes before the response of the pyramidal neuron to intrasomatic injection of a depolarizing current pulse was measured. In additional experiments, we manipulated oxygen concentration while keeping temperature constant by bubbling the solution with varying proportions of O2/CO2 (95%/5%) and N2/CO2 (95%/5%) gas. Each oxygen level was maintained for at least two minutes before the firing rate was measured.Figure S1+ entry ratio and temperature under control conditions (black), and when the Hodgkin-Huxley model of a cortical axon is run with an action potential wave form that is fixed to that occurring at 18°C (green) and 37°C (red). Note that in both experimental cases, using the fixed action potential waveform at colder temperatures results in a large increase in excess Na+ entry ratio, even though these spikes are shorter in duration than those that would have occurred at these colder temperatures. These results indicate that the decrease in Na+ entry ratio with temperature seen in control is not due to the shortening of the action potential duration.Relationship between excess Na(TIF)Click here for additional data file.Figure S2+ activation variable m (A), Na+ inactivation variable h (B) and K+ activation variable n (C) for temperature T = 18°C, and 36°C, respectively. Also plotted (black traces) are the steady state values of these variables . Note that although increasing temperature does increase the resemblance of the m versus V phase plot to the steady state relation, the same is not as true for Na+ channel inactivation (h) or K+ channel activation (n). Here, the phase plots still exhibit substantial deviations from steady state values during action potential generation.A–C. Phase plots of membrane potential V vs. Na(TIF)Click here for additional data file.Figure S3Increasing temperature increases firing rate during spontaneous Up states in entorhinal cortical slices. A. Example of an Up-state generated spontaneously in the medial entorhinal cortex at 23°C. B. Up states at 36°C. C. Up states at 42°C. Note the increase in Up rate, and increase in action potential discharge during each Up state. D–F. Action potential discharge rate (D), action potential duration (E), spike threshold (F) for action potentials occurring during Up states as a function of temperature. G. Resting membrane potential during the Down state at different temperatures. All data obtained from layer 2/3 pyramidal neurons (n = 6) in the mouse medial entorhinal cortex.(TIF)Click here for additional data file.Figure S4+ charge entry in response to a DC pulse (B).Effects of changing specific membrane resistance (decrease by 35%) and membrane potential (depolarization by 7 mV) on the relationship between temperature and firing rate in a HH model neuron (A) and the resulting effect on total Na(TIF)Click here for additional data file.Figure S5+ entry owing to changes in apparent input resistance and membrane time constant in a model neuron. A. Increasing specific membrane resistance from 20,000 to 60,000 only slightly decreases the excess Na+ entry ratio. B, C. Example action potentials with a specific membrane resistance of 18,000 and 66,000 Ohms. D, Relationship between membrane time constant and input resistance in the model. E. Excess Na+ entry ratio decreases only slightly by increasing membrane time constant. F. Firing rate of the neuron to a constant current pulse exhibits a small increase with a large increase input resistance.Increases in Na(TIF)Click here for additional data file.


The mechanisms of septic shock-associated adrenal insufficiency remain unclear. This study aimed at investigating the synthesis of corticotropin-releasing hormone (CRH) and vasopressin (AVP) by parvocellular neurons and the antehypophyseal expression of ACTH in human septic shock and in an experimental model of sepsis.To test the hypothesis that ACTH secretion is decreased secondarily to alteration of CRH or AVP synthesis, we undertook a neuropathological study of the antehypophyseal system in patients who had died from septic shock and rats with experimental faecal peritonitis.Brains obtained in 9 septic shock patients were compared to 10 nonseptic patients (controls). Parvocellular expression of AVP and CRH mRNA were evaluated by in situ hybridization. Antehypophyseal expression of ACTH, vasopressin V1b and CRH R1 receptors and parvocellular expression of iNOS in the PVN were evaluated by immunohistochemistry. The same experiments were carried out in a fecal peritonitis-induced model of sepsis. Data from septic rats with (n = 6) or without (n = 10) early death were compared to sham-operated (n = 8) animals.In patients and rats, septic shock was associated with a decreased expression of ACTH, unchanged expression of V1B receptor, CRHR1 and AVP mRNA, and increased expression of parvocellular iNOS compared to controls. Septic shock was also characterized by an increased expression of CRH mRNA in rats but not in patients, who notably had a greater duration of septic shock.The present study suggests that in humans and in rats, septic shock is associated with decreased ACTH synthesis that is not compensated by its two natural secretagogues, AVP and CRH. One underlying mechanism might be increased expression of iNOS in hypothalamic parvocellular neurons. About sixty percent of patients with septic shock develop adrenal insufficiency Most human studies of HPA axis during sepsis have relied on circulating hormonal levels and have never included neuropathological studies. Previous experimental studies investigated hypothalamo-pituitary structures and showed that endotoxin or cytokines stimulate CRH neurons In order to test the hypothesis that ACTH secretion is decreased secondarily to alteration of either CRH or AVP synthesis, we underwent a neuropathological study of the antehypophyseal system in patients who had died from septic shock and rats with experimental faecal peritonitis, which is considered one of the best model of human sepsis Experiments were carried out at University College London, with Home Office approval in accordance with the Animal Scientific Procedures Act . Wistar rats were purchased from Charles River laboratory . This model has been well described elsewhere in situ hybridization.Brain sampling was performed in eight sham, ten septic and six septic shock rats. Whole brains and pituitary glands were removed immediately after animal sacrifice by neck dislocation, and fixed by immersion in 10% formalin. Brain samples were processed as previously described Nine non-survivors from septic shock and ten non-septic patients who died from cardiogenic shock or an extracranial cause of sudden death were included. The characteristics of these patients have been reported elsewhere (10). The median age of patients who died from septic shock was 71 [58–76] years. Their SAPS-II score was 57 [35–90], the cumulative time of hypotension was 25 [12–36] h and duration of shock 6 [1–8] days. In patients who died from non-septic causes, SAPS-II score was higher (i.e. 70 [47–83]) but cumulative time of hypotension and duration of shock were shorter, namely 3 [2–5] h and 3 [1–4] days, respectively.in situ hybridization.Macroscopic examination of the brains was performed after 4 to 6 weeks of fixation in 10% formalin (vol./vol.). We took samples of the PVN at the level of the posterior hypothalamus. Pituitary glands were also sampled. Each sample was embedded in paraffin and cut in 4 µm serial sections with a Leica® microtome. For microscopic examination, one section on three was stained with haematoxylin and eosin; the others were processed for immunohistochemistry or Anatomopathological study of neuroendocrine and neurovegetative trouble during septic shock (n°PFS10-008) was approved by the French Biomedecine Agency.According to national regulations, brain samples were collected after obtaining informed written consent from patient's closest relatives. A protocol about sampling for research purpose by title Moreover, patients of our work from Raymond Poincaré Hospital (Garches) were enrolled in the CORTICUS study Immunohistochemistry was performed on deparaffinised formalin-fixed sections using an indirect ABC-peroxidase method revealed with 3-3′diaminobenzidine (DAB) or amino-ethyl-carbazol (AEC) as chromogenes Images were acquired using a Leitz Diaplan® microscope  equipped with a Nikon Coolpix 950® digital camera .In situ hybridization was performed using a 200 bp rat vasopressin cDNA probe  and 1 kb rat CRH cDNA probe  as described previously In situ hybridization was performed using the Ventana Discovery® . Slides were finally mounted with Vectashield mounting medium . The labelled cells were observed with an Axioplan 2 microscope  equipped with a video camera . Images were acquired with AxioVision acquisition and analysis software. Sections hybridized with sense probes showed no staining.in situ hybridization were assessed by two independent observers , blind to rats and patients' groups. Counting of parvocellular neurones was performed morphometrically using Image J 1.36b® software . For rat analyses, cell counts were performed on one 4 µm-treated slice containing the PVN , AVP (in-situ hybridization) and iNOS (immunohistochemistry) labelling of each neuron was semi-quantitatively scored as 0 (none), 1 (low), 2 (moderate) or 3 (high) in the PVN ((1*number (n) of low intensity cells + 2*n of moderate intensity cells +3*n of high intensity cells)/The intensity of CRH , depending on the global immunostaining intensity at the level of the section considered.th–75th percentile), respectively. Non parametric analyses were performed, using Kruskall-Wallis and Mann-Whitney test for comparison of quantitative variables between three and two groups, respectively. To account for multiplicity while testing the same physiological hypothesis in humans and rats, p-vlaues were adjusted using Holm's correction. A p-value less than 0.05 was deemed significant.Variables were expressed as median with interquartile ranges (25p = 0.65)  .p = 0.04), and was higher in septic shock than in sham (p = 0.02) and tended to be higher than septic animals (p = 0.08) (p = 0.21).By contrast, in rats the parvocellular expression of CRH mRNA differed significantly between groups ( = 0.08) . It did p = 1.00) (p = 1.00)  . Similar = 1.00) .p = 0.0008) (p = 0.007) (p = 0.01) animals and sham (p = 0.01) animals. It was not significantly different between septic and sham rats (p = 0.43).In patients, the index of parvocellular neurons immunoreactive for iNOS was significantly higher in septic shock than in controls ( 0.0008) . Similar= 0.007) . It was In parvocellular nuclei expression of CRH mRNA, AVP mRNA and iNOS in patients did not correlate with duration of septic shock. Evaluation of steroid effect was not possible because of the small number of patients treated.p = 0.04) (p = 0.002) (p = 0.0004) or sepsis (p = 0.01) (p = 0.19)  . Similar= 0.002) . It was  = 0.01) . It did  = 0.19) .p = 0.71, respectively). It was the same for V1b receptor (p = 0.44 for both), as shown in In patients and in rats, expression of CRHR1 did not differ between groups (p = 0.66 and In ante-hypophysis expression of CRH R1, V1b and ACTH in patients did not correlate with duration of septic shock. The small number of patients treated with steroids preclude to assess their effect.This study assessed sepsis-induced ACTH expression and expression of its main regulators, both in patients and in rats. Decreased ACTH and unchanged expression of AVP mRNA, CRHR1, and V1b receptors was a consistent finding in human and in rats. Conversely, CRH mRNA expression was increased in rats with septic shock but not in patients with septic shock. These results suggest that septic shock is associated with central impairment of the HPA, characterized by decreased ACTH synthesis while its two main regulators CRH and AVP remain roughly unchanged.Exposure to an acute stressful stimulus is followed by rapid and self-limited increase in plasmatic ACTH and corticosterone Our results show that HPA axis appeared non-reactive. It seems unlikely that sepsis induced by the surgical model was not enough severe to activate HPA axis. Faecal peritonitis constitutes a sustained stimulus sufficient to induce tissue inflammation and impairment of visceral sensory pathways Our study provides just a snapshot of the HPA axis's state at a given time of course of sepsis. Septic and septic shock animals were sacrificed or spontaneously died within 36 hours and patients died after several days of septic shock complicated by multiple organ failure. Thus, our description of HPA axis rather corresponds to the post-acute phase of sepsis in the experimental model, while that in septic patients to the ultimate phase. This difference may explain the finding that expression of CRH was greater in rats than in humans. Interestingly, a previous experimental study showed that parvocellular CRH expression peaked 24 hours after onset of CLP Another hypothesis to explain our results is based on the regulatory role of endogenous and exogenous glucocorticosteroids on HPA during stress. Indeed, cortisol exerts a negative feedback on PVN, reducing CRH and AVP mediated ACTH secretion Carlson and colleagues showed that plasma cortisol level decreased with time in CLP The role of iNOS on neuroendocrine modulation of axis is well recognized and his expression may contribute to stunting of HPA in sepsis. Most experimental studies reported that nitric oxide stimulates the expression of CRH In conclusion, the present study suggests that in humans and in rats, septic shock is associated with decreased ACTH synthesis that is not compensated by its two natural secretagogues AVP and CRH. One underlying mechanism might be increased expression of iNOS.Further studies are necessary to assess other mechanisms and to elucidate the role of HPA impairment on relative adrenal failure in sepsis.