We previously showed that P3 and alpha modulations occurred only when intentionally using rhythms to form predictions but not incidentally (in contrast to CNV effects that were also observed with passive exposure to nonpredictive rhythms, [34]).

Taken together, our current and previous results imply that the alpha-band and P3 effects reflect general mecha- nisms of intentional (rather than incidental) execution of temporal predictions, regardless of source.


Models of dynamic resource allocation argue for a dichotomy between temporally selective anticipation in rhythmic streams, mediated by oscillatory entrainment ("rhythmic" mode), and anticipation in nonrhythmic streams, which requires sustained vigilance ("vigilance" mode) [11].

These models leave out the well-documented memory-based resource allocation in time, which was shown to occur without rhythmic context [1,26].

Moreover, it was not clear whether rhythm- and memory-based temporal predictions are distinct at all, as interval-timing neural signatures were explained by oscillatory phase resetting [11], and, in contrast, seeming oscillations could be explained by repeated interval-timing [32] or even repeated evoked responses [72].

Similarly, imaging studies revealed that both rhythm- and memory-based tem- poral predictions activate similar regions (premotor and inferior parietal cortices, for a review see [73]).


Directly comparing conditions in which timing is predictable, based on memory, rhythm, and not at all, allows us to extend the dichotomy into a trichotomy: availability of temporal information, regardless of its source, allows the brain to shift from the "continuous" mode into a "temporally predictive" mode.

This is characterized by pretarget CNA (such as the CNV), anticipatory alpha reduction, and posttarget facilitation of processing of temporally expected events, reflected by earlier P3 latency and shortened RTs.

When anticipation is based on rhythms, the brain can further switch into a "rhythmic" mode, characterized by similar mani- festations but also by immediate cessation of facilitation and possibly inhibition once the expected time interval has elapsed, manifested by CNV immediate resolution and larger behavioral costs for unexpected events.

Methodologically, studies that aim to infer rhythm- specific mechanisms should use a less rhythmic predictive control similar to our Repeated- Interval condition, which would allow attributing the effects of rhythms to rhythmicity and not to general temporal prediction.


A central remaining question is whether the unique effects of rhythms are mediated by oscil- latory entrainment.

On one hand, this possibility is weakened by our finding that PC of low- frequency oscillations, which were usually conceived as a signature of rhythm-specific entrain- ment, was observed to the same extent in less-rhythmic streams.

On the other hand, oscilla- tions are characterized by resonance, that is, extension of several oscillatory cycles even when external phase-resetting input is terminated.

Findings fluctuations of several cycles in behav- ioral and physiological markers after stimulus termination was often interpreted as a direct evidence for the alignment of a self-sustaining oscillatory process to the external rhythm [74- 76].

In our data, the conspicuous and immediate return of the CNV to baseline after an omis- sion, in the absence of any external event to phase-reset it, represents the expected pattern of a resonance mechanism, consistent with entrainment.

It could also explain the increased cost of presenting targets outside the expected time window.


More than the ability to recruit resources, what seems to be unique to the rhythmic mode (and not to the anticipatory mode in general) is the tendency to "turn off" anticipation periodi- cally, consistent with the idea of energy efficiency [11].

While this mode is indeed efficient when the input maintains its predictability, the current findings also emphasize the inflexibility and resultant cost of this mode when events are temporally surprising.

This may be an unavoidable consequence of the oscillatory mode.


This study and all of its procedures, including subject recruitment methods, instructions, experimental task, apparatus, debriefing, and reimbursement, were approved by the local Eth- ics Committee of the Hebrew University of Jerusalem, Israel.

All participants provided written informed consent according to the local Ethics Committee and the Declaration of Helsinki.


Twenty-one students (11 females, mean age = 25, 19 right handed) were tested in return for course credit or payment.

Participants reported no history of neurological or psychiatric dis- ease, normal or corrected-to-normal vision, and no professional musical training or playing an instrument.


Stimuli were colored disks (diameter 1.2˚) presented for 100 ms.

Each trial began with a prepa- ratory sequence of stimuli, followed by a white WS and a green target to which participants had to respond with a speeded button press.

A fixation point (black "+", 0.6˚X 0.6˚) preceded each trial for 500 ms.


There were three conditions presented in different blocks (Fig 1A).

In "Rhythmic" blocks, the sequence consisted of four, five, or six (uniform probability) black sti- muli presented rhythmically with an SOA of 700 ms (1.43 Hz, short cue) or 1,300 ms (0.77 Hz, long cue).

The WS appeared in-phase with the rhythm.

The WS-Target SOA was identical to the rhythm SOA in valid trials or to the other SOA in invalid trials.


In the "Repeated-Interval" condition, the sequence consisted of four, five, or six (uniform probability) black-then-red pairs of stimuli.

The within-pair SOA was 700 and 1,300 ms in short and long cue trials, respectively, but the SOA between pairs was jittered (short trials: 1,500-1,900 ms, 50 ms steps; long trials: 1,900-2,700 ms, 100 ms steps, uniform distribution).

We assumed that this


Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 20 / 30 aperiodic sequence structure would reduce the ability of endogenous delta oscillators to main- tain a stable phase relation to the stream, as the first stimulus of each pair falls approximately on the opposite phase of the oscillation that is entrained by the previous pair.


The last pair of the sequence consisted of a white WS followed by a green target.

The SOA within this pair was the same as in the preceding pairs in valid trials and had the other SOA in invalid trials.


In both Rhythmic and Repeated-Interval conditions, the sequences were predictive of target tim- ing, as 75% of the trials within a block were valid and 12.5% invalid.


In the remaining 12.5% of the trials, in each condition no target appeared following the WS, in order to diminish expecta- tions based on conditional probability that the stimulus will occur given that it has not occurred yet [25].

In these "catch" trials, a blank screen was displayed after the WS for 1,500 ms.


Finally, in the "Random" condition, the sequence consisted of four, five, or six (uniform probability) stimuli whose SOAs were jittered around the average of 700 or 1,300 ms (−200 to +200 in 50 ms steps, uniform distribution).

Target SOAs were also drawn from these short and long distributions, independently from the average sequence SOA [34].


While the sequence was not predictive, for analysis purposes, "valid" trials were defined as those in which cue and target SOAs were drawn from the same distributions (e.g., short cue trials followed by short SOA targets, 43.75% of the trials) and "invalid" trials as those in which cue and target SOAs were drawn from different distributions (e.g., short cue trials followed by long SOA targets, 43.75% of the trials).

The other 12.5% of the trials were catch trials.


The experiment was conducted in a sound attenuated chamber (Eckel C-26, United King- dom).

Stimuli were presented at the center of a 17-in CRT screen (ViewSonic G75f, with 100 Hz refresh rate) on gray background (viewing distance = 90 cm).

Stimulus presentation and response acquisition were handled using Psychophysics toolbox [77,78] for MATLAB (version 7.5.0, Mathworks, Natick, Massachusetts).


Trials were presented in blocks of 32 trials.

The experiment started with two Random blocks.

Next, Repeated-Interval and Rhythmic blocks alternated (four each).

Which condition started was counter-balanced across participants.

Par- ticipants were encouraged to use the predictive information if available.

Eight practice trials were performed on first encounter with each condition and at least two trials for ensuing blocks.

Short breaks were given between blocks.


At the end of the experiment, participants were debriefed, verifying that they had used the predictive cues to create predictions.


Prior to the entire experiment, all participants conducted three blocks of a condition from our previous study ("Non-Informative" condition) [34] for replication purposes.

This condition and its results will not be presented in the main text (see S1 Text, and [34]).


For each participant, trials were discarded if the RT was larger than three standard deviations from the mean RT, separately for each condition and validity of the cue, or if the RT was shorter than 50 ms.

RTs were subjected to a repeated-measures ANOVA with factors condi- tion (Random/Rhythmic/Repeated-Interval), cue validity (valid/invalid), and target SOA (short/long).

Where the sphericity assumption was violated, we report Greenhouse-Geisser corrected p-value and uncorrected degrees of freedom.

Due to the small number of trials at the exact in-phase SOA in the Random condition, trials with SOA of up to 100 ms around the short and long target SOAs were used as representing these SOAs.

The range of this interval (50-150 ms) did not affect the pattern of results.

To examine the benefits and costs of having temporal predictions, we separately analyzed valid and invalid trials, conducting planned one- Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 21 / 30 tailed contrasts to compare each predictive condition to the random condition, and two-tailed contrasts to compare the predictive conditions.


EEG was recorded continuously from 64 preamplified Ag/AgCl electrodes, using an Active 2 system (BioSemi, The Netherlands) mounted on an elastic cap according to the extended 10- 20 system.

Additional electrodes were placed on the outer canthi of the right and left eyes and above and below the center of the right eye to track electro-oculographic activity, on the left and right mastoids, and near tip of the nose.

The EEG signal was sampled at a rate of 1,024 Hz (24 bits/channel) with an online anti-aliasing 204 Hz low-pass filter.

EEG preprocessing was conducted using BrainVision Analyzer 2.0 (Brain Products, Germany) using the following pipeline: referencing to the nose electrode; high-pass filtering using a zero-shift Butterworth filter with a cutoff of 0.1 Hz (24 dB/octave); correction of ocular artifacts using independent component analysis (ICA) [79] based on typical scalp topography and time course; elimination of epochs which contained other artifacts, defined as absolute activity larger than 100 μV, or a change of more than 100 μV in a 200-ms interval.

The data of two participants were rejected from EEG analyses due to excessive artifacts.


ERPs were analyzed separately for the preparatory period, i.e., during the presentation of the stimulus sequence, and the posttarget period.

For all ERP analyses, the data was re-referenced to the average of the left and right mastoids.

In the preparatory period, the analysis was con- ducted in two predefined electrode clusters: a central cluster (FC1, FCz, FC2, C1, Cz, C2, CP1, CPz, CP2), to test for modulations of the CNV potential, and an occipital cluster (PO3/PO4, PO7/PO8, O1/O2), to test oscillatory activity in visual circuits.

To visualize ongoing activity in these two clusters, segments extending from 5,600 or 3,200 ms before the target (for long and short SOA trials, respectively) to 1,000 ms after the target were averaged within condition across trials and subjects.


Two a priori hypotheses were tested regarding the CNV in the central electrode cluster.

First, we examined whether the modulation of the CNV buildup by the expected SOA (i.e., faster buildup as the expected SOA is shorter, [9,26]) differs between conditions.

For this, segments extending from 200 ms before to 1,300 ms after the WS were averaged within each condition for valid trials, separately for short and long cue SOA (in the Random condition according to the average of the sequence), with the period of 200 ms before the WS used as baseline.

In the Random condition, we only used trials in which the target did not inciden- tally appear in the first 700 or 1,300 ms after the WS, for short and long SOA trials, respec- tively.

Within each condition, the average CNV amplitude in a window of 400 to 700 ms after the WS was compared between expecting the target in short and long SOAs.

This time window was chosen to provide maximal differentiation, as it was at the end of the short inter- val but in the middle of the long interval.

Planned one-tailed contrasts were used to examine the hypothesis that the CNV will be more negative when expecting the target at a short SOA than long SOA.


Second, we compared the resolution of the CNV (i.e., return to baseline, [9,37,40]) between the different conditions in trials where an expected short SOA target was omitted (i.e., short- cue, invalid trials).

In this situation, the timed interval ends, but no target appears to end the anticipation.

For this, segments extending from 200 ms before to 1,400 ms after the WS were averaged separately for the Rhythmic and Repeated-Interval conditions, with the period of 200 ms before the WS used as baseline.

To quantify the CNV resolution, we fitted the amplitude of Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 22 / 30 the CNV waveform (averaged across participants) in each condition with a five-parameter continuous model, which allowed the CNV to buildup, remain at the peak amplitude for a flex- ible interval, and only then resolve (Fig 3C):


The free parameters of the model were the buildup initiation time (T 0 ), buildup slope (S 1 ), buildup termination time (T 1 ), resolution initiation time (T 2 ), and resolution slope (S 2 ).

The possibility for a variable delay between T 1 and T 2 allowed accurate estimation that would not be distorted by predefined choice of intervals and also reflected a cognitively plausible strategy of maintaining high preparation after target omission, in case the target appears at the long SOA.

Each parameter was compared between the Rhythmic and Repeated-Interval conditions using paired t tests using the jackknifing procedure (see S1 Text).

To verify that the five- parameter model does not overfit the data, this "resolution after delay" model was compared to a more restrictive four-parameter model, "immediate resolution," in which the transition between buildup termination and resolution initiation was immediate (T 1 = T 2 , see Fig 3D).

Model fitting was conducted using the "fminsearch" function in MATLAB (Nelder-Mead sim- plex optimization) with several initial values.

Model selection was based on calculation of the Bayesian Information Criterion (BIC, [80]).

For each condition, the model with the lowest BIC value was chosen (ΔBIC > 6 is considered strong evidence).


ERP analysis of posttarget activity focused on the P3 potential, which was analyzed at a cluster of midline-parietal electrodes (Pz, P1, P2).

We examined whether the modulation of the P3 latency by the validity of temporal prediction (shorter latency when the target appears at the expected SOA, [8,26]) differs between conditions.

For this, segments extending from 200 ms before to 700 ms after the target were averaged in each predictive condition, sepa- rately for trials with short or long target SOA and for valid or invalid cues.

As the pretarget CNV effect was expected to result in differences in the pretarget baseline in short versus long target SOAs, we used a period of 200 ms before the WS as baseline and analyzed each target SOA separately.

The time window for P3 analysis was 200-550 ms after target onset, based on unbiased visual inspection of average activity across all subjects and conditions.

The P3 latency was estimated by averaging the latencies in which the amplitude reached 30%, 50%, and 70% of the peak amplitude, allowing a stable estimation of the half-maximum point [81].

Reliable estimation of these latencies was achieved using a jackknifing procedure.

Within each condition and target SOA, the amplitudes and latencies of the P3 were each subjected to a repeated-measures ANOVA with factors condition (Rhythmic/Repeated Interval) and target validity (valid/invalid), and planned one-tailed contrasts were used to examine whether the P3 latency is shorter when the target SOA was expected compared to unexpected.


Following previous findings [9,34,35], we analyzed amplitude modulations of alpha-band activity (8-13 Hz) in occipital electrodes (PO3/PO4, PO7/PO8, O1/O2) by temporal


Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 23 / 30 predictions.

We compared the amplitude of alpha activity at the time of the short SOA target when expecting the target at the short versus long SOA.

In both short and long SOA trials, we only included trials in which the target actually appeared at the long SOA, avoiding contami- nation by target-evoked activity.

Thus, the only difference between the trials was the predicted target time.

We asked whether an amplitude reduction at the time of a temporally expected tar- get would differ between the different sequences.


The data were segmented from 1,400 ms before the WS to 1,800 ms after the target.

Instantaneous amplitudes were extracted using a complex Morlet wavelet transform (1-30 Hz, 1 Hz steps, ratio between the central frequency and the standard deviation of the Gaussian-shaped wavelet in the frequency domain = 8).

The first and last 1,000 ms of each segment absorbed edge artifacts and were discarded from analy- sis.

The resulting time-frequency representations were averaged across electrodes within each condition.


Baseline correction was performed using a range of 0-100 ms after the WS, as the occurrence of the WS is the only time point that is identical both in function and in perceptual input between the short and long SOA conditions (pre-WS intervals were contaminated with residual activity from previous stimuli, see [34]).

Average alpha-band amplitude (across 8-13 Hz and 700-900 ms after the WS) was subjected to a repeated-measures condition (Rhythmic/ Repeated-Interval) X expected SOA (short/long) ANOVA.

Within each condition, planned one-tailed contrasts examined whether the alpha-band amplitude is reduced when the target was expected at the short SOA compared to the long SOA.


We analyzed the effects of temporal predictions on the phase of delta-band activity in occipital electrodes in which these effects were expected to be maximal (PO3/PO4, PO7/PO8, O1/O2).

We examined whether increased PC and alignment to the optimal phase angle at target time [3,6,7,21,22] occur in the rhythmic and Repeated-Interval conditions.

Instantaneous phase of delta-band activity was extracted by band-pass filtering the unsegmented data in a frequency band of 0.5-3 Hz (zero-shift Butterworth filter, 24 dB/octave) and applying the Hilbert trans- form.

This was performed separately for each occipital electrode and then averaged (consider- ing the circularity of phase) across electrodes.

The delta-phase signal was segmented into epochs surrounding valid targets (−700 to 500 ms for short SOA targets and −1,300 to 500 ms for long SOA targets), separately for the Rhythmic, Repeated-Interval, and Random conditions and, for comparison, also surrounding the WS in the Rhythmic condition.

Analyses of circular variables were performed using the CircStat toolbox for MATLAB [82].


To examine whether certain phase angles were optimal for performance we tested the circu- lar-linear association between delta phase at target time and RTs in the random condition (as in the predictive conditions delta-phase was expected to have small variance due to PC).

We used a linear mixed-effect regression model to estimate this association at the group level, allowing each subject to have a different optimal value (see S1 Text).

Delta phase was linearized by taking its sine and cosine and using them as fixed effects along with target SOA.

The inter- cepts of participants, as well as by-participant random slopes for sine and cosine of the phase, were inserted as random effects.


Second, we examined delta PC at stimulus time (target/WS) by calculating the ITPC [83].

Across participants, these ITPC values were compared between conditions using an omnibus repeated-measures ANOVA with factors condition (Rhythmic/Repeated-Interval/Random/ WS-Control) and SOA (short/long), followed by subsidiary ANOVAs for comparing specific condition pairs.

Specifically, we examined the occurrence of ITPC in the predictive conditions by comparing them to the controls, and also compared the Rhythmic and the Repeated- Mechanisms of rhythm- and memory-based temporal prediction PLOS Biology | DOI:10.1371/journal.pbio.2001665 February 10, 2017 24 / 30 Interval conditions.

Due to the nonnormality of the ITPC data (bounded between zero and one), we used permutation-based ANOVAs (see S1 Text).


Finally, we examined whether delta phase at target time was aligned to the optimal phase angle, which was identified in each participant and each SOA by averaging the phases of the 33% fastest trials in the Random condition.

For each predictive condition, we then calculated for each participant the circular distance between the circular-averaged phase angle at target time and his\her optimal phase.

If the phase at target time was shifted to the optimal angle, the expectancy of these differences is zero, so they should be concentrated around zero across sub- jects.

We tested this using a V test, which contrasts a null hypothesis that phases are uniformly distributed to an alternative hypothesis that phases are unimodally distributed with an a priori specified mean angle, which, in the current case, was set to be zero (see [7]).

With the same approach, we also tested whether the phase angle at target time was identical between short and long SOA targets.


Neural oscillations align to external stimulus rhythms, such as the recurring onsets in rhythmic sequences, via neural entrainment—that is, adjustment to the oscillation’s phase and period.

As such, measures of neural phase coherence or “phase concentration” can inform us about the precision of entrainment.

Entrainment of ongoing neural oscillations is considered a potent mechanism that the brain utilizes to generate temporal predictions and to aid active perception, at least in sensory cortices.

However, as attempts to falsify or question this hypothesis are scarce, the idea that temporal predictions are instantiated in neural oscillatory entrainment should not be set in stone just yet.


For this reason, it is very important that a recent study by Breska and Deouell, published in PLOS Biology [1], set out to determine whether phase concentration at the time of a critical target stimulus would also be observed in instances when stimulus sequences are non-rhythmic, but targets are nonetheless temporally predictable [2].

Indeed, temporal predictions can certainly be formed on the basis of non-rhythmic information, such as the passage of time itself [3].

Breska and Deouell propose a “memory-based” prediction mechanism that would not be instrumented by entrainment but instead might utilize alternate, interval-based timing mechanisms [4,5].

While the study raises very important questions, logical and methodological considerations have motivated us to lay out a short guide for future experiments that aim to demonstrate oscillatory entrainment or the lack thereof.

Throughout, we support our arguments with simulations and supplemental audio, which can be found at https://osf.io/phs6e/.


Breska and Deouell aimed to assess behavioral and electrophysiological measures in contexts that vary in their rhythmicity, with the goal of exploring the bounding conditions of neural entrainment as well as the neural mechanisms that might support temporal predictions when entrainment is not possible.

The trouble with examining neural mechanisms in contexts with varying rhythmicity is that we are still lacking a good working definition of rhythm.

What does “rhythm” mean to a human or non-human brain, and to a perceiver more generally, and how variable does a sequence of events need to be so that our brains will cease to register it as rhythmic? Many researchers seem to use “rhythm” to refer to isochrony, that is, strict regularity, and contrast this with situations in which sequences are composed using variable stimulus onset asynchronies (SOAs; e.g., “repeated-interval” and “random” conditions in Breska and Deouell’s study).


However, there are many types of sequences that might be perceived as rhythmic despite being decidedly aperiodic and having relatively high variance in terms of the intervals making up the rhythm.

For example, metrical musical rhythms comprising intervals of different sizes (e.g., quarter notes, half notes, and dotted half notes) can give rise to a sense of regularity at a rate that is not actually represented by any of the intervals in the sequence (that is, at the whole note level).

In fact, many previous studies on neural entrainment did not even utilize the strictly isochronous sequences presented here as a hallmark of rhythm but instead allowed for considerable jitter between single stimuli (e.g., [6–9]).


Empirically, rhythmicity can be assessed best by asking individuals whether they perceive a sequence as rhythmic.

To this end, we transferred Breska and Deouell’s sequences to the auditory domain (arguably the more natural domain for assessing rhythm and entrainment) in an exercise that was quite informative: it turns out that their “repeated-interval” condition, conceived to reduce rhythmicity, feels very rhythmic indeed (see supplemental audio at https://osf.io/phs6e/).

Theoretically, rhythmicity can be assessed by attempting to align (that is, to entrain) a sinusoidal oscillation to a stimulus sequence (Fig 1), as Breska and Deouell have also done: if the oscillation aligns to the sequence such that its relative phase is sufficiently non-random relative to stimulus onsets, the sequence should be considered rhythmic from the perspective of the underlying oscillator.

Our attempts to do so suggest that the degree of jitter employed by Breska and Deouell, even in their “random” condition, would still allow for successful neural entrainment (Fig 1C).

We note that Breska and Deouell also report results of such a simulation and also show that, in particular for the long SOA condition, their “random” stimulation would have led to successful oscillatory entrainment.


An orthogonal issue contributing to Breska and Deouell’s largely similar phase-concentration results across “rhythmic” and “repeated-interval” conditions is the presence of large-amplitude contingent negative variation (CNV) and P3 components flanking the targets, which likely distorted the phase measures and resulted in artificially high phase concentrations (Fig 1D).

This suggestion (also put forward by Breska and Deouell) was confirmed by our simulations and accurately predicted lower phase concentration at the time of the warning signal compared to the time of the target, which would not be predicted by neural entrainment alone (Fig 5B of Breska and Deouell [1]).

The bottom line here has already been discussed at length in the field: phase concentration measures do not just index oscillations—they index the entire underlying signal—and therefore should be interpreted with utmost caution.


Nonetheless, to conclude that “phase concentration and alignment of slow EEG [electroencephalogram] oscillations are not a signature of rhythmic entrainment” ([1]; as in the title of their paper) is worrisome: Breska and Deouell seem only to consider the possibility that entrainment measures might not be good indicators of entrainment.

Potentially, however, their manipulation simply did not vary the intended independent measure (that is, rhythmicity) to a conclusive degree.


Our reservations notwithstanding, Breska and Deouell’s paper is an important reminder of how little we actually know about the way(s) in which rhythmic—or otherwise predictable—patterns in our environment are utilized by behaving organisms.

Accordingly, a hallmark contribution of their paper is to demonstrate the behavioral costs (in terms of response times) that can arise for a behaving organism from entrainment to a strictly periodic stimulus.


Let us conclude by briefly outlining requirements of an experiment that we think would settle the important questions raised by Breska and Deouell more conclusively.

First, the experiment would need to manipulate a perceiver’s ability to predict the precise timing of upcoming, behaviorally relevant events by truly varying rhythmicity.

That is, the presence of rhythmicity should be empirically verified by asking for information about the (non-)rhythmic percept.

One particularly promising approach is to manipulate meter: rhythms with different degrees of perceived rhythmicity can be constructed by reordering the exact same interval set, creating stimuli with identical statistical properties but different degrees of perceived regularity [13].

Such stimuli allow for a rigorous test of the effects of rhythmicity on neural entrainment.


Second, we suggest that unpredictable, near-threshold targets avoid as much as possible the contamination by large CNV and P3 components that can distort phase-concentration measures.

Inevitably, however, testing a memory-based prediction mechanism is contingent on a subject’s ability to predict when the target will occur—a process that can hardly escape large transient evoked responses like the CNV and so may be better studied using a psychophysically sound behavioral paradigm.

It remains to be conceded, however, that it is far from trivial to arbitrate between a (theoretically and empirically thus far substantiated) entrainment account on the one hand, and a potentially more parsimonious view that would subsume both periodic and aperiodic predictions but might challenge entrainment on the other.


So, what do we talk about when we talk about rhythm? As we have laid out here, regularity manipulations themselves need to be of sufficient magnitude and quality to modulate both percepts of rhythmicity and entrainment of a (neural or other) oscillator.

Even then, however, our beloved tools for dissecting rhythmic and non-rhythmic processes in the neural domain can be turned into rusty blades by notorious interpretational problems.

This should encourage us to humbly spell out our predictions in those domains where rhythm truly resides: in perception and behavior [14].


The idea of entrainment of neural oscillations to temporal structure has become a central theory for attentional selection in time [1,2].

In our study [3], we separated unique electroencephalogram (EEG) signatures of oscillatory entrainment to rhythmic streams from those (specifically, inter-trial phase coherence [ITPC]) that overlap with other mechanisms of prediction.

Obleser, Henry, and Lakatos’s (OHL’s) comment [4] highlights important issues regarding the study of entrainment mechanisms.

We share OHL’s main message: rhythms may be complex and not necessarily isochronous.

In fact, finding a stream devoid of any regularity is challenging.

This is probably why entrainment mechanisms are functionally important! Yet we should not see entrainment mechanisms as omnipresent, nor should we render them irrefutable.

Below, we reflect on some of OHL’s concerns regarding our study.


Oscillatory entrainment predicts increased ITPC for rhythms [2,5], as both our and OHL’s models show.

However, taking ITPC as evidence for entrainment, without ruling out other explanations, would be making the logical fallacy of "affirming the consequence" (a!b does not imply that b!a).

Our study [3] thus asked which electrophysiological phenomena are uniquely associated with entrainment to rhythms instead of with general temporal prediction mechanisms that also operate in rhythmic contexts [6].

We conjectured that if ITPC reflects entrainment it should be sensitive to the level of regularity.

However, we found that reducing the level of temporal regularity of streams did not reduce ITPC when predictability was preserved but only did so if predictability was also reduced.

OHL’s argument that this is explained by predictions-related climbing neuronal activity (CNA; e.g., the contingent negative variation) only recapitulates one of our main arguments [3].

Given this fact, can a repeated-CNA explanation be ruled out from previous ITPC findings, especially in designs with no warning signal?

Using near-threshold stimuli [7] reduces onset-responses–driven ITPC but not anticipatory activity.


OHL point out (like we did [3]) that our repeated-interval condition (RIC) was not arrhythmic, explaining observed ITPC levels.

Critically though, the RIC was not designed to be arrhythmic but to be less periodic than the isochronous condition (IsoC), which should diminish entrainment signatures.

For perceptual judgments evidence (cf.

OHL’s demo) that this was achieved, see Fig 1.


However, in accordance with OHL’s appropriate assertion that rhythmicity is ill defined, we worry that resorting to subjective judgment falls into the same ambiguity of periodicity/predictability that motivated our study.

Preferably, the degree to which a sequence is conducive to entrainment of a given oscillation, well defined by frequency and phase, could be objectively quantified.


In fact, OHL’s model, like ours, predicts a difference in phase (their figure 1b) and a lower ITPC (their figure 1c showing the effect of stimulus onset asynchrony [SOA] variance) in the RIC compared to IsoC. However, being a single-oscillator model with adjustable frequency, OHL’s model cannot attribute the ITPC to specific frequencies.

Our multi-oscillator model shows that the expected ITPC is in lower frequencies in the RIC than the IsoC, consistent with the RIC being by design destructive to entrainment at the IsoC frequency.


The predictions of both entrainment models are thus inconsistent with the EEG findings of similar ITPC, in the same frequency, and to the same (behaviorally optimal) phase angle in the 2 conditions.


For us, the strongest indication that the 2 sequences, while not differing in ITPC, were not as “rhythmic” (able to entrain) for the brain is the clear behavioral and electrophysiological differences they induced [3].

Crucially, these differences agreed with entrainment theories: the more rhythmic IsoC led to resonance and momentum after stream termination.

We emphasize again this overlooked point: our findings support, rather than falsify, entrainment theories.

However, they call for proper predictability controls when using ITPC during stream presentation as evidence for entrainment.

We join OHL in hoping for clearer definition and careful controls when dissecting predictive mechanisms.


An awake craniotomy (AC) is the gold standard for the resection of supra tentorial brain tumours in eloquent areas.

Intraoperative monitoring "ondemand" of essential eloquent brain functions and the increasing need to preserve higher intel lectual functions during surgery requires a unique anaesthetic approach during AC.

Dexmedetomidine is considered the firstchoice pharmacological agent for sedation during AC.


Twentysix patients with a single brain tumour located in areas of eloquent brain function were enrolled in this prospective study.

The patients underwent AC un der conscious sedation.

Motorevoked potentials and brainstemevoked auditory po tentials were measured using neurophysiological tests during surgery to assess brain potentials.

Intraoperative brain relaxation was reached using a modified Bristow scale.

Neuromonitoring and psychological tests were maintained until meningeal closure.


All operations were carried out successfully, and no reoperations were needed.

No significant impact on circulatory and respiratory parameters was observed during conscious sedation based on dexmedetomidine.

Neither instrumental airway support nor conversion to general anaesthesia was necessary.

Brain relaxation was good in 84% of cases.

Intraoperative epileptic episodes were observed in 15% of the patients.

Neuro logical and psychological monitoring was satisfactory.

Unaltered muscle force was ob served postoperatively in 88% of the patients.


AC performed under conscious sedation, and dexmedetomidine infu sion without instrumental airway support, was safe and welltolerated by patients with comfortable physiological sleep for most of the procedure.

This approach to AC was associated with minimal risk of perioperative adverse events and may be particularly beneficial in patients with severe comorbidities.


Bogusława Ewa Lechowicz-Głogowska, Agnieszka Uryga, Artur Weiser, et al. ation, significantly increases the chance of a good neurological and cognitive outcome [8].


Awake craniotomy (AC) is the gold standard for surgical removal of tumours in eloquent areas of the brain.

Maximal tumour resection with simultaneous preservation of brain functions such as movement, speech, and cognitive abilities are the goals of these procedures [9].

The joint use of brain-evoked poten- tials, neuropsychological tests, and intraoperative real-time imaging (magnetic resonance imaging [MRI], ultrasound, or computed tomography [CT]) increases the probability of maximal and functional- ly safe tumour resection [10-12].

However, the best method of anaesthesia for AC is still in question.


Various anaesthetic management protocols are used for AC, such as the asleep-awake-asleep tech- nique (AAA), monitored anaesthetic care (MAC), and CS with propofol, remifentanil, and infiltration anaes thesia [9, 10, 13, 14].

Most clinicians use the AAA technique with instrumental upper airway sup- port during the initial and closing phase.

The patient is awake only during neurological monitoring [15].

The intubation tube or the laryngeal mask inser- tion can be very difficult due to the patient's fixed head position in a Mayfield clamp and the surgical dressings that cover the head.

During intubation or extubation, various adverse effects can occur, such as prolonged apnoea, hypoventilation, and reten- tion of CO 2 .

All of these events can cause extensive brain oedema, epileptic episodes, additional brain ischaemia near the edge of the craniotomy, or in- tense bleeding in the operating field and may even prevent further surgery.

Sedation with propofol and opioids may lead to respiratory depression with hypercapnia, and hypoxia, which results in brain oedema.

Furthermore, other adverse events can occur during conventional sedation: vomiting, myo- clonus, uncontrolled movement of the patient, and non-cooperativeness [16-18].


Due to difficulties with AAA (agitation, difficult airways, uncooperativeness, sudden disagreement about being awake during surgery), another anaes- thesia protocol that is better tolerated is needed.

Dexmedetomidine, which is used as a basic seda- tive for AC, may be a promising solution.

This alpha2 adrenoceptor (a2-AR) agonist works as a sedative, light anxiolytic, and analgesic through the activa- tion of a2 brain receptors.

It does not depress res- piration and has no impact on the neurophysio logy of the brain.

In addition, it does not negatively in- fluence monitored brain-evoked potentials and can protect the brain.

Through activation of receptors located in the coeruleus locus area of the brain, dexmedetomidine mimics the natural physiologi- cal pattern of sleep without REM phase II/III [11-13].


In this study, the results of a CS research proto- col using dexmedetomidine infusion and a scalp block with ropivacaine are presented.

The objec- tive of this research was to evaluate the risk of re- spiratory and circulatory complications, changes in neurophysiological monitoring, and other adverse events during AC.

The vital element of this protocol was the psychological evaluation before, during, and after surgery.


This study was a single-centre prospective study without a control group for comparison, approved by the Bioethics Committee of the University Hos- pital in Wroclaw (10.10.2013) and by the Bioethics Committee of the Wrocław Medical University (KB - 279/2016 [02.06.2016]) in accordance with the Declaration of Helsinki.

Written informed consent was obtained from all subjects.


Thirty-one patients admitted to the University Hospital in Wroclaw between 2014 and 2018 were screened to ensure they met the study inclusion cri- teria.

The main inclusion criteria were a single brain tumour localized near or in the eloquent cortical areas (qualified for elective AC), good neurological state, age > 16 years, ASA 1-4, good patient cooper- ation, and good intellectual function confirmed by neuropsychological tests.

Exclusion criteria included alcohol or drug abuse, psychiatric disorders, severe anxiety, allergies to study medications, recent clini- cal manifestation of epilepsy, electrolyte or acid- base imbalance, significant glucose imbalance, low heart rate (< 55 bpm), arrhythmias and lack of neu- ropsychological evaluation.

Figure 1 presents a flow chart showing exclusion criteria and the experimen- tal design of the study.

Ultimately, 26 patients were enrolled in the study.


The preoperative patient evaluation was per- formed at least one day before surgery.

A neuropsy- chological evaluation, including a general assess- ment of neurological and psychological status, as well as an evaluation of the higher mental functions (such as speech, memory, thinking, perception, and attention), was performed using the following bat- tery of tests (before surgery): STAI - Inventory of State and Anxiety (C.D. Spielberger, R.L. Gorsuch, R.E. Lushene), Eysenck Personality Questionnaire-Revised Short Scale (EPQR-s), Hamilton Depression Rating Scale HAM-D, Numerical Rating Scale, Benton Visual Retention Test, Hodges J. Addenbrooke Cognitive Examination - ACR-III, Schmidt M. Rey Auditory Ver- bal Learning Test (RAVL); (intraoperatively): Hodges J. Addenbrooke Cognitive Examination -ACR-III, and W. Łucki questionnaire for the study of cognitive pro- cesses in patients with brain injuries.

Furthermore, each patient was informed of the operation in terms of decision-making and motivation for participation.

The issue of stress management, anxiety, and claus- trophobia was also discussed.

Patient treatment sat- isfaction was assessed using the Postoperative Short Psychological Questionnaire (see Supplemental File) and the following battery of tests: Addenbrooke's Cognitive Examination - ACE-III, Rey's Auditory Verbal Learning Test (RAVL), Benton's Visual Retention Test, and the numerical rating scale.


Premedications were not used, to avoid any pos- sible influence on cognition.

We avoided a loading dose of dexmedetomidine due to the risk of cardio- vascular depression.

We prefer a stable low-dose infusion of dexmedetomidine (0.1-1.0 µg kg -1 h -1 ).

The level of sedation was adjusted to the surgery phase to reach a level of sedation of -1/-3 on the Richmond Agitation Sedation Scale (RAAS).


After intraarterial cannulation under local anaes- thesia, the following parameters were monitored: electrocardiography, heart rate (HR), intraarterial blood pressure, bispectral index monitoring (BIS) (Integra, Ohio, USA) with electrodes typically fixed on the contralateral side of the craniotomy, satura- tion (Pulsoximetry, LG, Korea), respiratory frequency, and EtCO 2 with a nasal cannula.

Saturation, EtCO 2 , systolic arterial pressure (SAP), diastolic arterial pres- sure (DAP), mean arterial pressure (MAP), and HR were collected and analysed at baseline before dex- medetomidine infusion (T0), and then 5, 10, 30, and 60 minutes (T5, T10, T30, T60, respectively) after the start of dexmedetomidine infusion and 60 minutes after discontinuation of dexmedetomidine (Tpo).

Arterial blood gases (PaCO 2 , PaO 2 ), electrolytes, and glucose levels were collected and analysed at T0, T30, T60, and Tpo.

Furthermore, a modified Bristow Scale was used to assess brain relaxation after dura opening (quality of operative condition).


The standard analgesia plan was provided peri- operatively to our patients.

Paracetamol (acetamin- ophen) 1.0 g IV or metamizole 2.5 g IV was given before skin incision, and then both drugs - postope- ratively, in dose 1.0 g IV every 6 hours and 8 hours, respectively.

It was continued for 24-48 hours ac- cording to the requirements of individual patients.

There were no patients with an allergy to those drugs in the presented cohort of patients.

A scalp block was performed using 0.75% ropivacaine with adrenaline 1 : 200,000.

If a patient complained of pain, assessed on the Visual Analogue Scale (VAS), then additionally IV/SC oxycodone was used at a dose of 5-10 mg every 4 hours.


The small doses of propofol (10-20 mg IV) were used for scalp block, head fixation with a Mayfield clamp to the meningeal opening, and skull closure.

Some patients were also given small doses of mid- azolam (0.5-1.0 mg IV) after the neuromonitoring and psychological evaluation phase.

Before skull pin application, local skin was anaesthetized with 2-3 mL of 1% lidocaine and adrenaline 1 : 200,000 at each pin site.

Next, each patient received ondanse- tron (8 mg IV), omeprazole (40 mg IV), dexametha- sone (8-12 mg IV), and mannitol (0.5 to 1.0 g kg -1 ) within 20 to 30 minutes of skin incision.

The infusion was completed before opening the dura.

The dura was then flushed with 10 mL of a 1% lidocaine solu- tion before the incision.

In the event of an epileptic seizure, the following medications were prepared: clonazepam, valproate acid, midazolam, propofol, and ice-cold saline (to apply to the operating field).


Oxygen was provided through a mask at 2-4 L min -1 , and PaO 2 was maintained at a level between 100 and 160 mmHg.

Instrumental airway manage- ment and conversion to general anaesthesia were pre- pared (if necessary).

The patient was placed according to the surgeon's preference and checked to ensure safe positioning.

The neck and legs were supported with pillows as needed.

The patient's face was uncov- ered with surgical covers secured above the face.

Con- stant and clear access to the head was maintained for patient observation and to allow instrumental airway support if needed.

Before mapping the cortex and neurological tests, the infusion of dexmedetomidine was stopped.

During the post-monitoring closure phase, the sedation was deepened, if necessary, with an infusion of small doses of propofol, midazolam, or fentanyl.


During surgery, the following brain potentials and neurophysiological tests were performed: mo- tor-evoked potentials (MEP) - after direct electrical stimulation of the cortical and subcortical brain re- gions, brainstem-evoked auditory potentials (BEAP) Bogusława Ewa Lechowicz-Głogowska, Agnieszka Uryga, Artur Weiser, et al. - for the evaluation of brain function, sensation - as- sessed by a subjective feeling of sensory phenome- na by the patient.


Motor functions were evaluated based on electromyography (EMG) traces.


During the resection of the brain tumour, the patients performed motor tasks to engage specific groups of muscles necessary for the movement of the in- dicated limb.


Furthermore, the patients contracted the indicated muscles while needle electrodes were inserted.


The higher psychological functions, such as speech, memory, attention, and perception, were assessed with neuropsychological tests.


The entire exposed gyri were stimulated directly in 5-mm steps using a bipolar electrode with a tip distance of 5 mm.


During stimulation, patients were asked to perform either a counting task or a naming test.


All black and white pictures presented were famil- iar to the patient according to preoperative assess- ment, and each name was preceded by 'this is' [16].


Speech arrest or the appearance of semantic errors was considered a positive result of mapping and led the surgeon to mark it [17].


Statistical analyses were performed with Statis- tica software (v.13.1, StatSoft Inc.).

Descriptive sta- tistics are presented as mean ± SD unless otherwise indicated.

Statistical significance was assumed at a = 0.05.

Data distributions were tested using a Kol- mogorov-Smirnov test.

The hypothesis of normality was rejected for most of the analysed parameters; therefore, nonparametric tests were used.

To evalu- ate the differences between physiological param- eters at baseline before dexmedetomidine infusion (T0) and then 5, 10, 30, and 60 minutes (T5, T10, T30, T60, respectively) after beginning the dexme- detomidine infusion and 60 minutes after discon- tinuation of dexmedetomidine (Tpo), a Friedman nonparametric ANOVA test was applied.

Post-hoc analyses were performed using Wilcoxon signed- rank tests with Holm-Bonferroni corrections for multiple comparisons.

The Spearman rank correla- tion coefficient was used to examine the linear rela- tionships between BIS and RASS.


The group consisted of 16 men and 10 women, with a mean age of 51 ± 17 years (min 19 years, max 77 years).

The clinical characteristics of the patients are presented in Table 1.

The mean surgery time was 6 h 10 min ± 1 h 25 min.

The consumption of dexmedetomidine during the procedure was on average 711 ± 240 µg (min 400 µg, max 1200 µg).

The application of other drugs was marginal in the total group: propofol 36 ± 51 mg (max 150 mg), fen- tanyl 0.01 ± 0.03 mg (max 0.10 mg), and midazolam 0.08 ± 0.27 mg (max 1 mg).

No catecholamines were used intraoperatively.

Small doses of ephedrine were administered to 3 patients for transient blood pressure drops.

Significant (> 500 mL) intraopera- tive blood loss was noted in one patient (4%) with a large meningioma.


Intraoperative brain relaxation assessed using the modified Bristow scale was assessed as 2 points (a compliant brain within craniotomy edge) in 22 patients (84%), as 3 points (slightly tight brain, little above craniotomy edge) in 3 patients (12%), and as 4 points (tense brain, brain oedema, signifi- cantly above craniotomy edge) in one patient (4%).

Surgical conditions were excellent in 6 cases (23%), good in 16 cases (62%), and poor in 4 cases (15%; all with intraoperative epileptic episodes).


Intraoperative and postoperative complica- tions are presented in Table 2.

Intraoperative epi- leptic episodes were observed in 4 patients (15%).

The patients experienced generalized seizures caused by electrical stimulation of the motor cortex (EEG monitoring).

The seizures were observed in 31% of patients with an identified motor cortex.


The onset of seizures in 75% occurred during electrical stimu- lation of the cerebral cortex.

However, in one case, a generalized tonic-clonic seizure occurred during traction placed on brain tissue or the use of a Cavitron Ultrasonic Surgical Aspirator (CUSA).

All patients who experienced seizures during surgery presented tran- sient lower limb muscle force.

In one case, decreased consciousness without accompanying seizure was observed during motor cortex stimulation.


None of them showed sensory or speech disorders; however, no eloquent areas were identified in these patients.

In the event of a seizure, the surgical field was filled with ice-cold Ringer's solution, and in half of the cases, an additional 1 mg of clonazepam was administered intravenously, resulting in regression of convulsive activity and restoration of consciousness.


The following abnormalities were found on post- operative head CT (24 hours after surgery): haema- toma in 3 patients (12%) and venous stroke in one patient (4%).

No reoperation was needed.

Except for one ASA-4 patient, no patient required ICU observa- tion.

In this case, the patient had a severe chronic ob- structive pulmonary disease and pulmonary tumours compressing the aorta that required transfer to the ICU for 24 hours of observation.

Postoperative follow- up was good in all patients (including the ASA-4 case).

A death in the late postoperative period (7 th day after AC) was caused by a pulmonary embolism.


SAP, DAP, and MAP changed significantly during the analysed points in time (c 2 = 45.40, P < 0.001; c 2 = 33.56, P < 0.001; c 2 = 21.94, P < 0.001, respec- tively).

These alterations were within the clinically accepted range during the measurements and did not exceed 20% of the initial values.

SAP was lower at T30 (P = 0.001), T60 (P < 0.001), and Tpo (P < 0.001) vs. T0.

DAP decreased at T30 (P = 0.002), T60 (P < 0.001), and Tpo (P < 0.001) vs. T0.

MAP decreased at T60 (P = 0.001) vs. T0 (Figure 2A).

HR altered significantly at the analysed points of time (c 2 = 34.20, P < 0.001).

HR decreased at Tpo vs. T0 (P = 0.001) (Figure 2A).

Those changes were unim- portant (20% change from the initial value).


The saturation did not change significantly at the analysed points in time, and at no point in time was it less than 94%.

EtCO 2 did not change consid- erably during the analysed points of time.

PaCO 2 and PaO 2 increased over time (c 2 = 14.38, P = 0.002; c 2 = 9.70, P = 0.021); however, neither parameter


Intraoperative neurophysiological monitoring was performed in 96% of AC patients.

Electrical map- ping of the cerebral cortex allowed the identification of the motor cortex in 50% of patients and the sub- cortical structures in 12% of the cases.

Preoperatively, muscle force was 5/5 in 85% of patients and 4/5 in 2 patients, using the Lovett scale.

Unaltered muscle force was observed postoperatively in 88% of the pa- tients.


Postoperatively, one patient decreased from 5 to 4 on the Lovett scale and 2 others from 5 to 1.

Additionally, one patient with a score of 4/5 was tem- porarily paralyzed after surgery.

Sensory function measured after cortex or peripheral stimulation was evaluated in 62% of patients, and in 22% of cases the sensory cortex was successfully stimulated.

None of the operated patients reported quantitative or qualita- tive disturbances during surgery.


Brainstem auditory- evoked potentials were recorded in 15% of patients.

The latency and amplitude of peaks I-IV, II, III, and V, as well as the interpeak latency of I-III and III-V, were close to the initial values.

Of all patients eli- gible for AC, 73% had a previous diagnosis of epi- lepsy.

Suppressive treatment in 74% of the patients required an antiepileptic drug, and 26% required 2 different drugs.


AC is a technique developed for maximizing re- section while preserving neurological function [1].

Multiple studies have documented that CS based on low doses of dexmedetomidine, along with neuro monitoring, is a feasible approach [18-20].

Dexmedetomidine-based CS with a scalp block without instrumental airway support in conjunc- tion with psychologist support provides good qual- ity intraoperative sedation for AC, even during long surgical procedures requiring intraoperative mag- netic resonance imaging.


The protocol of anaes- thetic management proposed in this study allows one to achieve a good quality of analgesia while still maintaining sedation with fast recovery to full consciousness for neuropsychological monitoring after cessation of dexmedetomidine infusion.

It is comfortable and not too demanding for the pa- tient.

The clinical study showed that the anaesthe- tics used in this protocol did not significantly influ- ence cardiovascular and respiratory parameters.


Operative conditions were good according to the surgeon's opinion and the modified Bristow scale.

The quality of the monitored brain potentials, as well as neuropsychological monitoring, was satis- factory.

We did not observe symptoms of cough, nausea, vomiting, agitation, anxiety, brain swell- ing, or haemostatic disorders intraoperatively, as reported in the literature [18, 21].


Various anaesthetic management strategies for AC are being utilized; however, there are no clear recommendations [14].

Several authors have re- searched propofol-remifentanil and dexmedetomi- dine-based sedation, but only a few comparative studies have been performed [22-24].

Dexmedeto- midine has been reported to depress the cardio- vascular system by relaxing the autonomic system, increasing the risk of global or regional hypoper- fusion of the brain [25, 26].

However, in our study, no clinically significant influence on cardiovascular or respiratory parameters was observed.

SBP, DBP, MAP, and HR decreased significantly at T30 and T60 compared to initial values and continued at that level until 60 min after discontinuation of sedation.

It is noteworthy that these changes did not reach clinical significance.

Similar results were reported in another retrospective observation on a large cohort of patients, where the doses of dexmedetomidine used in that trial (up to 1.7 µg kg -1 h -1 ) were higher than what was proposed in this study [21].

However, due to the limited group of patients in our study, the impact of dexmedetomidine on the cardiovas- cular system requires further investigation.


We also marked that, except for one case, pa- tients were not admitted to the intensive care unit, due to stable immediate postoperative status.

Af- ter AC, patients were taken to a postoperative re- covery room for 2-3 hours and then transferred to the neurosurgery unit.

They were allowed to drink a few hours later.

The next day, they were mobilized after the completion of the postoperative head CT.

The duration of hospitalization was as short as 48 to 72 hours in 2 patients.

The remaining patients required longer hospitalizations due to neurologi- cal dysfunctions or fear of delayed new neurologi- cal complications and inadequate supervision after being sent home.

It is noteworthy that because of the short patient hospitalization after AC, the cost Bogusława Ewa Lechowicz-Głogowska, Agnieszka Uryga, Artur Weiser, et al. of the entire procedure would be greatly reduced compared to general anaesthesia [27, 28].


In this study protocol, a sedation level of -1/-3 on the RASS scale was maintained up to meningeal closure, and tests were frequently per- formed to warn the operator of possible white matter injury that could cause further emo- tional, memory, or character disorders [29].

The level of sedation was also monitored with BIS.

It is well documented that the RASS scale and BIS are correlated with the level of sedation [30, 31].

Such a correlation was also found in this study.

As reported previously [32], we did not observe a significant change in BIS values under seda- tion with dexmedetomidine RASS -1/-2, but we observed lower BIS values after a bolus of propo- fol and initiating deeper anaesthesia (RASS -3).

The time to achieve complete consciousness for a patient was 3-5 min (from RASS -1/-2), up to 10-13 min (RASS: -3) after stopping dexmedeto- midine, and is consistent with other research.

This study showed that RASS -2/-3, corresponding to BIS 98-50, is comfortable for the patient while al- lowing them to regain complete cooperativeness in a few minutes at any time during surgery to allow neuro and psychological evaluation.


The risk of seizures triggered by electrical brain stimulation is a common adverse event during brain mapping.

This study has shown that dexmedetomi- dine does not influence epileptiform discharges in EEG after IV infusion [33].

In cases without EEG, continuous free-running EMG recordings during the surgical removal procedure were successfully used to identify increased EMG activity and possible im- minent seizure.

The 15% incidence of intraoperative seizures experienced in our study was within the range described in the literature [34].

The seizures were predominantly caused by relatively high- intensity stimulation, which usually exceeds 10 mA despite the use of 50-Hz stimulation.

In 3 cases the seizures were generalized, and in one case it was a focal seizure, and all were stopped with irriga- tion with Ringer solution.

Half of the patients who experienced seizures had motor deterioration, but the motor functions of the remaining patients were stable despite preoperative weakness.


The quality of the monitored brain potentials did not change from the initial value.

It was pre- viously shown that appropriate dosing of dexme- detomidine is crucial to avoid its suppressive effect on motor function.

The motor function changes observed in our patients were a consequence of tumour removal manoeuvres rather than a direct pharmacological influence on cerebral blood flow that affected motor function [35].

Patients who regu larly performed simple and complex motor tasks to assess agility and muscle strength were well tolerated by them and did not alter the progress of the operation.

The results obtained from motor function monitoring were informative for the sur- geon and anaesthesiologist and consistent with the postoperative neurological achievements of the pa- tients [36].

Intraoperatively and postoperatively, all patients did not complain of sensory disturbance.


The indications for the use of CS for AC are in- creasing: all grade 1-2 WHO gliomas, basilar artery aneurysms, and other complex aneurysms, deep brain stimulation, cerebellopontine tumours, and pa- tients with complex comorbidities and obesity [29].

Furthermore, according to recent recommenda- tions, some of the patients included in this study would have been disqualified from using the AAA technique or any other type of CS for AC using pro- pofol, fentanyl, or remifentanil due to severe obe- sity, difficult airways, and severe respiratory and car- diovascular comorbidities due to an unacceptable increase in risks of severe intraoperative complica- tions.

Also, most anaesthetists would have excluded some of the surgical positions (park bench, sitting, prone) if a different type of sedation were used.

Pa- tients with severe comorbidities or obesity are the best candidates for the techniques presented in this study becuase many complications can be avoided.


Neuropsychological examination, which was not affected by dexmedetomidine, allowed efficient pro- tection of language and cognitive skills.

It was shown that dexmedetomidine did not block differentiation between congruent and incongruent language tasks based on the presence of N400 and is recommended during craniotomy [37].

It is worth mentioning that 50% of the patients had language disturbances before surgery, but only 30% of the electrical map- pings allowed the identification of cortical language epicentres.

After the operation, deterioration of the language was observed in 4 cases (15%).


The preoperative psychological evaluation was an important part of our study.

The patients in our study were evaluated by the psychologist not only before AC but also during the total procedure and 24-48 hours after AC.

The presence of a psycholo- gist during the whole procedure (in the operating room) is not a standard element of the procedure.

Previous studies have shown that hypnosis could be used successfully in the paediatric population dur- ing AC for the resection of brain tumours located close to eloquent areas [38] and during AC of low- grade gliomas [39].

Thus, we expected that, similarly to hypnosis, the presence of a psychologist would encourage patients to gain motivation and safety during AC and decrease the risk of conversion to general anaesthesia.

Our experience indicates that building patient trust and comfort can increase the chances of a positive patient experience during the long-lasting procedure and after hospital discharge.

The detailed psychological evaluation of patients in terms of early and late postoperative stress injury will be a matter of a separate report.


The age distribution in our group of patients was as follows: one case was < 20 years, 7 cases were between 20 and 40 years, 14 cases were between 40 and 65 years, and 4 cases were > 65 years.

The reac- tivity of sedative-analgesic agents could depend on age [40, 41].

However, the Kruskal-Wallis ANOVA test did not show significant differences in the total dose of dexmedetomidine (c2 = 2.65; P = 0.265), which is in line with other studies on the pharmacodynam- ics and pharmacokinetics of dexmedetomidine [42].


The anaesthetic protocol during AC is still under investigation.

Abazieu [43] reported that remifent- anil could be an independent risk factor for adverse events during AC.

Remifentanil can depress the re- spiratory centre in the brain and cause nausea and vomiting, leading to respiratory insufficiency, pro- longed hypoventilation, and conversion to general anaesthesia.

It should be noted that a combination of this opioid with propofol is generally recom- mended and used (including in the author's cen- tre) for the AAA protocol of anaesthesia [43].

This sedation procedure probably should be questioned, especially if we recognize that already impaired neuronal function in operated patients, due to any further imbalance, might be irreversibly damaged.

It has been shown that in older patients anaesthetized with remifentanil and propofol, there were higher incidences of cognitive deterioration.

Such mental disorders were not observed in patients sedated with dexmedetomidine and propofol [44].

Further- more, the other authors underlined the negative impact of propofol on cognitive functions, which can mask real neuronal dysfunction and cognitive impairment that must be minimized during AC [45].


This observational clinical study has limitations.

The procedures performed were long (about 5 hours on average), which can affect the patient's comfort.

However, dexmedetomidine-based sedation led to physiologically similar sleep with immediate "on- demand" arousal.

Other studies on dexmedetomi- dine sedation have analysed much shorter proce- dures in retrospective research, which could have an impact on the conclusions [25].


Secondly, we only analysed clinical parameters a few moments from the start of dexmedetomidine infusion to 60 minutes after infusion.

However, we noticed that all monitored parameters were stable from T60 until the end of the surgery.


Third, although anaes- thetic management was similar for all analysed pa- tients, some subjects required a bolus of propofol or mida zolam during head closure to maintain pa- tient comfort.

The synergistic effect of these chosen supplements deepens anaesthesia without the risk of adverse events.

In addition, the relaxation of the brain and the operative conditions were based on the observation of an experienced neurosurgeon.

The modified Bristow scale seems to be a suitable tool to access for brain relaxation assessment, but it is subjective.


Lastly, the character of this study was prospective without a control group.

However, the lack of adequate control was not perceived as a major deficiency in the study design.

This research has been designed as uncontrolled due to previous experiences in the AAA procedure that propofol and opioid sedation led to respiratory depression, uncon- trolled movements, poor arousal quality, long recov- ery to cooperative consciousness, and high risk of re- spiratory complications during AC.

Furthermore, the number of patients who met the inclusion criteria for AC was minimal at that time in our centre.

Given these limitations, a multicentre study with a control group would provide more reliable data and improve the generalizability of these observations.


CS with dexmedetomidine and scalp block can provide a safe and comfortable anaesthesia pro- cedure during AC for the surgeon and the patient.

It allows the implementation of advanced, multi- level intellectual tests, where perioperative psycho- logical support plays an important role.

Smooth in- duction and arousal with a cooperative patient are possible with the presented anaesthetic method, even during long-lasting procedures (5-6 hours).

Further observations and comparative trials are needed to conclude the best anaesthetic protocol for AC procedures.


Dear Editor, We read with great interest the recent study “Awake craniotomy with dexmedetomidine during resection of brain tumours located in eloquent regions” presented by Lechowicz-Głogowska et al. [ 1] and the method used to perform awake craniotomy (AC) in the authors’ centre.

Although AC is considered as a gold standard of management during resection of intra-axial lesions in proximity to eloquent cortical and subcortical regions, until now a uniform consensus on anaesthetic management has not been established, as the authors rightly pointed out.

Therefore, we would like to acknowledge and congratulate the authors for their effort to perform the study on such an interesting and still uncommonly used anaesthetic technique.

Proper selection of patients for elective AC in conscious sedation (CS) is a key factor determining the success of the whole procedure.

After reading the article we would like to raise a few issues concerning selection criteria and the standard of the AC procedure presented by the authors which need clarification.


Although the authors stated that the selection criterion for the operation was a good neurological status of the patient (unfortunately “good neurological status” was not defined precisely), data presented in Table 1 show that there were also people in the study group who were graded 13 points on the Glasgow Coma Scale (GCS) scale.

We have some doubts whether it was possible to explain the details of the operation to patients with such a low score and to ensure that the special requirements for the intraoperative interaction between the patient and the medical team were properly and reliably understood by the patient.

We have further doubts about selecting for ACs under CS patients with severe obesity, anticipated difficult airway, and severe respiratory (lung tumour compressing the aorta) and cardiovascular comorbidities.

In our opinion, such patients meet the criteria for a predicted anatomical and physiological difficult airway, and in the context of restricted access to the patient, emergency airway management would be extremely challenging.

The risk of severe complications related to airway management (including death and brain damage) was outlined in the excellent British NAP4 study, which showed the significance of proper identification of patients at risk of difficult airway and the need for advance planning to reduce the risk of a fatal outcome [2].


In Table 2, the authors indicate that they encountered four difficult intubations in 15% of operated patients but did not explain the reasons for instrumentation on the airways.

We also noticed that some patients were operated on in positions that could pose additional difficulties with access to the airways, if needed.

According to the information found in the article there was a group of patients who were operated on in a prone position.

Postoperative survey carried out in our centre revealed that one third of patients who were operated on in conscious sedation in a neutral or lateral position complained about discomfort.

We expect that staying in a prone position during lengthy (over 6 hours) procedures may be challenging for a conscious patient [3].


We strongly agree with the authors’ opinion that dexmedetomidine can be an excellent choice in AC surgery but have some concerns about the choice of less controllable midazolam and oxycodone as a supplement to sedation and analgesia.

We agree with the authors that further studies are needed to confirm the best and safest anaesthetic protocol for awake craniotomy.


Dear Editor, We are responding to the questions raised by our colleagues from Warsaw (commentary by Surówka et al.) [ 1] following the publication of our study “Awake craniotomy with dexmedetomidine during resection of brain tumours located in eloquent regions” [2].

We appreciate this discussion, as it could enhance the management of patients undergoing the awake craniotomy procedure.

The proper patient selection for awake craniotomy (AC) under conscious sedation (CS) is an important factor that influences possible adverse events during the procedure.

Therefore, in our study, a psychologist carefully evaluated each patient at least one day before the procedure to identify the risk factors of uncooperativeness, overwhelming stress, and an inability to understand and follow commands or answer questions.

Patients with significant intellectual or psychiatric disorders were disqualified from AC.

Some patients with somnolence, aphasia or motor impairment were recognised as candidates for AC under CS because they could cooperate and answer questions once aroused, which was confirmed by the psychologist.

In our study, we defined patients as having a “good neurological status” if they passed all psychological tests preoperatively.

Additionally, in our group, only one patient had a Glasgow Coma Scale (GCS) of 14 points, and the rest had a GCS of 15 points (Table 1 should show GCS 14–15: n = 26, and GCS 8–13, n = 0).

However, in our opinion, GCS is not appropriate and sufficient to describe a patient’s psychological and intellectual condition in the context of selection for AC, although commonly used to define neurological status [3, 4].

It may be surprising that even patients with previous psychiatric diseases, as well as those with a poor performance status (Karnofsky scale: < 70 points), were selected for AC and CS in other centres.

A significant correlation between an increase in intraoperative emotional intolerance, and further complications (uncooperativeness, respiratory insufficiency, conversion to general anaesthesia with intubation, or other instrumental upper airway support) during AC and CS, below 70 points of the Karnofsky scale has been noted [5].

This issue was addressed in other studies, which included patients with various neurosurgical procedures of AC/CS, based on dexmedetomidine sedation [5–7].


AC under dexmedetomidine-based CS is routinely performed in our centre.

To date, we have operated on about 70 patients for brain tumour resection, and we noted no adverse events.

All those procedures were completed successfully without conversion to general anaesthesia or any impact on respiration.

The psychological, psychiatric, and intellectual risk factors of adverse events associated with AC under CS were also of interest to us, but they will be presented in a separate publication.

Patients with severe comorbidities are at higher risk for conversion to general anaesthesia and instrumental airway support, during AC/CS.

This includes obese (BMI 30–35) and severely obese patients (BMI 35–40).

The issue is that surgery close to eloquent areas of the brain poses the risk of injury and postoperative disability.

AC increases the chance of the best postoperative neurological and intellectual result, preservation of multilingual skills, instrument playing, artistic skills and personality structure.

To date, only intraoperative performance helps to preserve these skills.

It is better to open up the chance for the patient than to risk irreversible disability and social disclosure.

The study by Garavaglia et al. [ 8] presented three cases of successful AC under CS: one with severe obesity, one with asthma and one with a lung tumour.

Recently other authors [9] presented a unique case of a patient with Eisenmenger syndrome who underwent two operations within a short period: the first under general anaesthesia and the second, successful AC/CS for resecting a large cyst in the third ventricle that was causing obstructive hydrocephalus and a risk of a brain inclination.


We agree that “difficult airways” is the “pearl” of AC.

The head fixed in the Mayfield clamp makes it challenging to instrumentally support the upper airways.

This procedure requires constant high attention, preparation and training of all surgical team members.

Only staff (such as the neuro-anaesthesiologist and anaesthetic nurses) who are aware and experienced in AC/CS nuances can perform such procedures.

Manual support through a face mask, nasopharyngeal, oropharyngeal tubes, laryngeal mask airway (LMA), videolaryngoscope, and fibrescope must be on hand, ready to be used by trained personnel, if needed.

This is recommended in all anticipated and unexpected difficult intubations [10].

In the presented cohort of patients, we based our anticipation of difficult intubation on standard criteria (Mallampati classification, thyromental distance, head and neck movement, inter incisor gap) and the above-mentioned instrumentation was always prepared.

To date, there has been no need to support upper airways in presented cases, as well as in all other AC/CS cases for brain tumour resection in our centre.

A report by Gruenbaum et al. [ 11] studied and presented the successful, fast, and useful ways of upper airway support during AC.

This report showed that the most successful and fastest method (32 seconds) was LMA (laryngeal mask), followed by intubation on bougie/through LMA insertion.

Videolaryngoscopic intubation and nasal/fibrescope intubation took longer (60–90 seconds and 70–120 seconds, respectively).

Cricothyroidotomy was effective only in 40% of cases in the context of ventilation, whereas tracheotomy done by an experienced surgeon was more effective [12].

Respiratory insufficiency, even asystole, was reported by some authors, during AC [12, 13].

Various factors can cause respiratory insufficiency, not only the sedative drugs, as commonly believed, but electrical stimulation, neurophysiological monitoring, aspiration, epilepsy with trismus and tongue biting with bleeding, severe comorbidities, obesity, other rear diseases, and medications [13, 14] All the above aspects should be analysed individually for candidates for AC [15].


Patient positioning for neurosurgical procedures is dictated by the need to assess brain lesions and the neurosurgeon’s preference.

In some cases, one can discuss altered positioning, if it is possible.

Lateral, park bench, prone, semi-sitting, and sitting positions are commonly used in neurosurgery.

The need for upper airway instrumentation in prone and park bench positions is of the highest risk of failed intubation and ventilation.

In our centre, no patients were operated on in the prone position.

In the paper, it was given as the concern of anaesthesiologists.

However, the study of Takami et al. [ 5] presented cases in lateral, park bench, sitting and semi-sitting positions for AC/CS.


In our study [1], small doses of fentanyl or midazolam were used for patient comfort only for the closing phase of the surgery if needed.

Five patients were given 0.05–0.10 mg of fentanyl in boluses of 0.05 mg and two patients received 1.0 mg of midazolam when neurophysiological and psychological assessments were finished.

No impact on respiration was observed.

Other reports on AC/CS show no adverse impact on such management [5, 9].

We did not use oxycodone during the surgery; it was used for postoperative pain control, but from observations on ropivacaine with adrenaline scalp block, excellent pain control up to 8–12 hours was possible.


We believe that the development of robotic surgery opens a new approach to anaesthetic management, and AC/CS is becoming common for more neurosurgical procedures.

Even recently, CS has been used for other surgery, such as deep brain stimulus: for Parkinson’s disease, obsessive-compulsive disorder, severe obesity, depression, psychiatric diseases, and many others.

The indications for intraoperative neuro-psycho-monitoring are still increasing.

There is an urgent need to develop a mode of anaesthetic management that is safe and comfortable for the patient, and that is suitable for all groups of patients, including those with severe comorbidities.

We hope that our study is a small step in this direction.


Citrus, like many other woody species, flower profusely.

In the Mediterranean area, flower induction takes place in autumn/ winter and the main blossoming in spring (Sherman and Beckman, 2003).


Current knowledge discriminates between exogenous and endogenous components for regulating flower- ing biology in citrus (Krajewski and Rabe, 1995; Martínez-Fuentes et al., 2004).

The former mainly includes climatic factors.

Thus, environmental cues such as temperature or photoperiod, and stress conditions (e.g. water deficit or sal- inity), have been reported as modulators of flowering responses (Agustí, 1999; Valiente and Albrigo, 2004).


On the other hand, endogenous factors are essentially genetic and/or hormonal.

Phytohormones, such as cytokinins, polya- mines or gibberellins, are known to participate to a certain extent in the physiological processes regulating flower induc- tion or differentiation (Koshita et al., 1999).

In particular, gibberellins have been traditionally considered as essential inhibitors of flower bud induction (Mutasa-Gottgens and Hedden, 2009).


In addition, the presence of fruit has also been found to be an inhibitor of flowering in fruit trees.

The response of flower- ing to fruit load has been reported in numerous species (Garner and Lovatt, 2008; Spinelli et al., 2009; Rosenstock et al., 2010), including citrus (Monselise and Goldschmidt, 1982).

This behaviour basically consists in the fluctuating production between heavy fruit yields (on year) followed by scarce ones (off year).


Moreover, the inhibitory effect of fruit in flower for- mation depends on both the number of fruits developed and the harvest date (El-Otmani et al., 2004; Martínez-Fuentes et al., 2010).

Hormonal factors, competition for nutrients or even changes in carbohydrate and mineral status (Goldschmidt et al., 1985; Valiente and Albrigo, 2004; Baninasab et al., 2007; Rohla et al., 2007) appear to participate in the regulation processes, although the way in which the presence of fruit affects flowering and the nature of the regulatory mechanisms remains unknown.


Recently, the development of genomic and transcriptomic tools has contributed to a better understanding of the metabolic and molecular processes involved in floral biology.

Most of our knowledge about flower induction has come from studying flowering regulatory genes in Arabidopsis thaliana (Komeda, 2004).

These genes appear to be extraordinarily conserved in woody species (Brunner and Nilsson, 2004), and previous research has demonstrated that many of them even share roles and/or metabolic pathways (Greenup et al., 2009).

Moreover, current evidence assumes that flowering is the result of complex interactions at the metabolic and molecular level involving multiple promoter and inhibitor genes (Moon et al., 2005; Michaels and Michaels, 2009).

Regarding citrus, several studies have recently been conducted to eluci- date the molecular mechanisms involved in flower formation and differentiation.

A first approach on citrus was developed by Peña et al. ( 2001), who demonstrated through plant trans- formation the role of APETALA1 (AP1) and LEAFY (LFY) genes in juvenile phase development.

Later studies have ident- ified and/or isolated in this species these (CsAP1, CsLFY; Pillitteri et al., 2004b) and other regulatory genes involved in both the determination of the flowering time and floral iden- tity processes (see Dornelas et al., 2007).


The regulatory role of the FLOWERING LOCUS T (FT) gene has been identified in numerous species (Turk et al., 2008; Zhang et al., 2010) including citrus (Endo et al., 2005; Nishikawa et al., 2007), and the encoded protein (FT protein) associated with the mythic 'florigen' (Yu et al., 2006).

Other genes, like CONSTANS (CO) or SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) have been reported to integrate signals from different pathways and, at least in Arabidopsis, are involved in promoting vegeta- tive to reproductive tissues (Borner et al., 2000; Lee et al., 2000; Onouchi et al., 2000; Komeda, 2004).

Specifically, CO participates in upstream and complex regulatory pathways, whereas SOC1 appears to act downstream (Dornelas et al., 2007).


In contrast, FLOWERING LOCUS C (FLC) represses flowering; high transcript levels have been found in late- flowering mutants, suggesting a central role of this gene in the control of flowering time, mainly through the vernalization pathway (Sheldon et al., 2000; Michaels et al., 2005).


Even though other repressor genes of flowering, like TERMINAL FLOWER 1 (TFL1), have been isolated from 'Washington' navel sweet orange (CsTFL; Pillitteri et al., 2004a) and corre- lated positively with juvenility, rigorous information about the function of FLC in citrus is not yet available.


In summary, alternate bearing is the result of complex meta- bolic and molecular regulatory pathways affecting flowering induction and floral identity.

The phenomenon has been largely explained as a crop load dependency and, although the factors modulating the processes involved have recently been studied for diverse species and key genes regulating flow- ering identified, we lack knowledge about the effects of crop load and the molecular mechanisms involved.

In this work, for the first time, the effects of fruit on the expression of puta- tive homologues of genes involved in flowering pathways were analysed to provide insight into the molecular mechanisms underlying alternate bearing in citrus due to crop load.


This study involved field grown 12-year-old trees of 'Moncada' mandarin [Clementina 'Oroval' (Citrus clementina Hort. ex Tan.) × 'Kara' mandarin [C. unshiu (Swingle) Marcow. × C. nobilis Lour.] trees, grafted onto 'Carrizo' citrange [C. sinensis Osbeck × Poncirus trifoliata (L.) Raf.] rootstock, planted 5 m × 5 m apart.

Experimental fields were located in the IVIA Research Station (Moncada, Spain).

Trees of this cultivar exhibit a marked alternate-bearing behaviour.


To examine the effect of fruit load on flowering, 25 trees were randomly selected for their uniformity in size and vigour at spring.

Total yield per tree was determined by count- ing and weighing all fruits at harvest (April), and flowering intensity was evaluated in spring as follows.

Four branches per tree of three ages (late spring, summer and autumn sprouts) with some 300 nodes per branch were previously selected.

Both the number of sprouted nodes and sprouts were counted.

The flowers per sprout were also counted, obtaining the results as the number of flowers per 100 nodes to compensate for the differences in size of the selected branches.


Defruiting experiments were performed on another set of 24 trees using four levels of fruit removal (0, 33 %, 66 % and 100 %).

Treatments were performed at the onset of stage II of fruit development (July).

A randomized complete-block design was used in the experiments.


From early September to the end of February, 30 fully developed mature adult leaves per tree from on (fully loaded) and off trees (without fruits) were collected for RNA extractions.

Samples were pooled into three groups, and immediately ground and stored at - 80 8C until analysed.


Total RNA was isolated from frozen tissue using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany).

RNA samples were treated with RNase free DNase (Qiagen) through column purification following the manufacturer's instructions.

RNA quality was tested by OD 260 /OD 280 ratio and gel electrophoresis.

RNA concentration was determined by fluorometric assays with the RiboGreen dye (Molecular Probes, Eugene, OR, USA) according to manufacturer's instructions.

Three fluorometric assays per RNA sample were performed.


Quantitative real-time RT-PCR was performed with a LightCycler 2 . 0 Instrument (Roche Diagnostics, Basel, Switzerland) equipped with LightCycler Software version 4 . 0.

One-step RT-PCR was carried out.

Reactions con- tained 2 . 5 units of MultiScribe Reverse Transcriptase (Applied Biosystems, Carlsbad, CA, USA), 1 unit of RNase Inhibitor (Applied Biosystems), 2 mL LC FastStart DNA MasterPLUS SYBRGreen I (Roche Diagnostics, Basel, Switzerland), 25 ng total RNA and 250 nM of the specific forward and reverse primers of each gene in a total volume of 10 mL. Incubations were carried out at 48 8C for 30 min, 95 8C for Muñoz-Fambuena et al. - Fruit regulation of flowering gene expression in citrus 512 10 min followed by 45 cycles at 95 8C for 2 s, 58 8C for 8 s and 72 8C for 8 s. Fluorescent intensity data were acquired during the 72 8C-extension step and transformed into relative mRNA values using a 10-fold dilution series of an RNA sample as a standard curve.

Relative mRNA levels were then normalized to total RNA amounts as previously described (Bustin, 2002; Hashimoto et al., 2004) and an expression value of 1 was arbi- trarily assigned to the first sample of the on trees.

Specificity of the amplification reactions was assessed by post-amplification dissociation curves and by sequencing the reaction product.


Putative genes were identified through homology search with related genes from an EST database of a random 5 ′ 'Clemenules' mandarin (C. clementina Hort.

ex Tan.) full- length cDNA library (Terol et al., 2007).

Synthetic oligonu- cleotides were designed to amplify the gene from the selected clones and, as stated before, sequenced for confirmation.

Details about the forward and reverse primers are listed in Table 1.


Parameters were statistically tested by analyses of variance (ANOVA), using the least significant differences (LSD) test for means separation.

The experimental data were analysed with Statgraphics Plus 5 .

1 software (Statistical Graphics, Englewood Cliffs, NJ, USA).


A significant inverse relationship between preceeding fruit load and flowering intensity was found in 'Moncada' mandarin (r ¼ -0 . 93; P ≤ 0 .

05; n ¼ 25; Fig. 1).

Considering the results, the larger the crop load the lower the flowering intensity, with a breaking point of about 50 kg tree 21 .

Above this fruiting value, flowering was independent of crop load and paralleled nearly nil values.


Although statistically significant, this relationship does not imply causality, therefore further experiments were carried out to demonstrate the direct effect of fruit removal upon flow- ering.

As expected, control on trees (non-defruited, fully loaded) showed the lowest number of flowers (0 . 5 flowers/ 100 nodes, on average), in comparison with those completely defruited, which presented the highest number of flowers (142 flowers/100 nodes).

Intermediate fruit loads (33 % and 66 % defruited trees) resulted in intermediate flowering intensities (Fig. 2).


The effect of fruit removal on flowering was also evident on fruit set and yield assessment.

The higher the flowering inten- sity, the larger the crop load (data not shown).

The magnitude of the response, however, depended on the time of fruit removal.

Thus, removal of all fruits in August, September or October did not affect flowering in spring (100 - 135 flowers/ 100 nodes; Fig. 3); however, when fruit removal was per- formed from November onwards, a dramatic inhibition of flowering was observed the following spring (,10 flowers/ 100 nodes).


The time-course of CiFT expression in leaves throughout the study was strongly affected by fruit load (Fig. 4).

Significant differences in mRNA transcripts were detected between on and off trees from October onwards.

The expression in off tree leaves increased progressively, becoming more than 10-fold higher than that of on tree leaves in December, and remaining almost constant up to late in February.

CiFT tran- scripts in on tree leaves did not significantly vary within the study period.


The time-course of SOC1 relative expression in leaves was also higher in off trees than in on trees (Fig. 5).

Differences were statistically significant from early September to mid-December, becoming highest in November (close to 50 % higher for off trees).

From early January onwards, differ- ences between treatments were not statistically significant.


Figure 6 shows the time-course of FLC expression in leaves from on and off trees.

From September to mid-December, no differences in gene expression between treatments were found.

In January, however, levels of mRNA transcripts mark- edly increased in on trees.

From January until the end of the study, expression in on tree leaves remained between 1 .

6- and 2 .

8-fold higher than in off tree leaves.


Expression of CsTFL in leaves was slightly reduced from September to mid-November; afterwards, it continuously increased up to the end of February, when it was .3 times the amount measured in November (Fig. 7).

No significant differences were found between leaves from on and off trees during the study period.


CsAP1 and CsLFY revealed different patterns of change in response to fruit load (Fig. 8).

CsAP1 expression remained almost constant from early October up to the middle of December.

During this period, expression values were 3 - 5 times lower in on trees than in off trees.

From December onwards expression increased in both cases and, although minor in magnitude, significant differences between on and off trees were also found until the end of February.


On the other hand, CsLFY expression progressively increased from September to the middle of December (15- to 20-fold).

After a transitory reduction in January, expression considerably aug- mented towards the end of February, reaching values between 30 and 50 times those registered in September.

Differences between on and off trees were only significant at the end of the study.

In late February, CsLFY expression in off trees was significantly higher than that found in on trees (close to 20 %).


Alternate bearing has been associated primarily with the pres- ence of fruit (Verreynne and Lovatt, 2009; Martínez-Fuentes et al., 2010).

According to the literature, it is assumed that crop load alters both flower induction and floral bud differen- tiation (Valiente and Albrigo, 2004).

Particularly, in citrus, flower induction has been reported to occur late in autumn, whereas differentiation occurs afterwards (Iglesias et al., 2007).

Recent molecular approaches support this scheme, dis- tinguishing between genes regulating flowering induction and those regulating floral differentiation processes (Tadeo et al., 2008).

The present results indicate that genes regulating flower induction are strongly related to fruit load in alternate- bearing citrus trees.


Fruit load acts as a strong inhibitor of flowering in numerous woody fruit tree species including citrus, although the responses vary among species and cultivars (Monselise and Goldschmidt, 1982; Valiente and Albrigo, 2004; Albrigo and Galán, 2004).

The present data obtained for 'Moncada' man- darin confirmed, once again, a non-linear fruit load - flowering intensity relationship (Fig. 1).

However, since this relationship does not necessarily imply causality, convincing evidence that fruit load is directly responsible for reducing flowering inten- sity was provided by means of defruiting treatments.


Trees maintaining all developed fruits up to maturation hardly flow- ered (5 flowers/100 nodes, on average), whereas those fully defruited at the onset of stage II of fruit development flowered profusely (142 flowers/100 nodes, on average).

Trees defruited at an intermediate intensity (33 % and 66 % of developing fruitlets) flowered intermediately as well.

However, because no differences were found between 33 % and 66 % of fruit removal (16 and 22 flowers/100 nodes, respectively) the effect does not appear to be strictly quantitative.


These results support the hypothesis that the fruit load - flower- ing intensity relationship is not a linear function and this coincides with previous reports suggesting that there might be a threshold value for crop load, dependent on the variety and physiological status, above which flowering is strongly inhibited (Agustí et al., 1992; Martínez-Fuentes et al., 2010).


It has been previously reported that flower inhibition due to crop load also depends on the length of time the fruit remains on the tree (see review by Monselise and Goldschmidt, 1982).


Considering the present data for 'Moncada' mandarin, fruit Sep Oct Nov Dec Month Relative CsTFL expression Jan Feb 1 2 On Off Muñoz-Fambuena et al. - Fruit regulation of flowering gene expression in citrus 515 remaining on the tree did not affect return bloom (100 - 120 flowers/100 nodes) up to November; however, fruit remaining on the tree from November onwards dramatically reduced flowering (values close to 0).

Therefore, effective inhibition time of fruit load began between October and November.

This period has been associated with the fruit reaching its maximum size (Martínez-Fuentes et al., 2010), or with peel ripening (García-Luis et al., 1986).

In any event, fruit drasti- cally inhibits flowering from November onwards, suggesting that there is a point of no return for induction of quiescent buds, and that some irreversible physiological, metabolic or molecular events induced by presence of fruit must be respon- sible for inhibition.

This hypothesis is supported by the fact that bud sensitivity to gibberellic acid (GA 3 ) inhibiting flower- ing occurs at this moment (García-Luis et al., 1986).


The mechanism whereby fruit load affects flowering intensity is not completely understood, although several regulatory factors have been described.

Early observations linked carbo- hydrate and nitrogen metabolism to the process (Goldschmidt et al., 1985; Lovatt et al., 1988); however, recent studies demon- strated that carbohydrate or nitrogen status are involved in nutri- tional or storage adjustments rather than in the floral process directly (Reig et al., 2006; Martínez-Fuentes et al., 2010).

Several studies have linked flowering intensity to bud sprouting, showing that changes in flowering intensity paralleled changes in the summer/autumn shoot number (Verreynne and Lovatt, 2009; Martínez-Fuentes et al., 2010), and also illustrated that the higher the fruit load the lower the number of sprouted nodes in spring (Martínez-Fuentes et al., 2010).

Additionally, environmental factors can modulate flowering through modifications in the physiology of shoot development or even in key metabolic path- ways (Agustí, 1999).

Nonetheless, knowledge about factors affecting flowering has considerably increased, but data do not provide enough information to understand the mechanisms through which the fruit controls the process of flowering.