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. 2018 Aug 22;120(4):1906–1913. doi: 10.1152/jn.00318.2018

The activity of discrete sets of neurons in the posterior insula correlates with the behavioral expression and extinction of conditioned fear

José Patricio Casanova 1,2,3,*,✉, Marcelo Aguilar-Rivera 4,*, María de los Ángeles Rodríguez 1, Todd P Coleman 4, Fernando Torrealba 1
PMCID: PMC6230801  PMID: 30133379

Abstract

The interoceptive insular cortex is known to be involved in the perception of bodily states and emotions. Increasing evidence points to an additional role for the insula in the storage of fear memories. However, the activity of the insula during fear expression has not been studied. We addressed this issue by recording single units from the posterior insular cortex (pIC) of awake behaving rats expressing conditioned fear during its extinction. We found a set of pIC units showing either significant increase or decrease in activity during high fear expression to the auditory cue (“freezing units”). Firing rate of freezing units showed high correlation with freezing and outlasted the duration of the auditory cue. In turn, a different set of units showed either significant increase or decrease in activity during low fear state (“extinction units”). These findings show that expression of conditioned freezing is accompanied with changes in pIC neural activity and suggest that the pIC is important to regulate the behavioral expression of fear memory.

NEW & NOTEWORTHY Here, we show novel single-unit data from the interoceptive insula underlying the behavioral expression of fear. We show that different populations of neurons in the insula codify expression and extinction of conditioned fear. Our data add further support for the insula as an important player in the regulation of emotions.

Keywords: conditioned fear, insular cortex, single-unit recording

INTRODUCTION

The insular cortex (IC) is a visceral sensory area since it is the main recipient of interoceptive information from the thalamus through its granular region (Allen et al. 1991; Saper 1982). Together with the adjacent agranular and disgranular regions, they form the posterior IC (pIC). The pIC is in a key position to distribute sensory information to the anterior (higher order) IC, which, in turn, is connected with other structures, such as the amygdala and prefrontal cortex (Allen et al. 1991; Cechetto and Saper 1987; Shi and Cassell 1998), well known to be involved in the processing of cognitive and emotional states. The role of the interoceptive pIC in bodily mapping (Cechetto and Saper 1987) and its connections with crucial structures of the emotion circuit (Burgos-Robles et al. 2009; Duvarci et al. 2011) makes the pIC a strong candidate structure for behavioral and emotional regulation.

It has been reported that the activity of the pIC, with its different sensory modalities, is important for the acquisition of spatial memory (Nerad et al. 1996), consolidation of taste-based memory (Berman and Dudai 2001), and for the processing of diverse stimulus-outcome associations (Contreras et al. 2007, 2012; Gardner and Fontanini 2014). However, there are conflicting results regarding the role of the IC in fear memory (Alves et al. 2013; Brunzell and Kim 2001; Shi and Davis 1999). We have hypothesized that differences in methods and the specific sensory modalities of the pIC in which they are focused may account for this discrepancy (Casanova et al. 2016). We recently reported that the interoceptive pIC is involved in the consolidation of auditory conditioned fear and that its reactivation is accompanied by an increase in pIC activity, evidenced as an enhanced expression of the early genes c-fos and zif268 (Casanova et al. 2016). Accordingly, a study conducted by Brydges et al. (2013) showed a marked increase in activity, measured by fMRI of the granular pIC in response to a fear-conditioned stimulus. However, the electrical activity pattern of the pIC during the expression of conditioned fear remains to be studied. In the present work, we aim to address the question of whether the behavioral expression and extinction of conditioned fear is accompanied by changes in pIC neural activity that may signal these states. We hypothesized that different populations of single units in the pIC could code high and low fear states. To this end, we carefully designed an experimental setup that allowed us to record the activity of pIC units during both fear states. We present evidence supporting a role for the pIC in the regulation and expression of fear memories. Notably, we found different sets of pIC units showing excitatory or inhibitory responses during either fear expression or extinction, which were correlated with freezing time course.

MATERIALS AND METHODS

Study design.

Our main goal was to study how the activity of single neurons in the pIC represents the expression of fear. To this end, we ran a protocol of auditory fear conditioning in rats. Then we simultaneously recorded fear behavior and single-unit activity from the pIC during extinction of conditioned fear.

Animals.

Male Sprague-Dawley rats weighing ~280 g were housed in individual cages, fed with standard chow diet and water ad libitum, and kept on a 12:12-h light-dark cycle. Room temperature was kept between 23°C and 25°C. All the experimental procedures were approved by the Bio-Ethical Committee from the Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, and they were performed according to the National Institutes of Healthʼs “Guide for the Care and Use of Laboratory Animals.”

Surgery.

Rats were deeply anesthetized with a mixture of ketamine 100 mg/kg ip (Imalgene; Rhodia Merieux, Santiago, Chile) and xylazine 20 mg/kg ip (Rompun; Bayer, Santiago, Chile) and were placed in a stereotaxic apparatus. A small craniotomy was made to target the left pIC according to the following coordinates: 0.51 mm posterior to bregma and 6.0 mm from the midline (Swanson 1998). An array of six independently movable custom-made tetrodes (wires of 17-µm diameter; Stablohm, California Fine Wire, Grover Beach, CA) was lowered into the pIC. The tip of each wire was gold-plated by passing a DC current of 1 µA measured at 1 kHz to reduce impedances to 1.5 MΩ. Immediately after surgery, rats received ketoprofen 0.2 mg/kg (Rhodia Merieux) and enrofloxacin 20 mg/kg ip (Bayer). Rats were allowed to recover for 6–7 days before manipulations. Tetrodes were slowly lowered to reach the pIC, placed ~4 mm under the dura (Swanson 1998).

Fear conditioning.

Rats were conditioned in a standard conditioning chamber (Harvard Apparatus Model LE1005; context A), equipped with a floor of steel rods to deliver electric foot shocks. Extinction and recordings took place in a different Plexiglas chamber of 64 × 38 × 30 cm (context B), situated in a different room, to rule out environmental cues, so conditioned responses were restricted to the auditory conditioned stimulus (CS). The training (day 1) consisted of presenting five habituation tones (CS; 80 dB, 5 kHz, 20 s), followed by a sequence of five tones that coterminated with a mild foot shock (0.5 mA, 0.5 s). Two minutes after the last CS-US pairing, rats were taken back to their home cages. On day 2, rats were placed into context B, where the CS was presented 15 times (extinction) while neural activity was recorded. Behavior was recorded using a webcam plugged to a computer, where videos were stored for analysis off-line.

Single-unit recording and spike sorting.

Tetrodes were connected to a headstage preamplifier containing 36 channels of unity-gain amplification (HS-36, Neuralynx). The headstage was connected to a 32-channel, computer-controlled system (four Lynx-8 amplifier units, Neuralynx). Signals exceeding a voltage threshold of two standard deviations from the noise floor were amplified (15.000×), bandpass filtered (0.6 kHz–6 kHz), sampled at 32 kHz, stored, and sorted off-line using MClust (A.D. Redish, University of Minneapolis, MN). Clusters formed by spikes of individual neurons were further analyzed using the custom MATLAB script KlustaKwik (Kadir et al. 2014). Using the extracted spikes, we reconstructed the spike trains across time. We calculated the interspike interval (ISI) as the time difference between two consecutive spikes. Clusters for which less than 1% of an ISI was less than 2 ms were declared multiunit; otherwise, they were declared single units. Recordings were performed on day 2.

Behavioral analysis.

Behavioral expression of fear was quantified as freezing, defined as the absence of movement except for breathing (Blanchard and Blanchard 1969). Videos were scored by an observer using a stopwatch. Data were expressed as a percentage of time spent in freezing during a tone. Freezing data were analyzed with repeated-measures ANOVA. The null hypothesis that the percentage of freezing during each tone was the same across conditioning was rejected with a P < 0.05. The same null hypothesis was tested during extinction.

Spike data analysis.

A total of 100 pIC units from nine successfully implanted rats was included in the analysis. To better understand the role of different cell types in the expression of freezing, we classified the set of pIC neurons into putative pyramidal and putative interneurons. Toward this end, peak to trough and repolarization at 460 μs after the peak were considered (Vinck et al. 2016).

We next analyzed modulation of firing rate (FR) of each neuron in response to the CS during the early extinction period, when freezing is at its highest, and during the late extinction period, when freezing is at its lowest level. Toward this end, FR was computed in 2-s bins from 20 s before to 20 s after CS onset.

To obtain a single time course of average FR for early extinction in each neuron, the first three conditioned stimuli were collapsed to calculate a mean FR across them.

The first three CS (early extinction) were collapsed to calculate a mean FR across them, obtaining a single time course of averaged FR for early extinction per unit. Next, we calculated an averaged FR and a standard deviation for the first 10 bins (20 s of baseline). We then z-scored the 20 bins to obtain a measure of variation in FR. Modulation of FR associated to the last 3 CS (late extinction) were measured in the same way.

Neurons with three or more bins above or below 1.96 SD (P < 0.05) after tone onset were classified as excited or inhibited by the CS, respectively, like analysis reported elsewhere (Burgos-Robles et al. 2009; Duvarci et al. 2011). Wilcoxon matched-pairs signed rank test was used to detect differences in units basal FR during early vs. late extinction.

The test of Pearson (GraphPad Prism software, La Jolla, CA) was used to establish the correlation between freezing behavior and the activity of pIC single units. For this analysis, bins of 5 s were used to measure the percentage of freezing during 20 s before and 20 s after CS onset. The FR was computed in 5-s bins with a custom MATLAB code. The null hypothesis that there was not dependence between freezing behavior and FR of pIC neurons was rejected with a P < 0.01 or P < 0.05 for grouped and individual units, respectively.

Histology.

Upon completion of experiments, rats were anesthetized, and electrolytic lesions were made by applying anodic current of 25 µA for 20 s through two wires in each tetrode and the animal tail. Two days afterward, rats were perfused transcardially with 500 ml of saline followed by 500 ml of 10% formalin. Brains were removed, left in postfixation in 10% formalin for 2 h, and then stored in 30% sucrose with 0.02% sodium azide in PBS until they sank. Brains were sectioned frozen under dry ice in the coronal plane, at 50-µm thickness, using a sliding microtome. The sections were stained with cresyl violet and examined by light microscopy to determine tetrode placement (Fig. 1A).

Fig. 1.

Fig. 1.

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Recordings from the posterior insular cortex of rats during expression of conditioned fear. A: left: photomicrograph of a Nissl-stained section; the electrolytic lesion is indicated with an asterisk. Scale bar: 500 μm. Right: anatomical drawing adapted from Swanson (1998) (with permission from the author). CP, caudate putamen; pIC, posterior insular cortex; rf, rhinal fissure. B: percentage of time rats spent in freezing during tone at different stages of the behavioral protocol. Gray and black solid circles indicate which conditioned stimuli were used to measure neuronal activity. Cond, conditioning; Hab, habituation. Data are expressed as means ± SE.

RESULTS

Fear conditioning and extinction.

On day 1, rats were conditioned to an auditory cue paired with a footshock in context A. Conditioned fear was rapidly acquired (Fig. 1B) and was evidenced as a significant increase in the percentage of time during tone spent in freezing (one-way ANOVA, F4,32 = 28.16, P < 0.0001). On day 2, rats were transferred to context B for extinction of conditioned fear, while neural activity was recorded. At the beginning of extinction, rats showed a high level of conditioned freezing, which was significantly reduced in response to subsequent tone presentations, evidencing the extinction of the conditioned fear (one-way ANOVA, F14,112 = 14.17, P < 0.0001).

Electrophysiological characterization and response of pIC neurons to CS during extinction training.

Recordings were carried out during extinction training. The median basal FR was 0.88 spikes/s, with a range between 0.1 and 10.33 spikes/s, which agreed with other studies (Hanamori 2005). The median action potential duration (peak to valley) of all recorded units was 0.4 ms. Distribution of action potential (AP) duration was bimodal (Hartigan dip test, P < 0.05), suggesting that two populations of units were recorded. In fact, distribution of units in AP duration versus repolarization time at 0.46 ms evidenced two clusters, which we designated as putative pyramidal (92 units) and putative interneurons (8 units; Fig. 2, A and B), as proposed elsewhere (Homayoun and Moghaddam 2007). Compared with putative pyramidal, putative interneurons showed fast hyperpolarization, shorter AP duration (P < 0.0001; Fig. 2C), a trend to lower mean interspike interval (ISI; P = 0.06, Fig. 2D) and higher basal FR (P = 0.086; Fig. 2E), with a significantly lower local variation (LV) in firing (P = 0.045; Fig. 2F). Taken together, these findings indicate that two different populations of neurons were obtained from pIC recordings and validate our pIC spike data set.

Fig. 2.

Fig. 2.

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Electrophysiological characterization of posterior insular cortex (pIC) neurons. A: scatterplot for action potential (AP) duration vs. waveform amplitude at 0.46 ms. Clusters were gray scale-coded and classified as putative pyramidal (gray) and putative interneurons (black). B: average waveform aligned at peak for each unit. Putative interneurons showed shorter AP duration (C) and lower firing irregularity (F) than putative pyramidal neurons, whereas no significant differences were observed in mean interspike interval (ISI) or firing rate (FR) (D and E). Bars represent means + SD; putative pyramidal units, n = 92; putative interneurons, n = 8. *P < 0.05.

Units were classified as excited or inhibited by the CS if their z-score was above or below 1.96 SD, respectively, in three or more bins following tone onset (see materials and methods). Using this criterion, we found that a discrete population of units (24 units, 24%; Fig. 3) showed significant modulation of FR, elicited by the CS during early extinction, which was significantly reduced to 11% during late extinction (Fig. 3, χ2 = 4.987; P = 0.0255), suggesting attenuation in response of pIC units as extinction progresses.

Fig. 3.

Fig. 3.

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The pattern of activity of the posterior insular cortex (pIC) changes with extinction training. Pie charts represent percentage of units showing increase, decrease, and no change in activity (see methods for criterion).

Next, we analyzed the response of each of these units in both stages to test whether units showing significant modulation in FR to CS during early extinction persist in their response during late extinction. We found that most units (22 out of 24), met our criterion for significant change in FR exclusively during early extinction and, therefore, were classified as freezing units. This population was composed of four putative interneurons and 18 putative pyramidal neurons. Accordingly, 9 out of 11 units showed significant modulation of FR exclusively during late extinction and, therefore, were classified as extinction units, all of which correspond to putative pyramidal neurons. Figure 4 shows the time course of FR from freezing and extinction units centered at CS onset during early and late extinction. Representative examples of single units’ response to CS are also shown. Basal FR of either fear (P = 0.85) or extinction units (P = 0.57) was not modified as extinction progresses (Fig. 4C). Taken together, these findings indicate that extinction training modifies the pattern of activity of pIC units and suggest that changes in pIC activity accompany the recall and extinction of auditory conditioned fear.

Fig. 4.

Fig. 4.

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Fear and extinction are codified by different sets of posterior insular cortex (pIC) units. A: heat plots showing units that respond with significant change in firing rate (FR) exclusively during early (top: freezing units) or late extinction (bottom: extinction units). B: representative response of two freezing units and one extinction unit. Top: raster plots representing spike trains during early and late extinction. Bottom: peristimulus time histograms for the same units, showing firing rate as z-score (2-s bins). Inset: average waveform for each unit. C: basal FR does not change for either freezing units (top) or extinction units (bottom) as extinction progresses. Bars represent means + SD.

Correlation between pIC activity and freezing time course.

Next, the relation between activity and freezing behavior was further analyzed for each unit. Freezing and neuronal activity of the pIC were computed in 5-s bins, 20 s before, and 20 s after CS onset. Intersubject variability regarding freezing response allowed us to explore activity-behavior relations for each unit recorded from the same rat. Unit-by-unit analysis revealed that 20 units have a significant correlation (either positive or negative) between freezing and activity (P < 0.05) during early extinction, from which 15 units were previously classified as freezing units. Representative examples of this effect are shown in Fig. 5.

Fig. 5.

Fig. 5.

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Activity of posterior insular cortex (pIC) neurons parallels freezing time course. Representative examples of single-unit activity-behavior correlations. Black line depicts firing rate (FR) during early extinction. Bars represent percentage of time spent in freezing during early extinction. Both, positive (top) and negative (bottom) correlations were observed.

Accordingly, the grouped activity of all freezing units parallels freezing time course, evidenced as a significant correlation between both variables (Fig. 6). The grouped activity of pIC freezing units classified as excited was positively correlated with freezing time course (r = 0.96; P = 0.0002), whereas inhibited units showed a negative correlation (r = −0.96; P = 0.0002). No significant correlation was observed for either excited (r = 0.70; P = 0.06) or inhibited (r = −0.81; P = 0.02) freezing units during late extinction. On the other hand, excited extinction units showed significant negative correlation with freezing during early extinction (r = −0.87; P = 0.005), but not during late extinction (r = 0.67; P = 0.07). No significant correlation was found for inhibited “extinction units” either during early (r = −0.45; P = 0.26) or late extinction (r = −0.65; P = 0.08). These findings further support the role of the pIC in the expression of conditioned fear.

Fig. 6.

Fig. 6.

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Activity of freezing and extinction units correlates with freezing during early but not late extinction. A: freezing time course during early (left) and late extinction (right) assessed in 5-s blocks. B: correlation between freezing time course (A) and average response to conditioned stimulus (CS) of posterior insular cortex (pIC) freezing (top) and extinction units (bottom) classified as excited and inhibited during early (left) and late extinction (right). Data are expressed as means ± SE. Freezing units: excited, n = 8; inhibited, n = 7. Extinction units: excited, n = 7; inhibited, n = 2.

In most freezing units (15 out of 22) and almost half of extinction units (four out of nine), changes in FR outlasted the duration of the CS, suggesting that these responses are related to fear expression rather than to an auditory response. Representative examples of freezing units showing this effect are shown in Fig. 7. Taken together, these findings suggest that pIC neural responses were attributable to emotional processing associated with the CS.

Fig. 7.

Fig. 7.

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Responses of posterior insular cortex (pIC) neurons are related to the emotional valence of the auditory conditioned stimulus (CS). Freezing and change in activity outlast the duration of the tone. The activity of three representative units, along with freezing time course during early extinction, is shown. Freezing (bars) was assessed in 5-s blocks and unit activity (black line) was plotted in 5-s bins.

DISCUSSION

The neural activity of the pIC during expression of conditioned fear had not been previously studied, thus, motivating the present work to explore the activity of pIC neurons underlying fear. We used single-unit recordings from the pIC during expression of conditioned fear in rats undergoing extinction training. We found that pIC neurons significantly modified their FR in the presence of the conditioned-fear stimulus. Also, this pattern of activity changes as extinction progresses; while a population of neurons modifies their FR during early but not late extinction (freezing units), another population modifies their activity only during late extinction (extinction units). Moreover, we found a population of pIC units whose activity is highly correlated with freezing time course. Taken together, these findings suggest that pIC activity is important for the regulation and expression of fear memories.

The IC has been described as a site for long-term storage of different memories, including conditioned taste aversion (Berman and Dudai 2001), object recognition (Bermudez-Rattoni 2014), and drug-related memory (Contreras et al. 2012). Also, it has been demonstrated that IC inactivation interferes with fear expression in both context (Alves et al. 2013) and auditory conditioned fear (Brydges et al. 2013). The use of single-unit recording in conditioned-fear rats has allowed us to measure the activity of pIC neurons during the behavioral expression of fear memory. In the present work, we found a greater proportion of units with significant changes in FR during high-fear states (early extinction) compared with low-fear states (late extinction). Changes in pIC activity during high-fear states may account for the recall of a fear memory that is stored in the pIC, which, in turn, allows its behavioral expression. On the other hand, units showing significant changes in activity only during late extinction might be signaling the development of new learning, i.e., the tone does not predict the aversive outcome, as has been observed for neurons of the amygdala (Herry et al. 2008).

The progressive decrease in conditioned freezing is accompanied by lower changes in FR, which may account for a modulatory role of the pIC in extinction of conditioned fear. This effect is consistent with previous findings showing extinction of conditioned taste aversion (Berman and Dudai 2001) and amphetamine-induced conditioned place preference (Contreras et al. 2007) by pharmacological manipulation of the IC. Supporting this idea, reactivation of conditioned fear is accompanied by an increase in Zif268 expression in the IC (Casanova et al. 2016), a protein crucially involved in neural plasticity (Chaudhuri and Zangenehpour 2002). These results suggest that pIC activity may also be involved in the formation and storage of extinction memory. Further experiments involving transient pharmacological inactivation or inhibition of protein synthesis following extinction are necessary to further understand this issue.

We found two units that showed significant responses during both early and late extinction. An auditory domain within the pIC has been described (Rodgers et al. 2008), which might explain the persistence in the response of pIC neurons to the tone. However, this seems unlikely since in both cases, the responses were sustained or even outlasted the duration of the tone in contrast to the rapid (short latency) and brief response to auditory stimulus reported previously in the insula (Kimura et al. 2010). On the other hand, persistence in response might represent the persistence in fear memory despite extinction training. It has been described that extinction of conditioned fear implies the formation of a new memory, which, in turn, competes with the previous one, and does not erase it (Dudai 2012). Neurons showing persistent firing despite extinction might be important for the persistence of fear memory, which can be reinstalled afterward (Bouton 2002).

Neurons of the IC can be excited or inhibited by a conditioned stimulus predicting the availability of food (Gardner and Fontanini 2014; Kusumoto-Yoshida et al. 2015). Here, we extended these observations to an aversive/negative emotion-related behavior since we found a population of pIC neurons that markedly increased or decreased their FR during the fear-conditioned stimulus presentation. Also, these changes were blunted as extinction progressed. Taken together, these findings suggest a modulation of fear expression by IC activity.

We previously suggested that the pIC may be involved in controlling the expression of conditioned fear. Accordingly, it has been previously shown with fMRI that activity of the pIC increases during the presentation of a fear-conditioned stimulus (Brydges et al. 2013). However, a description of single neuron activity-behavior relations was lacking. Taking advantage of recordings from awake-behaving rats and the duration of the CS, we were able to find pIC units whose activity was significantly correlated with conditioned freezing expressed by the rat. Correlations between pIC activity and freezing time course further support modulation of fear memory expression.

Modulation of behavior by IC activity in rats has been proposed on the basis of its connections: a strong visceral input from the thalamus (Cechetto and Saper 1987), along with key structures mediating conditioned-fear responses. The IC has strong connections with the periaqueductal gray (PAG) (Floyd et al. 2000), which is the main structure generating freezing. PAG, in turn, receives direct projections from the medial prefrontal cortex and from the central amygdala, both structures showing neural activity that parallels freezing behavior (Burgos-Robles et al. 2009; Duvarci et al. 2011). Moreover, the IC is connected to the central amygdala (Shi and Cassell 1998), which is also critical in mediating autonomic correlates of fear (LeDoux et al. 1988). This connectivity pattern posits the IC as a plausible site mediating autonomic and behavioral responses to CS (Cechetto and Saper 1987).

Although a detailed correspondence between the human and rat insula is still lacking (Craig 2009), it is worth mentioning evidence linking the activity of the insula with emotion perception. Evidence from fMRI shows that both an increase and decrease of IC activity can be observed when subjects remember different emotions (Damasio et al. 2000). Also, sustained activation of the IC is observed in subjects experiencing conditioned fear (Alvarez et al. 2008; Phelps et al. 2001). Accordingly, the continuous monitoring of the physiological condition of the body, exerted by the interoceptive system in the rat (Cechetto and Saper 1987; Hanamori 2005), suggests that the pIC is an important site for emotional processing, as several physiological changes accompany emotion. The integration of an emotionally competent stimulus and interoceptive information might be important for emotion perception, as proposed first by William James (Barbalet 1999).

In summary, we have found a population of pIC neurons showing a consistent pattern of activity during freezing behavior, which is modified as extinction progresses. Also, excitatory and inhibitory responses of pIC units were correlated with the time course of freezing behavior. These findings further support a role for the pIC in the regulation and expression of fear memories.

GRANTS

This study was financed by Anillo ACT-66, Fondecyt 1130042. J. P. Casanova was partially supported by the Millennium Nucleus Nu-MIND (NC16 130011) and Fondecyt Postdoctoral Grant 3170497. J. P. Casanova also thanks the International Society for Neurochemistry for a CAEN travel grant. T. P. Coleman was partially supported by National Science Foundation Center for Science of Information CCF-0939370 and National Institutes of Health Grant 1 R01MH-110514.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.P.C. and F.T. conceived and designed research; J.P.C. and M.A.R. performed experiments; J.P.C. and M.A.-R. analyzed data; J.P.C., M.A.-R., and F.T. interpreted results of experiments; J.P.C. prepared figures; J.P.C. drafted manuscript; J.P.C., M.A.-R., T.C., and F.T. edited and revised manuscript; J.P.C., M.A.-R., M.A.R., T.C., and F.T. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Dr. Dhakshin Ramanathan for providing comments on this manuscript.

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