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# A cognitive account of trace conditioning in insects

Marco Paoli, Catherine Macri, Martin Giurfa

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Marco Paoli, Catherine Macri, Martin Giurfa. A cognitive account of trace conditioning in insects. Current Opinion in Insect Science, 2023, 57, pp.101034. 10.1016/j.cois.2023.101034. hal-04125507

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# A cognitive account of trace conditioning in insects

Marco Paoli<fnref n="1" />,*, Catherine Macri<fnref n="1" />,\* and Martin Giurfa<fnref n="1" />,<fnref n="2" />

<figure><img src="image_5.png" /></figure>

Trace conditioning is a form of Pavlovian learning in which the conditioned stimulus (CS) and the unconditioned stimulus (US) are separated by a temporal gap. Insects learn trace associations of variable nature (appetitive, aversive) and involving CSs of different sensory modalities (olfactory, visual). The accessibility of the insect neural system in behaving animals allowed identifying neural processes driving trace conditioning: the existence of prolonged neural responses to the CS after stimulus offset and the anticipation of US responses during the free-stimulus gap. Specific brain structures, such as the mushroom bodies seem to be allocated to this learning form. Here, we posit that a further component facilitating trace conditioning in insects relates to neuromodulatory mechanisms underlying enhanced attention. We thus propose a model based on different types of mushroom-body neurons, which provides a cognitive account of trace conditioning in insects.

## Addresses

Corresponding author: Giurfa, Martin (martin.giurfa@univ-tlse3.fr)

**Current Opinion in Insect Science** 2023, 57:101034

This review comes from a themed issue on Neuroscience

Edited by Makoto Mizunami and Nobuhiro Yamagata

Available online 10 April 2023

https://doi.org/10.1016/j.cois.2023.101034

2214-5745/© 2023 The Authors. Published by Elsevier Inc.

## Introduction

Since Ivan Pavlov established the principles of Pavlovian conditioning [1], this learning form became one of the most systematically studied phenomena in the fields of psychology and neurosciences. Pavlovian learning is the process by which an association between an originally neutral-conditioned stimulus (CS) and a biologically relevant unconditioned stimulus (US) is established, leading to a novel conditioned response to the CS in anticipation of the US [2]. Many researchers view

Pavlovian conditioning as a 'simple' form of learning, distant from higher-order cognitive processes. Yet, Robert Rescorla, who devoted his life to the study of multiple aspects of Pavlovian conditioning, stated that "Pavlovian conditioning is not a stupid process by which the organism willy-nilly forms associations between any two stimuli that happen to co-occur. Rather, the organism is better seen as an information seeker using logical and perceptual relations among events, along with its own preconceptions, to form a sophisticated representation of its world" [3].

Cumulating evidence shows that some forms of Pavlovian conditioning may require higher-order cognitive processes, such as attention and awareness of the predictive relationship between CS and US. These arguments apply to trace conditioning, a form of learning in which the CS and the US are separated by a temporal gap [4] (Figure 1a). Trace conditioning is different from traditional Pavlovian conditioning (i.e. the so-called delay conditioning) in which the CS and US exhibit some degree of temporal overlapping. An example of this situation is provided by studies on eyeblink conditioning in humans [5]. In this protocol, a tone (CS) is presented just before a mild air puff directed to the eye (US), provoking an unconditioned eyeblink response (Figure 1b). After training, individuals exhibit a conditioned response to the tone that anticipates the air puff. Characterization of the neural structures mediating trace and delay eyeblink conditioning revealed the involvement of different substrates: whereas delay conditioning depends on the cerebellum and brainstem and remains intact in amnesia, trace conditioning depends also on the hippocampus [6,7] and neocortex and is impaired in amnesia [8]. Moreover, trace (but not delay) conditioning can be disrupted by a distractor provided during the gap separating the CS and the US. This result, reproduced also in mice [9], raises the question of whether trace conditioning may reveal the presence of attention and even forms of awareness or conscious processing [4,10,11] as necessary states to bridge the gap between the CS and the US.

This question can be extended to insects, which have acquired the status of useful models for the study of learning and memory [12–16] based on the existence of controlled learning protocols that allow dissecting the rules and mechanisms of these abilities at different scales. In spite of this status, analyses of trace conditioning in insects are scarce and have been limited to bees [17••], moths [18•], and fruit flies [19••–24••].

<footnote n="1">¹Centre de Recherches sur la Cognition Animale, Centre de Biologie Intégrative (CBI), University of Toulouse, CNRS, UPS, 31062 Toulouse cedex 9, France</footnote>

<footnote n="2">²Institut Universitaire de France (IUF), Paris, France</footnote>

<footnote>\*Equal contribution.</footnote>
<figure><img src="image_6.png" /><figcaption>Figure 1</figcaption></figure>

Delay and trace conditioning: experimental design. (a) In delay conditioning (left), the US is partially overlapping with the CS, whereas in trace conditioning (right), CS and US are separated by a stimulus-free temporal gap. The interstimulus interval is defined as the time between the CS onset and the US onset. (b) During eyeblink conditioning in humans [6], a tone (CS) is presented shortly before an air puff (US) eliciting a reflexive eyeblink. A successful association is assessed by the conditioned eyeblink to the tone alone. (c) During olfactory PER conditioning in honey bee, an odorant (CS) is presented shortly before delivering a sucrose solution reward (US). A successful association has occurred when the odorant alone can induce PER (photographs by R. Girling, C. Reitmayer) [27]. (d) During conditioning in an olfactory T-maze, flies are freely flying in a chamber in which odorants will be delivered. One odorant (CS+) will be followed by an aversive electric shock (US), the other will be presented alone (CS-). During a memory test, a performance index will be computed to assess the flies' odorant choice in the absence of the negative reinforcement [29]. (e) Visual conditioning in tethered flight developed by Grover et al. [23••]. During conditioning, flies are presented with two visual cues: one is negatively reinforced (CS+), the other one is not (CS-). A heat shock was used as negative reinforcement (US). Because flies were not allowed to move along the vertical axis, turning intentionality in response to the visual stimuli was calculated using the angular inclination of the wings.

Trace conditioning studies in insects

Olfactory trace conditioning in moths

Ito et al. [18•] investigated the neural representations of
odorants in the moth *Manduca sexta*. They focused on
the mushroom body (MB), a higher-order brain structure
associated with learning and memory [25,26], and re-
corded the activity of its constitutive neurons, the
Kenyon cells (KCs), upon olfactory stimulations of
variable duration. With this approach, they defined the
precise time window during which KC activity takes
place after reception of an odorant. Using this knowl-
edge, they conditioned moths during five trials and de-
termined if the coincident activation of the CS and US
pathways — here, KCs and sucrose reward, respectively

— leads to appetitive olfactory learning. Moths were able to do trace learning and the CS–US association did not require the temporal coincidence between CS- and US-induced spiking activity at the level of KCs. Thus, it was suggested that longer-lasting biochemical changes — for example, signaling pathways, second messengers, or temporal alternation of membrane electrical properties induced by the spiking activity — could mediate the CS and US association in trace conditioning [18•].

Olfactory trace conditioning in honey bees

Trace learning in honey bees was studied using the ol-
factory conditioning of the proboscis extension response
(PER) [27], an appetitive response triggered by the
contact of sucrose solution (US) with the antennae. Odorants used as CS can trigger PER after being paired with the US [27,28] (Figure 1c). Using different CS-US contingencies, Szyszka et al. [17••] showed that trace learning could be induced with a gap up to 6 s separating the CS and US. Performance was successful after one or six conditioning trials. Because both delay and trace conditioning elicit comparable memory retention and response generalization to novel odorants [17••], it was proposed that both learning processes may rely on similar molecular mechanisms. Adding an olfactory stimulus within the gap improved trace learning, suggesting that the new odorant may act as a contextual bridge allowing temporal coupling between the CS and the US [17••].

Coincidence between the CS and the US neural re-
presentations could be achieved by maintaining a trace
of the CS after its termination. Calcium imaging in the
antennal lobe (AL), the primary olfactory processing
center in the insect brain, revealed a CS-specific after-
odor activity in the projection neurons (PNs) conveying
the olfactory information from the AL to higher-order
brain centers, such as the MB. However, while PN re-
sponses *during* olfactory stimulations correlated with
behavioral responses (i.e. perceived odor quality), *post-
stimulus* activity did not coincide with odor similarity
relations deduced from behavioral tests. This suggested
that the PNs do not provide the neural substrate for an
olfactory trace, which could be located downstream in
the olfactory circuit [17••].

Alternatively, the associative link between a CS and a
late US could be promoted by the anticipation of US-
related neural activity to the delivery of the US itself.
This would require multiple conditioning trials to de-
velop and does not account for successful trace con-
ditioning after a single conditioning trial, which in the
case of PER conditioning, was enough to mediate
learning [17••]. Yet, in different animal species and trace
conditioning protocols, more trials may be required to
observe both the extension of CS-induced neural ac-
tivity and the anticipation of US-related activity [23••]
(see below).

Olfactory trace conditioning in fruit flies

Aversive olfactory trace learning in *Drosophila* was stu-
died using the traditional T-maze, in which groups of
freely moving flies are trained with an odorant (CS+)
paired with electrical shock (US) and with a different
odorant (CS-) delivered without shock [19**] (Figure
1d). During a memory test, flies that learned the dis-
crimination avoided the arm containing the punished
odorant [29]. Using this protocol, Galili et al. [19**]
found that flies could hold an aversive memory trace of a
punished 10-s odor presentation for up to 15 s after sti-
mulus termination. Although learning efficiency was

lower for trace conditioning than for delay conditioning,
in particular, when the number of conditioning trials
varied from 1 to 4, both protocols yielded a similar
learning rate and comparable memory retention and
generalization scores when more conditioning trials were
added (from 8 to 12) [19••]. These similarities suggested
that both delay and trace conditioning may rely on the
same neural representation of the CS, and that the de-
creased efficiency of trace learning depends on the
temporal decay of such representation during the gap.

*In vivo* calcium imaging of AL input neurons (i.e. the olfactory sensory neurons, OSNs) during odor stimulation revealed a persistent stimulus-specific post-odor activity, which was different from odor-induced activity. Yet, as in the case of bee PNs (see above) [17••], similarity profiles of post-odor OSN responses did not correlate with perceptual odor similarity profiles deduced from generalization tests. Thus, neither the post-odor activity observed in OSNs provided support for trace learning [19••].

A further study investigated the neural representation of
the CS and the US across MB compartments [20••].
During aversive trace conditioning, an odorant that in-
duces the activation of a specific population of KCs, was
paired with an electric shock, which activated dopami-
nergic neurons (DANs) projecting to the MB lobes.
Calcium imaging recordings of these neural populations
revealed that olfactory stimulation (CS) induced KCs
and DAN activity across multiple MB compartments,
while the electric shock (US) induced a specific strong
response in DANs, with almost no activity in KCs.
During trace conditioning, CS-induced activity in DANs
was enhanced, while KC responses were reduced in
comparison to untrained flies. These observations raised
the question of the molecular mechanisms allowing the
link between CS and US in a trace conditioning para-
digm. The rutabaga adenylyl cyclase (Rut-AC), which
acts as a molecular coincidence detector between CS and
US signaling [30,31], could be excluded as the in-
tracellular calcium concentration of CS-recruited KC re-
turns to baseline levels before US arrival. Thus, it was
suggested that CS-US coincidence detection in trace
learning may rely on alternative biochemical effectors,
for example, a non-Rut-AC or a protein kinase C [20••].

To investigate the molecular underpinnings of trace
learning, Shuai et al. [22•] combined aversive olfactory
conditioning and mutagenesis. By pairing an odorant
with an electric shock delivered simultaneously or sev-
eral seconds later in a single conditioning trial, they
confirmed that flies could associate an odor with a shock
in a trace conditioning paradigm. Then, they used mu-
tants to show that Rut-AC was not necessary for trace
learning (as opposed to its essential role in delay con-
ditioning), and that inhibiting the activity of the G-
coupled protein Rac in the MB, but not in the AL, enhanced trace conditioning [32]. These results support the MB as the neuroanatomical locus for trace learning. They also postulate the existence of a CS trace, which is both sensitive to Rac activity and Rut-AC-independent. Thus, it is possible that Rac inhibition enhances or prolongs the biochemical alterations triggered by the CS presentation and that unknown molecular detectors other than Rut-AC [21] mediate the association between the post-CS and the US neural signals. Other forms of signaling may be relevant for trace conditioning as besides cholinergic receptors, KCs have also slow muscarinic receptors, which could mediate long-lasting neural signaling after odor offset [33]. Similar results may be obtained *via* the release of the short neuropeptide F by KCs [34], which seems to act in a slower way and be less temporally confined than fast neurotransmitters.

## Visual trace conditioning in fruit flies

Trace conditioning in the visual modality was recently studied in fruit flies [23••]. This study focused on the question of whether distractors from a modality different from that of the CS may disrupt trace conditioning. This is a relevant question given that in mammals (see above [5,9,35]), the negative effect on learning performances of a distractor was interpreted as an indication of the presence of an attentional component, and even awareness or conscious information processing during trace learning [4,10,11]. If similar effects were found in the small brain of insects, questions about the existence of these processes would require an answer [36].

To study trace conditioning in the visual modality, Grover et al. [23••] conceived a protocol in which a tethered fly flies stationary without the possibility of rotating along its vertical axis. The fly is passively exposed to a negatively reinforced (CS+) or to a neutral/unpunished (CS−) visual cue for seven conditioning trials, and successively subject to a 1-min memory test (Figure 1e). As a measure of learning, the angle of inclination of its wings was used to score CS+ avoidance and CS− preference. The study shows that individual flies learned to associate the visual stimulus with a heat shock even when separated by 20 s (Figure 2b–e). This finding is consistent with a previous attempt to study the effect of stimulus timing on aversive color learning (pairing of a color with electric shocks) in groups of flies, which also used nonoverlapping stimulus presentations as in trace conditioning [37]. Similar to findings reported for mammals [5,9], an air puff distractor impaired trace but not delay conditioning. These results were further strengthened by applying a distractor from another modality (optogenetic activation of auditory sensory neurons) [23••].

Neuronal silencing experiments revealed that the ring neurons of the ellipsoid body [38], a subregion of the

central complex involved in visual processing, are necessary for both delay and trace conditioning. Calcium imaging analyses showed that both protocols increased the oscillatory activity of two types of ring neurons (R2 and R4m) (Figure 2b). Yet, only the oscillatory pattern induced by trace conditioning was more persistent and negatively affected by distractors. Thus, trace learning induces longer-lasting CS-specific neural activity, which may help bridge the gap between CS and US, and distractors impair trace conditioning by interfering with this long-lasting activity [23••].

Differences were also found in terms of US (heat) processing. Silencing and activating a cluster of DANs projecting to the central complex blocked and facilitated, respectively, both delay and trace conditioning. This effect is consistent with the role of these DANs in mediating the negative reinforcement. In addition, although DANs were initially recruited only by the heat shock (US), they acquired the capacity to respond also to the visual stimuli (CS) during the learning process, as predicted by the substitution principle of the Pavlovian theory [39] (Figure 2c–e). Yet, in trace learning, the visual CS induced a stronger and anticipated activation of DANs during the gap. Once again, this effect was sensitive to distractors, which thus affected both CS and US signaling pathways [23••]. This result shows that besides a prolongation of the CS neural signal, a neural anticipation of the reinforcement may also contribute to trace learning.

## A cognitive, mechanistic account of trace conditioning

The previous studies show that insects (moths, bees, and flies) learn CS-US associations in trace conditioning protocols of variable nature (appetitive, aversive) and with CSs of different sensory modality (olfactory, visual) despite the temporal separation between CS and US. Although differences in performances may depend on protocols and species-specific features [21], the demonstration that the neural processes underlying delay and trace conditioning are not always the same raises the question of the mechanistic difference between these two forms of learning.

Studies available uncovered two nonexclusive neural processes driving trace conditioning: the existence of prolonged neural responses to the CS after stimulus offset and the anticipation of the US response. Both effects may help bridging the CS-US gap. Neural analyses discarded OSNs and PNs as possible neural substrates of the olfactory stimulus trace [17••,19••,40]. Local interneurons in the AL — located upstream of PNs — also seem an improbable candidate for keeping the stimulus trace. Conversely, molecular evidence in fruit flies indicated that the MBs could host the CS trace
<figure><img src="image_7.png" /><figcaption>Figure 2</figcaption></figure>

A distractor negatively affects trace learning. (a) Human subjects were conditioned to six blocks of 20 trials of eyeblink trace conditioning. The blue dataset (n = 7) shows that subjects were able to learn the CS-US contiguity, while the presence of a distractor (red dataset, n = 9) negatively affected learning efficiency. Error bars show standard errors. (b) Trace conditioning induces an increasing and distraction-sensitive oscillatory component in ellipsoid-body ring neurons of fruit flies [23••]. The boxplot shows the oscillatory activity during the trace intervals at successive conditioning trials with (red dataset, n = 16) and without heat shock distractor (blue dataset, n = 16). (c-e) Calcium signal of ellipsoid-body-projecting DANs across conditioning trials. Response is initially US specific (e). However, trace learning induces an anticipation of DAN activity, which becomes active during CS arrival (c) and during the CS-US gap (d). This effect is predicted by the substitution principle of the Pavlovian theory and occurs independently of the presence of a distractor (blue dataset, no distractor, n = 5; red dataset, distractor, n = 5).

(a) Plots are adapted from Ref. [53]. Plots (b)–(e) adapted from Ref. [23••].

[20••,22••,32,41]. In addition, calcium imaging record-
ings revealed that post-odor responses of KC somata
carry information about the identity of the odor pre-
viously perceived, thus potentially serving as a basis for
association with a delayed US [40].

Distractors provided during the gap in a trace con-
ditioning protocol seem to have different effects ac-
cording to their sensory modality relative to that of the
CS. In olfactory trace learning, the presence of a dif-
ferent odorant within the gap improved learning [17••].
Importantly, the delivery of a second olfactory stimulus
after odor offset modifies the post-CS activity profile. To

understand the mechanisms leading to an improved
learning, it would be crucial to determine if the odorant
provided during the gap does not in fact elicit a neural
response that is closer to the CS representation, thus
facilitating associative trace learning. Alternatively, it
may be possible that, because of the temporal coherence
of the CS and the distractor, associative learning may
rely on the identification of the CS distractor as a single
olfactory entity, or on second-order association per-
formed between CS, distractor, and US. Conversely,
distractors of different modalities impair trace but not
delay conditioning [23••]. Increased attention may pro-
vide improved salience to a CS signal, which decays
<figure><img src="image_8.png" /><figcaption>Figure 3</figcaption></figure>

A quadripartite STDP model of fruit fly olfactory trace conditioning. (a) The model incorporates the KCs of the MBs and the MBONs as components of the CS circuit, a reinforcement dopaminergic signal from DANs acting on the synapses between KCs and MBONs to provide US instruction, and a neuromodulatory signal from a serotonergic (5-HT) DPM neuron [24••], which could represent an attentional component. s, synaptic strength; c, eligibility trace; d, dopamine signal. References in support of the connectivity model are indicated between square brackets. (b) Temporal coincidence between presynaptic decaying odor responses of KCs and MBON activity within a short temporal window of a few ms induces STDP, tagging the synapses where this coincidence occurs and generating an eligibility trace [43]. Synapses with such a trace would be further strengthened by the dopamine (DA) US signal from DANs coinciding with the residuals of the decaying eligibility trace, thus providing a basis for CS-US association. The serotonergic (5-HT) DPM neuron is reciprocally connected to KCs so that acetylcholine (Ach) release by KCs activates the DPM neuron, which in turn provides 5-HT inhibitory feedback to KCs [24••]. An increase of 5-HT released by the DPM neuron prolongs the coincidence window and helps to bridge the CS-US temporal gap. A prolongation of the eligibility trace enables its interaction with the DA reinforcement signal at a later time. Thus, enhanced attention could eventually favor trace learning.

Adapted from Refs. [21,24••,42].

during the gap, thus facilitating trace conditioning. It
may also explain why bees and flies that were success-
fully trained in a trace learning paradigm exhibit fa-
cilitated trace learning with longer and initially
unsolvable gaps [17••,19••]. Attentional networks may
thus provide modulatory mechanisms accounting for the
selective changes reported for both CS and US signaling.

Such neuromodulatory processes may change the sy-
naptic strength between the neural circuits mediating
CS and US representation. They can be seen as an ad-
ditional layer in learning models of spike timing-de-
pendent plasticity (STDP) that have been proposed to
account for the linkage of a CS neural signal and a de-
layed US signal [42]. In locusts, a well-studied species
with respect to the dynamics of neural responses in ol-
factory circuits, an STDP-based model of olfactory
learning posits that coincident activation of the KCs and
the mushroom-body output neurons (MBONs) may
provide the recruited synapses with an eligibility trace
[43]. The nature of such a trace remains evasive, but it
may rely on slow biochemical modifications of the sy-
naptic environment, which may prime the CS-recruited
synapses to be modified upon US signal arrival. Thus,
synapses with this trace are fit to be modulated by the

arrival of the US signal, which in fruit flies are mediated
by DANs [44,45], thus changing synaptic strength and
providing a basis for CS-US associations.

A quadripartite STDP model that incorporates a neuro-
modulatory attentional signal besides CS (KCs and
MBONs) and US elements (DANs) could modify the
length of the eligibility trace, acting as a regulator of the
CS-US coincidence time window, that is, the maximal
interval that allows these two stimuli to be associated in
Pavlovian learning. Such a model has recently been
corroborated in fruit flies [24••], where the nature of the
regulatory signal is provided by the serotonergic (5-HT)
dorsal-paired medial (DPM) neuron (Figure 3a) and can
modulate trace learning already after one or five con-
ditioning trials. In this quadripartite model, recruited
KCs release acetylcholine that activates locally the DPM
neuron. The DPM neuron releases in turn 5-HT that
binds to 5-HT1A receptors on KCs. These receptors are
coupled to the inhibitory Gαi pathway, which reduces
cAMP intracellular concentration and, as a consequence,
KC excitability. The modulation of adenylate cyclase
activity and of cAMP levels have been implicated in
learning in the MB [21,22]. DPM activity controls KCs'
tonic level of cAMP, providing a neural signature lasting
after CS offset [24••]. Although the molecular cascade
leading from 5-HT modulation of cAMP levels to trace
learning-induced plasticity remains unclear, serotonergic
neuromodulation helps bridging the CS–US temporal
gap and provides a first mechanistic explanation for as-
sociative links between a CS and a late US in trace
conditioning [24••]. The model provides a parsimonious
account in the case of a complex network rich in feed-
forward and feed-backward connections, for example, of
KC and MBON to DANs [46–48]. In terms of the STDP
model, extending the coincidence time window could
have the consequence of prolonging the eligibility trace
so that the reinforcement signal would interact with a
more salient trace (Figure 3b). Importantly, in this
model, there is no attempt at capturing the specific
nature of changes in synaptic strength. Although in fruit
flies the responses of MBONs to the CS decrease after
learning, suggesting CS-specific depression in recruited
synapses [49], no assumptions are made on this point in
the absence of quantifications of CS- and US-related
spike dynamics (which depending on their temporal
relationship may lead to potentiation or depression).

Interestingly, in reporting the action of the DPM neuron,
no mention to attentional processes was made [24••].
Natural variation in 5-HT signals from this neuron was
referred to variation in factors such as stress, sociality, and
aging, arguing that flies with different brain stages due to
these factors would have different coincidence time win-
dows [24••]. Yet, 5-HT plays a major role in the modula-
tion of behavior and cognition both in vertebrates and
invertebrates [50], enhancing attention and arousal at
multiple levels [51,52]. This is consistent with the fact that
upregulation of 5-HT levels increases the coincidence time
window and facilitates trace learning in fruit flies [24••]. In
other insect species and behavioral contexts, different
neuromodulators could participate to achieve similar ef-
fects, yet, the quadripartite model involving a neuromo-
dulatory attentional signal besides CS (KCs and MBONs)
and US elements (DANs) may still be maintained.

Overall, these considerations underline the fact that suc-
cessful trace conditioning in insects goes beyond simple
associative learning as it may recruit additional processes
on top of the CS and US neural signaling. While Hebbian
theory accounts for delay conditioning based on the co-
incident overlap between activation of CS and US neural
circuits, trace conditioning may require further compo-
nents leading to changes in these circuits to ‘fill the gap’.
The fact that cognitive processes such as attention or
awareness, relying on 5-HT signaling or on other forms of
neural signaling, might be involved in insect trace con-
ditioning, highlights the interest of this learning form to
study the complexity of insect minds.

Data Availability

No data were used for the research described in the ar-
ticle.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

We thank the ERC (European Research Council) for generous support through the Advanced Research Grant COGNIBRAINS (Grant agreement ID: 835032) to M.G.

**References and recommended reading**

Papers of particular interest, published within the period of review, have
been highlighted as:

• of special interest

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