Вы находитесь на странице: 1из 14

Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Neurobiology of Learning and Memory

journal homepage: www.elsevier.com/locate/ynlme

Neural representations of time-linked memory

Maryna Pilkiwa, Kaori Takehara-Nishiuchia,b,c,
Department of Cell and Systems Biology, University of Toronto, Toronto M5S 3G3, Canada
Department of Psychology, University of Toronto, Toronto M5S 3G3, Canada
Neuroscience Program, University of Toronto, Toronto M5S 3G3, Canada


Keywords: Many cognitive processes, such as episodic memory and decision making, rely on the ability to form associations
Time between two events that occur separately in time. The formation of such temporal associations depends on
Episodic memory neural representations of three types of information: what has been presented (trace holding), what will follow
Temporal association (temporal expectation), and when the following event will occur (explicit timing). The present review seeks to
Neural code
link these representations with firing patterns of single neurons recorded while rodents and non-human primates
Single-unit activity
associate stimuli, outcomes, and motor responses over time intervals. Across these studies, two distinct firing
Ensemble decoding
patterns were observed in the hippocampus, neocortex, and striatum: some neurons change firing rates during or
shortly after the stimulus presentation and sustain the firing rate stably or sidlingly during the subsequent
intervals (tonic firings). Other neurons transiently change firing rates during a specific moment within the time
intervals (phasic firings), and as a group, they form a sequential firing pattern that covers the entire interval.
Clever task designs used in some of these studies collectively provide evidence that both tonic and phasic firing
responses represent trace holding, temporal expectation, and explicit timing. Subsequently, we applied machine-
learning based classification approaches to the two firing patterns within the same dataset collected from rat
medial prefrontal cortex during trace eyeblink conditioning. This quantitative analysis revealed that phasic-
firing patterns showed greater selectivity for stimulus identity and temporal position than tonic-firing patterns.
Our summary illuminates distributed neural representations of temporal association in the forebrain and gen-
erates several ideas for future investigations.

1. Introduction linked memories. Parallel neurophysiological studies reported several

unique firing patterns of single neurons during intervals separating two
When you hear a ringtone for email notification, you expect to see a events. This review provides a concise summary of these studies with
new email on the screen of your phone. When you hear the sound of the aim of linking specific neuron firing patterns in a given brain region
skidding tires on a busy street, you feel a sense of dread for an imminent with the information types required for the formation of time-linked
car crash. When you see the tail lights of the car in front of yours go off, memory. In the following section, we will first outline several beha-
you prepare to release your break. All of these behaviors are the out- vioral paradigms used to study the association formed between stimuli,
comes of processes through which the brain formed association be- outcomes, and motor responses across time. A particular emphasis will
tween two arbitrary events that took place separately within a short be placed on their differences in the relative reliance on trace holding,
time window. The formation of these time-linked memories depends on temporal expectation, and explicit timing. Subsequently, we will review
neural representations of at least three types of information. First, the neurophysiological studies that characterized neuron firing patterns
brain must continue representing events after they have passed (trace during time intervals separating two events. We will highlight the
holding). Second, expectation signals need to be formed regarding studies whose clever design uncovers the selectivity of specific neural
which event would follow the first event (temporal expectation). Third, firing patterns for the three information types required for time-linked
the brain needs to represent the exact timing at which the subsequent memory. The subsequent section will expand the discussion with the
event would take place (explicit timing). result of quantitative decoding analysis applied to two largely non-
Over the past decades, a considerable number of studies has been overlapping groups of neurons with distinct firing profiles in rat medial
conducted to identify brain regions necessary for the formation of time- prefrontal cortex. We believe that the thorough survey of available

Corresponding author at: Department of Psychology, University of Toronto, Toronto M5S 3G3, Canada
E-mail address: takehara@psych.utoronto.ca (K. Takehara-Nishiuchi).

Received 29 October 2017; Received in revised form 29 March 2018; Accepted 30 March 2018
1074-7427/ © 2018 Elsevier Inc. All rights reserved.

Please cite this article as: Pilkiw, M., Neurobiology of Learning and Memory (2018), https://doi.org/10.1016/j.nlm.2018.03.024
M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

literature will contribute to (1) uncovering a potential functional dif- with monkeys (Murray et al., 1993). In contrast, rats with damage to
ference, or absence thereof, among the variety of neural firing patterns the dorsal hippocampus are able to acquire odor-odor associations;
observed during the interval between temporally discontinuous events however, they are not able to correctly respond to an odor from a pair
and (2) providing ideas for future experiments. when it is presented in a temporal order reversed from the one used
during training (Bunsey & Eichenbaum, 1996).
2. Behavioral paradigms used to study time-linked memory In a different version of the task, a cue object is presented for
1.2–3 sec, followed by a 10 sec delay and a presentation of a container
Various behavioral paradigms have been used to study mechanisms filled with scented sand (Kesner, Hunsaker, & Gilbert, 2005). If the
underlying time-linked memory. Based on their design, these paradigms scent was previously associated with the cue object, the animal must
can be categorized into three groups. In some paradigms, like delayed respond by digging in the sand to find a reward, or otherwise, it must
paired-associate tasks, associations are formed between sensory stimuli, withhold the response. Notably, the duration of a delay period in this
such as images, objects, or object-location pairs presented with a tem- task is ∼10 sec, which is longer than the primate version of the delayed
poral gap (stimulus-stimulus association). In others, like classical trace paired-associate task with visual stimuli (a few seconds). Rats with le-
conditioning, subjects are trained to associate a sensory stimulus with sions of the CA1 region of the dorsal hippocampus cannot form the
reward or punishment that occurs after a temporal delay (stimulus- association between an object and an odor stimulus over the 10-sec
outcome association). In another set of paradigms, such as conditional delay while lesions of the CA3 region do not have any effect on the task
visuomotor learning and interval timing tasks, animals need to form performance (Kesner et al., 2005). Notably, lesions of the CA3, but not
association between a sensory stimulus and a particular motor response CA1, impair the task performance when the cue and choice phases are
over a fixed time interval (stimulus-response association). This section not separated by the delay (Gilbert & Kesner, 2003), suggesting the
summarizes the design of these behavioral paradigms with an emphasis unique involvement of the CA1 region for trace holding, temporal ex-
on their differences in the degree of dependence on trace holding, pectation, or both.
temporal expectation, and explicit timing.
2.2. Stimulus-outcome associations
2.1. Stimulus-stimulus associations
Other commonly used paradigms to study time-linked memory are
One paradigm traditionally used to characterize neural firing pat- the trace paradigms of classical conditioning, such as trace eyeblink
terns underlying time-linked memory is a delayed paired-associate task. conditioning and trace fear conditioning. In these paradigms, a neutral
In the learning phase of this task, animals are repeatedly presented with conditioned stimulus (CS) is followed by an aversive stimulus that is
the pairs of arbitrary neutral stimuli. After the subjects have reached a presented after a temporal gap, called a trace interval. To associate the
behavioral criterion, their memory for associations is tested. First, one CS with the US, the brain must maintain the “trace” of the CS during the
stimulus from a pair is presented as a cue, followed by a delay period. temporal gap (Graves & Solomon, 1985; Kamin & Schaub, 1963;
Then, the animal is presented with a choice of two stimuli, one of which Pavlov, 1927). Before conditioning, the US induces a specific reflex
was previously paired with the cue and another one from a different response (unconditioned response, UR), whereas the CS does not. Re-
pair. To receive a reward, the subject must choose the stimulus which peated pairings of the CS and US build the association between them,
completes the pair. To successfully perform this task, animals must not resulting in the development of anticipatory behavioral responses that
only maintain the identity of the cue during the delay period (trace precede the onset of US (conditioned response, CR). The CR reaches the
holding) but also prospectively retrieve a stimulus that was paired with maximum intensity at the expected onset of the US, suggesting that
the cue during the learning phase (temporal expectation). This latter animals not only acquire the association between the CS and US but
requirement makes the paired-associate tasks different from working also the timing of the US presentation. Therefore, for successful
memory tasks, in which correct responses depend solely on the cue learning in the trace paradigms, animals must maintain the re-
maintenance over the delay period. The successful performance does presentation of the CS during the trace interval (trace holding), expect
not require the subject to track the duration of the delay period (explicit the upcoming US (temporal expectation), and estimate precisely when
timing) because the end of the delay period is explicitly signaled by the the US will be presented (explicit timing).
presentation of the choice stimuli. Trace fear conditioning presents footshock as the US after a
In primates, pairs of visual stimuli, such as unrelated color images of 20–30 sec delay from the offset of the CS (Marlin, 1981; McEchron,
scenes and objects, or monochrome geometric patterns (Fourier de- Bouwmeester, Tseng, Weiss, & Disterhoft, 1998). Rats, mice or rabbits
scriptors) are frequently used (Fujimichi et al., 2010; Higuchi & acquire the conditioned response, such as freezing and heart rate in-
Miyashita, 1996; Naya, Yoshida, & Miyashita, 2003, 2001; Rainer, Rao, crease, in one session that contains 5–16 CS-US pairings. The dorsal
& Miller, 1999; Sakai & Miyashita, 1991). In these studies, a monkey hippocampus plays a critical role in the acquisition and expression of
typically forms the paired association after a few hundred presentations the association, but its involvement is limited to the paradigm with a
per stimulus pair (Higuchi & Miyashita, 1996; Sakai & Miyashita, trace interval longer than 10 sec (Bangasser, Waxler, Santollo, & Shors,
1991). The duration of a cue period is 0.5–1 sec, followed by a delay 2006; Burman, Starr, & Gewirtz, 2006; Chowdhury, Quinn, & Fanselow,
period of 1–4 sec. Murray, Gaffan, and Mishkin (1993) showed that 2005; Misane et al., 2005). In the hippocampus-dependent version of
combined bilateral removal of the perirhinal and entorhinal cortices this task, many additional brain regions are involved in acquisition and
impairs the retrieval of previously acquired paired associates as well as retrieval. Those include the ventral hippocampus (Burman et al., 2006;
the acquisition of new associates. Yoon & Otto, 2007), prelimbic cortex (Gilmartin & Helmstetter, 2010;
In rodents, the delayed paired-associate task pairs two arbitrary Runyan, Moore, & Dash, 2004), anterior cingulate cortex (Han et al.,
odors (Bunsey & Eichenbaum, 1996) or an object and an odor 2003), perirhinal cortex (Kholodar-Smith, Boguszewski, & Brown,
(MacDonald, Lepage, Eden, & Eichenbaum, 2011). For example, when a 2008), entorhinal cortex (Esclassan, Coutureau, Di Scala, & Marchand,
rat has placed its nose in a port, a cue odor is presented for 0.75 sec, 2009), and amygdala (Gilmartin, Kwapis, & Helmstetter, 2012; Kwapis,
followed by a 0.5 sec delay with no stimuli, and another odor for Jarome, Schiff, & Helmstetter, 2011; Selden, Everitt, Jarrard, &
0.75 sec which is an associate of either the cue or a different odor. If the Robbins, 1991 but see Raybuck & Lattal, 2011). Moreover, the acqui-
second odor is paired with the cue, the rat has to approach a water port sition of trace fear memory is impaired by optogenetic inhibition of
to receive a reward. Combined lesions to the entorhinal and perirhinal projections from the medial entorhinal cortex (MEC) to the dorsal CA1
cortices before learning prevent rats from forming associations between region during the trace interval, but not during the CS (Kitamura et al.,
the odors (Bunsey & Eichenbaum, 1993), a result comparable to that 2014). Similarly, photoinhibition of the prelimbic region of the medial

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

prefrontal cortex during the trace interval, but not during the CS, pre- response (temporal expectation); however, the subject does not need to
vents rats from forming the CS-US association (Gilmartin, Miyawaki, estimate the elapsed time from the stimulus offset (explicit timing). The
Helmstetter, & Diba, 2013). These results suggest that the formation of acquisition of new sensorimotor associations is impaired by combined
CS-US association depends on the proper activity of the MEC-CA1 ablation of the hippocampus and subjacent cortex (Murray & Wise,
projections and the prelimbic region during the trace interval. 1996), the dorsolateral prefrontal cortex (Petrides, 1982), and the dis-
In trace eyeblink conditioning, the US is applied as an air-puff to the connection of these regions (Eacott & Gaffan, 1992; Gaffan & Harrison,
cornea or an electric shock near the eyelid. After 300–800 presentations 1988; Parker & Gaffan, 1998).
of the CS-US pairings over several days, animals form anticipatory Another version of stimulus-response association tasks, the interval
blinking responses whose intensity peaks at the expected timing of the timing task, requires subjects to learn the correct timing at which they
US (Moyer, Deyo, & Disterhoft, 1990; Weiss, et al., 1999). The duration need to initiate a motor response. A subject is presented with a cue
of trace interval is 250–500 msec in rats (Beylin et al., 2001; Weiss stimulus, such as a light or sound, and it must make a motor response
et al., 1999) and mice (Kishimoto, Kawahara, Mori, Mishina, & Kirino, no sooner than a determined time interval after the cue presentation
2001; Takehara, Kawahara, Takatsuki, & Kirino, 2002; Tseng, Guan, (usually 12–60 sec). With learning, the timing at which animals initiate
Disterhoft, & Weiss, 2004) and 250–750 msec in rabbits (Graves & the response comes to center around the expected timing of the reward
Solomon, 1985; Hoehler & Thompson, 1980; Solomon, Vander Schaaf, delivery. The acquisition of the well-timed response is impaired with
Thompson, & Weisz, 1986). Most mice and rats cannot form the CS-US lesions to the dorsal hippocampus (Tam & Bonardi, 2012; Yin & Meck,
association if the duration of the trace interval exceeds 250 and 500 ms, 2014), ventral hippocampus (Yin & Meck, 2014), medial frontal cortex
respectively (Tseng et al., 2004; Volle et al., 2016). Memory acquisition (Narayanan, Horst, & Laubach, 2006; Xu, Zhang, Dan, & Poo, 2014),
in trace eyeblink task requires an intact hippocampus (Beylin et al., and caudate putamen (Meck, 2006). Furthermore, pharmacological
2001; Moyer et al., 1990; Solomon et al., 1986), medial prefrontal inhibition of the D1 receptor activity within the medial prefrontal
cortex (Kronforst-Collins & Disterhoft, 1998; Takehara-Nishiuchi, cortex makes the timing of behavioral response less accurate and more
Kawahara, & Kirino, 2005; Weible, McEchron, & Disterhoft, 2000), variable (Narayanan, Land, Solder, Deisseroth, & DiLeone, 2012;
entorhinal cortex (Ryou, Cho, & Kim, 2001; Tanninen et al., 2015), Parker, Chen, Kingyon, Cavanagh, & Narayanan, 2014). In parallel,
perirhinal, postrhinal cortices (Suter, Weiss, & Disterhoft, 2013), med- optogenetic inhibition of prefrontal neurons expressing D1-receptors
iodorsal thalamus (Powell & Churchwell, 2002), caudate nucleus impairs well-timed behavioral responses, whereas optogenetic activa-
(Flores & Disterhoft, 2009), and cerebellum (Pakaprot, Kim, & tion during the waiting period improves the temporal accuracy of the
Thompson, 2009; Woodruff-Pak, Lavond, & Thompson, 1985). Notably, response (Narayanan et al., 2012).
a dysfunction of these forebrain regions does not impair delay eyeblink
conditioning in which the CS co-terminates with the US (Berger & Orr,
1983; Morrissey, Maal-Bared, Brady, & Takehara-Nishiuchi, 2012; 2.4. Difference and commonality in brain regions involved in paradigms
Schmaltz & Theios, 1972; Takehara-Nishiuchi et al., 2005). Also, within assessing time-linked memory
trace eyeblink paradigms, lesions to the entorhinal cortex do not impair
acquisition of CS-US association in rabbits if the trace interval is 300 ms The survey of available behavioral studies highlights remarkable
(Ryou et al., 2001). The rats with the hippocampal lesion can learn the similarity in the network structure underlying the formation of sti-
association if the trace interval is reduced to 50 ms (Walker & mulus-stimulus, stimulus-outcome, and stimulus-response associations
Steinmetz, 2008). These observations suggest that the forebrain regions over time. The hippocampus is required for all three types of associa-
are engaged in learning only when the association needs to be formed tions. In the case of trace conditioning, the necessity of the hippo-
across a sufficiently long time interval. campus for memory acquisition is contingent on the presence of suffi-
As reviewed above, trace fear and trace eyeblink conditioning share ciently long temporal gaps between two paired stimuli. These results
many common components in the underlying network structure. They, suggest that the hippocampus likely mediates trace holding, temporal
however, differ in the number of CS-US pairings required for the ac- expectation, or both, which are required for all paradigms. The pre-
quisition of CRs: animals are able to acquire CRs in trace fear con- frontal cortex (dorsolateral parts in primates and medial parts in ro-
ditioning with a few CS-US pairings while CR acquisition in trace dents) is implicated in trace conditioning, conditional visuomotor
eyeblink conditioning requires a few hundreds CS-US pairings. There learning, and interval timing tasks, in which neutral stimuli gain
are two factors that may account for this difference. Firstly, the US in emotional or motivational salience. Therefore, the prefrontal cortex
trace fear conditioning (footshock) is more aversive than the US in trace may play a role in the assignment of emotional or motivational values
eyeblink conditioning (mild electric shock to the eyelid or corneal air- to the stimulus that was presented a moment ago, which is a case of
puff). Secondly, CRs in trace fear conditioning, such as freezing re- temporal expectation. Delayed paired-associate tasks, on the other
sponses and heart rate changes, depend on the periaqueductal grey and hand, involve the formation of multiple associations between arbitrary,
lateral hypothalamus (LeDoux, Iwata, Cicchetti, & Reis, 1988) while behaviourally irrelevant stimuli. This particular task feature demands
CRs in trace eyeblink conditioning, such as the movement of eyelid and accurate discrimination between multiple perceptually similar visual
nictitating membrane depend on the cerebellum (Hu et al., 2010; stimuli or objects and precise maintenance of the stimulus identity over
Pakaprot et al., 2009; Woodruff-Pak et al., 1985). Plasticity in these the temporal delay. This heightened demand on trace holding may re-
brain regions may develop with different speed. late to the unique involvement of the inferior temporal cortices in these
paradigms. Lastly, among the memory paradigms reviewed above, the
2.3. Stimulus-response associations involvement of the cerebellum, caudate putamen, and dopamine system
is needed for trace eyeblink conditioning and interval timing tasks, but
Another set of paradigms requires subjects to link a sensory stimulus not for paired associates tasks or conditional visuomotor learning tasks.
with a specific motor response over a temporal interval. In one version The difference between the two sets of the paradigms is the dependence
of these tasks, primate conditional visuomotor learning tasks, subjects on the accurate estimation of the time elapsed after stimulus onset.
are trained to associate an arbitrary visual stimulus with a movement of These regions, therefore, may be uniquely involved in representations
the eye or hand in a particular direction. The subjects need to withhold and computations of explicit timing, as was previously proposed (Coull
the motor response until the fixation point disappears from the screen & Nobre, 2008; Ivry & Spencer, 2004; Petter, Lusk, Hesslow, & Meck,
after the fixed duration of a few seconds. Successful performance in this 2016).
task depends on the maintenance of the visual stimulus during the
fixation period (trace holding) or the preparation for the correct motor

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

3. Neural activity patterns observed during the paradigms of time- CS while the remaining 60% sustain CS-evoked firing rate only after
linked memory one of the two CS (Morrissey et al., 2017). Together, these results in-
dicate that persistent activity in the prelimbic region serves as a re-
By using various behavioral paradigms reviewed in the previous presentation of trace holding and temporal expectation.
section, a considerable number of studies characterized firing patterns During interval timing tasks, on the other hand, gradual, ramping
of single neurons during intervals between sensory stimuli, outcomes, changes in firing rates are reported in the anterior cingulate cortex
and motor responses. Collectively, these studies reported two types of (Emmons et al., 2017; Matell, Meck, & Nicolelis, 2003; Parker,
firing patterns during the interval between events. One is the tonic Ruggiero, & Narayanan, 2015; Parker, Chen, Kingyon, Cavanagh, &
firing responses to the first event which are initiated during or after the Narayanan, 2014) and the prelimbic region of the mPFC (Xu et al.,
event, persisting into the subsequent interval until the presentation of 2014) in rats. When the duration of the interval is modified, the slope of
the second event. The other is phasic firing changes that occur during a ramping firing patterns is adjusted accordingly so that the firing rate of
specific moment within the inter-event interval. Below we review stu- each neuron reaches the maximum at the new expected end of the in-
dies separately for each of the two firing patterns and further divide terval (Emmons et al., 2017; Xu et al., 2014). Classification analysis
them based on the recording locations. A special focus is placed on the demonstrated that the exact timing within the interval can be decoded
studies that allow for inferring the types of information represented by from population activity of neurons with ramping firing patterns, but
these firing patterns. not from neurons without the ramping patterns (Emmons et al., 2017),
providing evidence that the slope of ramping firing patterns represents
3.1. Tonic firings explicit timing.

3.1.1. Prefrontal cortex 3.1.2. Inferior temporal cortex

During working memory paradigms some neurons in monkey dor- Persistent firings are also reported in the inferior temporal (IT)
solateral prefrontal cortex change firing rates in response to a stimulus cortex in primates during delayed paired-associate tasks. The IT cortex
and sustain the firing rate during a subsequent interval until the sti- can be divided into the Areas 35, 36 (a.k.a., perirhinal cortex) and Area
mulus information is no longer needed (Fuster & Alexander, 1971). TE, all of which contain neurons that show selective firing patterns to
Similar persistent firing patterns are observed in the lateral prefrontal specific visual stimuli (for reviews, see Rolls, 2000; Tanaka, 1996).
cortex during the interval between the cue and target stimulus in the During the delayed paired-associate task with 24 arbitrary visual sti-
delayed paired-associate tasks (Rainer et al., 1999). The study utilizes a muli, the majority of IT neurons show the strongest firing rate increase
unique design which interleaves delayed paired-associate (DPA) trials to one of these stimuli (“preferred stimulus,” perceptual selectivity). In
with delayed-match-to-sample (DMS) trials. Both types of the trials well-trained monkeys, these neurons also show a weaker phasic re-
include different images as the cue but share the same image as the sponse to a stimulus paired with the preferred stimulus when it was
target. About 25% of prefrontal neurons differentiate firing rates during presented as a cue (pair selectivity, Naya et al., 2003, 2001; Sakai &
the cue-target interval between DPA and DMS trials, suggesting that Miyashita, 1991). Following the initial phasic response, some of these
these neurons maintain the representation of the image used as the cue. neurons gradually increase firing rates and maintain the stable rate
In parallel, ∼30% of neurons show a comparable firing rate between until their preferred stimulus is presented as a target stimulus (Naya
the two trial types, suggesting that these neurons prospectively signal et al., 2003, 2001; Sakai & Miyashita, 1991). In Area 36, the difference
the image to be presented as the target moments later. Toward the end in persistent firing responses across 24 stimuli is correlated with the
of the interval, ensemble firing patterns come to signal the upcoming perceptual and pair selectivity at the equal strength (Naya et al., 2003,
image more strongly than the preceding image, suggesting the priority see also Erickson & Desimone, 1999). In contrast, in Area TE, the firing
from trace holing to temporal expectation. Neurons selective for the differences are correlated with the pair selectivity more strongly than
identity of the cue or target were also found in the lateral prefrontal the perceptual selectivity. These patterns suggest that while persistent
cortex during the conditional visuomotor learning task in which each firings in Ares 36 represent the cue and target stimuli with comparable
image is associated with a certain direction of the eye movement strength, those in the TE are more selective for the impending target
(Asaad, Rainer, & Miller, 1998; Pasupathy & Miller, 2005). Collectively, stimulus than the cue stimulus. In addition, the pair, but not perceptual,
these findings suggest that neurons in the lateral prefrontal cortex re- selectivity of TE neurons is attenuated by lesions of the perirhinal and
present trace holding as well as temporal expectation for impending entorhinal cortex (Higuchi & Miyashita, 1996), suggesting that the pair
sensory stimuli and movement. selectivity of TE neurons is driven by the activity of the rhinal regions
In rats and rabbits, persistent firings are observed in the medial while their perceptual selectivity is likely driven by early visual areas
prefrontal cortex (mPFC) during trace eyeblink conditioning (Hattori, (see also, Naya, Yoshida, & Miyashita, 2001; Takeda, Naya, Fujimichi,
Yoon, Disterhoft, & Weiss, 2014; Siegel, 2014, 2016; Siegel, Kalmbach, Takeuchi, & Miyashita, 2005).
Chitwood, & Mauk, 2012; Siegel & Mauk, 2013; Siegel et al., 2015; An interesting contrast is found in the selectivity of neurons in the
Takehara-Nishiuchi and McNaughton, 2008) and trace fear con- perirhinal cortex during the conditional visuomotor learning task, in
ditioning (Baeg et al., 2001; Gilmartin & McEchron, 2005). Typically, which monkeys associate an image with one of four directions of sac-
neurons show abrupt changes in firing rate during the conditioned cade responses (Yanike, Wirth, Smith, Brown, & Suzuki, 2009). About
stimulus (CS) and sustain the firing rates during subsequent intervals 68% of neurons with image-evoked firing responses are selective for the
until the unconditioned stimulus (US) is presented. These sustained identity of the image. In contrast, virtually no neurons show a com-
firing patterns are observed in animals that received paired presenta- parable response to two images associated with the same rewarded
tions of the CS and US, but not animals that received unpaired pre- saccade direction. Collectively, these findings suggest that neurons in
sentations (Gilmartin & McEchron, 2005; Hattori et al., 2014). More- the IT cortex represent trace holding and temporal expectation, but its
over, in the trace eyeblink conditioning, ∼25% of neurons in the contribution to temporal expectation appears to be limited to im-
prelimbic region of the mPFC maintain CS-evoked firing changes during pending sensory stimuli.
the subsequent interval only when the CS is predictive of the upcoming
US (Takehara-Nishiuchi & McNaughton, 2008). Furthermore, when rats 3.1.3. Hippocampus
are trained to form the association between an auditory CS and eyelid In primates conditional visuomotor learning tasks, a sizable pro-
shock in parallel to the association between a visual CS and the same portion of neurons in the hippocampus change firing rates during the
eyelid shock, ∼40% of persistent-firing neurons show comparable presentation of the cue, the subsequent cue-response interval, or both
firing rates during CS-shock intervals regardless of the modality of the (Wirth et al., 2003; Yanike, Wirth, & Suzuki, 2004). The majority of

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

these neurons differentiate cue-evoked firing rates depending on the et al., 2017; Xu et al., 2014), the slope of ramping pattern changes
identity of the stimulus (∼70%) while the proportion of neurons dif- depending on the interval duration. Notably, the ramping firing
ferentiating firing rates for the specific saccade direction is small changes in MSNs are attenuated when the mPFC is pharmacologically
(∼10%; Wirth et al., 2003; Yanike et al., 2004). These patterns raise a inactivated (Emmons et al., 2017), suggesting that the explicit timing
possibility that cue-evoked firing rates in the primate hippocampus play signal in the striatum depends on the integrity of the mPFC.
a more important role for trace holding than temporal expectation. In
parallel to these cue-responding neurons, other neurons gradually 3.2. Phasic firing responses
change firing rates during the cue-response interval regardless of the
identity of the cue or response (Sakon, Naya, Wirth, & Suzuki, 2014). 3.2.1. Hippocampus
These observations, along with similar incremental firing patterns ob- Past neurophysiological investigations of the rodent hippocampus
served during a temporal order paradigm (Naya & Suzuki, 2011), sug- primarily focused on the characterization of neurons which fire at a
gest that the primate hippocampus contains a specialized class of particular location within the environment (Hartley, Lever, Burgess, &
neurons that signal explicit timing and that these neurons exist in O’Keefe, 2014; Moser, Kropff, & Moser, 2008). In the past ten years, a
parallel to neurons representing trace holding and temporal expecta- growing number of studies has characterized the selectivity of hippo-
tion. campal neurons for the temporal information. In rat working memory
Similar stimulus-evoked firing patterns are also reported in the paradigms, some neurons in the hippocampus phasically fire during a
hippocampus of rabbits and cats during trace eyeblink conditioning specific moment within the delay period between the sample and re-
(Hattori, Chen, Weiss, & Disterhoft, 2015; McEchron & Disterhoft, sponse phases (MacDonald, Carrow, Place, & Eichenbaum, 2013;
1997; McEchron, Weible, & Disterhoft, 2001; Munera, Gruart, Munoz, Pastalkova, Itskov, Amarasingham, & Buzsáki, 2008; Salz et al., 2016).
Fernandez-Mas, & Delgado-Garcia, 2001). The proportion of CS-re- MacDonald et al. (2011) extended the finding to time-linked memory
sponding neurons is higher in animals which received pairings of the CS by using a delayed paired associate task, in which rats associate objects
with corneal air-puff (US) over a fixed interval than in animals which with odor stimuli over a 10-sec interval. About half of individual neu-
received the presentation of the CS and US with a random interval. rons in the CA1 region of the dorsal hippocampus show a phasic in-
Although these patterns suggest that some neurons in the hippocampus crease in firing rates during a specific moment within the interval
signal temporal expectation, it is important to note that hippocampal period. Because the exact timing at which each of these neurons
neurons show similar firing responses even in delay eyeblink con- changes the firing rate varies, the group of these “time cells” forms a
ditioning which does not include the temporal separation of the CS and sequence firing pattern spanning the entire interval. The selectivity for
US. Neurons in the hippocampus of rabbits robustly increase firing rates a specific moment within the interval is preserved even after removing
upon the CS when it co-terminates with the US but not when the CS is the influence of head direction, speed, distance traveled and rat’s po-
presented alone (Berger, Alger, & Thompson, 1976; Berger, Laham, & sition analytically (MacDonald et al., 2011) or experimentally (Kraus,
Thompson, 1980; Berger & Thompson, 1978). As a group of neurons, Robinson, White, Eichenbaum, & Hasselmo, 2013). When a different
the temporal profile of firing responses closely models the amplitude object is presented, ∼31% of time cells change the magnitude or
and time course of conditioned responses. These findings suggest that temporal pattern of firing rates during the subsequent interval, sug-
the CS-evoked firing rate changes are selective for CS-US associations, gesting that these neurons retrospectively signal the identity of the
thereby providing further support for the role of the stimulus-evoked object presented a moment ago (MacDonald et al., 2011). In parallel,
firings in the hippocampus in representations of temporal expectation. when the duration of the interval is abruptly altered, about half of time
The hippocampus, however, appears to play this role even when two cells cease to fire or become active when they were previously inactive
events are not separated by an intervening temporal gap. (MacDonald et al., 2011). In parallel, a small proportion of time cells
(5%) shifts the temporal firing position relative to the new duration of
3.1.4. Striatum the interval while the remaining time cells do not change firing pat-
During the conditional visuomotor tasks, some neurons in the pri- terns, suggesting that these neurons signal relative and absolute time
mate striatum (putamen and caudate nucleus) show phasic responses to elapsed from interval onset, respectively. These patterns suggest that
the cue while others show tonic responses that persist into the cue- the sequential firings of time cells represent trace holding and explicit
response intervals (Brasted & Wise, 2004; Pasupathy & Miller, 2005; timing.
Tremblay, Hollerman, & Schultz, 1998). The majority of neurons with A question remains open as to whether hippocampal sequential
persistent firings do not differentiate tonic firing responses depending firing patterns also signal temporal expectation. In a task similar to the
on the direction of reaching response until immediately before monkeys one used in MacDonald et al. (2011), rats’ performance is significantly
execute the correct response (Brasted & Wise, 2004). With learning, this attenuated when the medial entorhinal cortex is optogenetically in-
direction-selectivity comes to appear in an earlier part of the delay hibited for several seconds in the middle of the inter-event interval
period (Pasupathy & Miller, 2005), suggesting that striatum neurons (Robinson et al., 2017). The same manipulation disorganizes the se-
develop the selectivity for temporal expectation with learning. quential firing patterns in the CA1 region during the interval, but it
Similar stimulus-evoked firing patterns are also observed while does not affect CA1 neuron selectivity for objects or locations within the
rabbits receive trace eyeblink conditioning. Some putative medium environment. Further evidence for the link between the hippocampal
spiny neurons (MSNs) in the caudate nucleus increase firing rates sequential firing pattern and temporal expectation comes from firing
during the CS, the subsequent interval, or both (Flores & Disterhoft, sequences of hippocampal neurons during trace eyeblink conditioning
2009, 2013). The proportion of these CS-responding neurons is higher (Modi, Dhawale, & Bhalla, 2014). The two-photon calcium imaging of
in rabbits which received paired presentations of the CS and US than CA1 neurons shows that as immobilized mice associate an auditory
those which received unpaired presentations. Similarly, during an ap- stimulus (CS) and an air-puff to the eye (US) over a 250-msec interval,
petitive version of classical trace conditioning, some putative MSNs individual neurons become active for ∼100 msec at a various temporal
differentially respond to the CS depending on whether it is predictive of position within the interval. Prior to learning, individual neurons show
reward or not (Bakhurin, Mac, Golshani, & Masmanidis, 2016). Col- calcium responses at apparently random moments within the interval.
lectively, these findings provide additional evidence that links MSN After learning, however, neurons come to reliably show calcium re-
persistent firings with temporal expectation. sponses at fixed time-points within the CS-US interval. The enhanced
In contrast, during interval timing tasks, MSNs show a ramping reliability of calcium responses is related to the CS-US association be-
increase or decrease of firing rates over 3–40 sec intervals (Emmons cause it is not observed in mice which receive unpaired presentations of
et al., 2017; Matell et al., 2003). Like neurons in the mPFC (Emmons the CS and US or those which do not acquire the CS-US association.

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

Collectively, these findings provide evidence that along with their role rats increased the frequency of anticipatory blinking responses that
in trace holding and explicit timing, hippocampal sequential firing occurred immediately before the expected onset of the US (CR%,
patterns also serve as a representation of temporal expectation. Fig. 1B) in the block of CS-US paired trials, but not in the block of CS
alone trials. By using chronically implanted array of tetrodes, we col-
3.2.2. Striatum lected firing patterns of total 2077 neurons across four rats.
Phasic firing changes are also reported in the striatum during an To screen neurons which sustained CS-evoked firing responses to
interval timing task, in which a reward becomes available 2.5 sec after the subsequent interval, the difference in averaged firing rates during
the offset of an odor stimulus (Bakhurin et al., 2017). Like hippocampal CS-US intervals and pre-CS periods (900–400 ms before CS onset) was
neurons, some putative medium spiny neurons change firing rates calculated in each neuron. The same procedures were repeated 1000
during a specific moment within the stimulus-reward interval and as a times by randomly assigning each firing rate to either the CS-US or the
group, they form a sequential firing pattern that covers the entire in- pre-CS period to estimate the distribution of firing difference at chance.
terval. With a group of these neurons, machine learning classification The firing rate difference with the correct period label was considered
algorithms are able to decode time elapsed from the onset of the odor as significant when it fell within the 5% upper tail of the chance dis-
stimulus. They are also able to decode when mice would start antici- tribution. We separately applied this analysis to firing patterns during
patory licking responses to receive the reward. These findings suggest the auditory CS-US pairings and those during the visual CS-US pairings
that phasic-firing responses in the striatum encode the temporal in- to screen “persistent-firing” neurons in each trial block.
formation of stimuli as well as behavioral expression of memory. Si- The above mentioned criteria would overlook neurons which tran-
milar patterns are also observed in a paradigm in which rats need to siently changed firing rates during a specific phase of the CS-US inter-
estimate when the reward becomes available after they have collected vals due to the averaging of firing rates across the entire duration of CS-
the reward in a previous trial (Mello, Soares, & Paton, 2015). When the US intervals. To screen these “phasic-firing” neurons, we quantified the
duration of the interval is altered within a range from 12 to 60 sec, degree of firing rate changes within the CS-US interval by using mutual
∼68% of striatum neurons maintain the selectivity for the temporal information as a measure. In each neuron, firing rates during a CS-US
position relative to the duration of the interval, thereby maintaining interval were binned into five 100-msec time bins in each trial. The
their ordinal position in time across the neuron population. This result firing rate in each time bin across all trials was binned into 10 bins to
contrasts with the very small proportion of hippocampal neurons se- describe the probability distribution. Mutual information was calcu-
lective for relative timing (∼5%; MacDonald et al., 2011), raising a lated as follows:
possibility that the sequential firing patterns in the two regions re-
present explicit timing differently. Specifically, in the striatum, each P (i,j ) ⎞
Mutual information = ∑ P (i,j ) ∗log ⎛⎜ ⎟

neuron maintains a stable ordinal position in time and scales the firing i,j ⎝ P (i) P (j ) ⎠
duration relative to the duration of the interval. In the hippocampus, on
the other hand, the ordinal position of each neuron is flexible, resulting where P(i,j) is the joint probability distribution of time bin “i” and firing
in orthogonal sequential patterns for different interval duration. rate “j”, P(j)is the marginal probability distribution of firing rates,
averaged across time bins, and P(i) is the marginal distribution of firing
4. Distributed representations of associations between temporally rate in time bin “i”. To assess the significance of selectivity, permutation
discontiguous events in prefrontal neural ensembles tests were performed for each neuron using the exact same procedure as
above, after assigning randomized labels to each time bin. This proce-
The across-study comparisons conducted in the previous section dure, repeated 1000 times, yielded the distribution of the chance level
illuminate some similarities and differences in the selectivity of tonic of mutual information values. An observed mutual information value
and phasic firing patterns. Yet, the source of these differences is difficult with the correct time bin labels was considered as significant when it
to decipher because these studies were conducted in different para- fell in the 5% upper tail of its corresponding chance distribution. We
digms and brain regions. By using a decoding approach with machine separately applied this analysis to firing patterns during the auditory
learning algorithms, we here compare the selectivity for trace holding, CS-US pairings and those during the visual CS-US pairings to screen
temporal expectation, and explicit timing between tonic and phasic “phasic-firing” neurons in each trial block.
firing patterns within the same dataset recorded from the prelimbic To quantify the selectivity of neuron ensembles for the identity of
regions of the medial prefrontal cortex (mPFC) while rats received trace the CS (trace holding), the CS-US relationship (temporal expectation),
eyeblink conditioning (Morrissey et al., 2017). This quantitative ana- and the temporal position within the CS-US interval (explicit timing),
lysis uncovers that although both firing patterns are selective for all we used a machine learning algorithm, a Support Vector Machine
three types of information, phasic firing patterns outperform tonic (SVM) classifier (Chang & Lin, 2011; Cortes & Vapnik, 1995). SVM
firing patterns in trace holding and explicit timing. classification was performed in MATLAB (Mathworks, Natick, MA,
USA) with the algorithms from the open source LIBSVM library (Chang
4.1. Data analysis & Lin, 2011). The classifier constructed a model with the Gaussian ra-
dial basis function kernels. To maximize the decoding accuracy, two
The present study analyzed action potentials of individual neurons SVM parameters, cost and gamma, were identified by performing a grid
recorded from the prelimbic region of the mPFC while four male Long- search over a range of values, using the firing rates from all available
Evans rats received two epochs of trace eyeblink conditioning trials for a given target value. Then, a new population firing rate matrix
(Morrissey et al., 2017). Details of the materials and methods for data was generated from a subset of trials in the original population firing
acquisition are found in our previous publication with this dataset matrix. These trials were randomly drawn, without replacement, from
(Morrissey et al., 2017). Briefly, daily recording sessions included two all trials in a given trial block. Then, in each neuron, the firing rate in
epochs of trace eyeblink conditioning in which a neutral conditioned each trial was divided by the maximum firing rate of the neuron among
stimulus (CS, 100 msec, auditory or visual stimulus) was paired with a all selected trials. All of the trials except one trial were then used to
mild electric shock near the eyelid (US, 100 msec) which was presented train the SVM classifier with the parameters selected by the grid search.
500 msec after the offset of the CS. In each epoch, the CS was presented This was done by using a 5-fold cross-validation procedure to minimize
by itself during the first 20 trials (CS alone trials) while the CS was over-fitting. The one trial was then used to test the decoding accuracy
paired with the US during the latter 80 trials (CS-US paired trials; after training. The accuracy was defined as a proportion of correct
Fig. 1A). Intervals between trials were pseudorandomized between 20 predictions out of all test trials. Note that the neurons were recorded in
and 40 sec. Over the course of two weeks of conditioning sessions, the separate sessions from four rats, and thus we ignored any correlated

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

Fig. 1. Behavior and single neuron firing patterns during trace eyeblink conditioning. A. Daily recording sessions included two epochs of trace eyeblink conditioning
with either an auditory or visual stimulus as the conditioned stimulus (CS). In the first 20 trials, the CS was presented alone while in the latter 80 trials, the CS was
paired with mild electric shock near the eyelid (US). Two epochs took place in an identical conditioning chamber. B. Over the course of two weeks of daily
conditioning, four rats gradually increased the frequency of anticipatory blinking responses (CR%, mean ± s.e.m.) in trials of paired presentations of the CS and US
(red, blue) but not in trials of CS alone presentations (pink, cyan). C–E. Representative raster plots and peri-stimulus time histograms (1-ms bins, smoothed with a 50-
ms Hanning window) of firing responses to the auditory CS (red) or the visual CS (blue) during CS-US paired trials. Some neurons changed firing rates during the CS
and stably sustained the firing rate until the US was presented (C). Other neurons show a transient increase in firing rates during a specific phase of the CS-US interval
(D). Other neurons show ramping increase/decrease of firing rates over the CS-US interval (E).

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

activity between neurons. firing” neurons was comparable between the ACS-US paired block and
To quantify the selectivity of ensemble firing patterns for the tem- the VCS-US block, and ∼34% of them passed the criteria for both of the
poral position within CS-US intervals, we followed the strategy used in two CS. These neurons maintained CS-evoked firing increase or de-
Bakhurin et al. (2017). Each run of multiclass SVM analysis used a set of crease stably until US onset (Neurons 1–4; Fig. 1C).
200 randomly subsampled cells (total 20 runs). We chose this sample In parallel, 449 neurons (21.6%) were classified as significantly
size because classification accuracy reaches asymptote with 200 neu- changing firing rates across five time bins during the CS-US interval
rons or greater (Morrissey et al., 2017). Population firing rate vectors (Table 1). Most of these “phasic-firing” cells showed bi-phasic firing
were constructed by concatenating five 100-msec binned firing rates of rate changes, with an initial dip followed by a sharp, transient increase
each neuron during CS-US intervals (200 neuron × 5 time bins × 80 of firing rates (Fig. 1D). Some neurons increased firing rates im-
trials). All trials except one trial were used as training trials (200 mediately after the offset of the CS (Neurons 5, 9) while others in-
neurons × 5 time bins × 79 trials), and classifier was tested on the creased firing rates in the middle of CS-US intervals (Neurons 6, 10, 11).
remaining trial (200 neurons × 5 time bins × 1 trial). This procedure Others showed a sharp increase of the firing rate toward the expected
yielded 400 predictions (5 time bins × 80 trials). To quantify the ac- onset of the US (Neurons 7, 8, 12). The proportion of these neurons with
curacy of predictions, we calculated Pearson correlation coefficient (r) temporally modulated firing patterns was comparable between the
between 400 predictions and their corresponding true temporal posi- ACS-US paired block and the VCS-US block; however, most of these
tions. These procedures were applied separately to four different cells were judged to be selective during either one of the CS types
neuron types, phasic-firing neurons in the auditory CS-US pairings, (Table 1).
phasic-firing neurons in the visual CS-US pairings, persistent-firing A small proportion of neurons (∼7%) passed the criterion for the
neurons in the auditory CS-US pairings, and persistent-firing neurons in persistent and phasic firings. These neurons showed a steep ramping of
the visual CS-US pairings. Because the r values distributed normally firing rate decrease (Neurons 13, 14) or increase (Neurons 15, 16)
(Lilliefors test), their difference between phasic- and persistent-firing during CS-US intervals. In a subsequent decoding analysis, these neu-
neurons were examined with t-test. rons were included to both persistent- and phasic-firing neurons. This
To quantify the selectivity of ensemble firing patterns for the CS-US allowed for increasing the number of phasic-firing neurons usable for
relationships, we randomly selected 20 trials out of 80 CS-US paired the decoding analysis, which made it reach the minimum number of
trials and constructed a population firing rate matrix by concatenating neurons required for stable decoding performance (Morrissey et al.,
five 100-msec binned firing rates of each neuron during CS-US intervals 2017).
(200 neuron × 5 time bins × 20 trials). We also constructed a popu-
lation firing rate matrix of five 100-msec firing rates of the same set of 4.2.2. Selectivity of neuron ensembles for CS identity, CS-US association,
neurons during CS alone trials (200 neurons × 5 time bins × 20 trials). and temporal position within CS-US intervals
To quantify the selectivity for CS-US relationships, SVM classifiers were Similar to time cells in the hippocampus (MacDonald et al., 2011;
trained to discriminate CS-US paired trials from corresponding CS alone Modi et al., 2014), each phasic-firing cell reached the peak firing rate at
trials. To quantify the selectivity of ensemble firing patterns for the various time points spanning across the entire CS-US interval (Fig. 2A,
modality of the CS, SVM classifiers were trained to discriminate audi- left). On the other hand, most persistent-firing cells reached the peak
tory CS-US paired trials from visual CS-US paired trials. In both cases, firing rate either during the CS or the first 100-msec period immediately
all trials except one trial were used as training trials (200 neurons × 5 after CS offset (Fig. 2B, left). In both cases, the order of the peak firing
time bins × 39 trials), and classifier was tested on the remaining trial timing became disorganized during the corresponding interval when
(200 neurons × 5 time bins × 1 trial). This procedure yielded 200 the same CS was presented alone (middle) and during the interval when
predictions (5 time bins × 40 trials). This procedure was repeated the other CS was paired with the US (right).
twenty times, each of which used 200 sub-sampled neurons. Because To test whether the differentiation of sequential ensemble firing
classification accuracy was not distributed normally in most cases patterns between the trial types was robust at the single-trial level, we
(Lilliefors test), we used the Wilcoxon signed-rank test to test whether used a Support Vector Machine (SVM) classifier to decode the temporal
classification accuracy was better than chance (i.e., 50%). Also, the position within the trace interval, the identity of the CS (ACS-US or
Wilcoxon ranked sum test was used to test whether classification ac- VCS-US paired trials), or stimulus relationships (CS-US paired or CS
curacy significantly differed between persistent- and phasic-firing alone trials) from binned ensemble firing patterns. With a group of
neurons as well as between the CS identity and CS-US relationship. phasic-firing neurons, classifiers were able to decode the first bin almost
perfectly, whereas the accuracy dropped in the subsequent bins
4.2. Results (Fig. 2C, left). Most inaccurate classifications were due to mis-
classifications of an adjacent time bin, suggesting that ensemble firing
4.2.1. Firing profiles of single neurons patterns were similar between two time points closest in time. In con-
Among 2077 neurons recorded across four rats, 1248 neurons trast, with a group of persistent-firing neurons, misclassifications spread
(60.1%) were classified as having significant firing rate difference across time bins regardless of temporal positions of the bins (Fig. 2C,
during the CS and subsequent interval relative to those during the right), suggesting weaker relationships between ensemble similarity
period before CS onset (Table 1). The proportion of these “persistent- and the temporal proximity of the time bins. In both phasic- and per-
sistent-firing neurons, the predicted time bin was positively correlated
Table 1 with the correct time bin (Fig. 2D). The degree of correlation, however,
Number and percentage of CS-selective neurons with persistent and phasic was significantly higher in phasic-firing neurons than in persistent-
firing patterns. Table summarizes the number of neurons with persistent and firing neurons (t test, ps < 0.001, in both ACS-US and VCS-US trials, 20
phasic firing responses in auditory (A), visual (V) CS-US paired trial, or both. SVM runs with 200 randomly subsampled neurons). These results
The values in parentheses show the percentage of neurons to the total number suggest that phasic-firing neuron ensembles showed stronger selectivity
of recorded neurons. for the temporal position within the CS-US interval than persistent-
ACS-US VCS-US Both Total recorded firing neuron ensembles.
neurons We then compared the selectivity of two neuron types for CS
identity and CS-US relationship. In both phasic- and persistent-firing
Persistent 1034 922 (44%) 708 (34%) 2077
neurons, classification accuracy for the stimulus identity and relation-
neurons (50%)
Phasic neurons 243 (12%) 268 (13%) 62 (3%) 2077 ship was significantly better than chance (i.e., 50%) in all five 100-msec
bins during the ACS-US and VCS-US intervals (Fig. 2E; Wilcoxon signed-

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

(caption on next page)

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

Fig. 2. Ensemble firing patterns and their selectivity for task variables and temporal positions within CS-US intervals. A. The firing patterns of all identified neurons
with phasic-firing responses during the visual CS-US paired trials. Each row represents the normalized firing rate of one neuron over the duration from the onset of
the visual CS to the onset of the US, and the order of neurons presented is determined by the timing at which each neuron reached the maximum firing rate (peak
time, left). The same order was used to depict firing patterns of the same set of neurons during visual CS alone trials (middle) and auditory CS-US paired trials (right).
B. The firing patterns of all identified neurons with persistent-firing responses during the visual CS-US paired trials, as shown in A. C. Confusion matrices visualizing
SVM classifiers’ performance on the decoding of the temporal position within the CS-US interval from ensembles of phasic- (left) and persistent-firing neurons (right).
Each row represents the proportion of predictions for firing patterns during the corresponding correct time bins (y-axis). D. Correlation coefficients (mean ± s.e.m.)
between predicted and correct time bins across 20 classification runs with 200 randomly subsampled neurons. ***, p < 0.001, t-test. E. Classification accuracy for
binary discrimination between CS-US paired trials versus CS along trials (red, blue) as well as between auditory versus visual CS trials (pink, cyan). Each dot displays
classification accuracy observed in one of 20 classification runs with 200 randomly subsampled neurons, and lines connect their median. ***, **, *, p < 0.001, 0.01,
0.05, between two classification types, Wilcoxon Rank-Sum test.

rank test, α = 0.05/15 comparisons, all ps < 0.001). In phasic-firing of neurons in the orbitofrontal cortex and striatum during the interval
neurons, classification accuracy for stimulus identity was greater than timing task with a short interval (1.5 sec, Bakhurin et al., 2017). On the
that for stimulus relationship during the first 100-msec period after CS other hand, another firing pattern associated with explicit timing
offset (Wilcoxon Rank-Sum test, ACS-US and VCS-US paired trials, during interval timing tasks, ramping firings (Emmons et al., 2017;
ps < 0.002). The pattern became reversed in the middle of the CS-US Matell et al., 2003) were observed in less than 10% of neurons in our
interval, and during the last bin, classification accuracy for the CS-US dataset (i.e., neurons passing the criteria for both persistent and phasic
relationship became greater than that for the CS identity in ACS-US firings). This may be due to the difference in the duration of time in-
trials (p = 0.0004). A similar trend was found during VCS-US trials tervals between trace eyeblink conditioning (< 1 sec) and interval
(p = 0.027). Such time-dependent changes in the selectivity were not timing tasks used in these studies (from 3 to 20 sec), raising a possibility
observed in persistent-firing neurons (all ps > 0.003). The comparison that the mPFC neurons may use different firing profiles to encode in-
of the selectivity between two neuron types revealed that during the tervals of milliseconds from those of seconds.
first 100-msec bin, the selectivity for the CS modality was higher in A strong selectivity for the CS identity and the upcoming US was
phasic-firing neurons than persistent-firing neurons (Wilcoxon Rank- observed in both persistent-firing and phasic-firing neurons, and the
Sum test, p < 0.001 in ACS-US trials, p = 0.002 in VCS-US trials) degree of selectivity was mostly comparable between the two groups of
while the selectivity for the CS-US relationship was comparable be- neurons. Notably, phasic-firing neurons differed from persistent-firing
tween the two neuron types in all time bins (ps > 0.003). These results neurons in that they changed the relative strength of selectivity for
suggest that phasic-firing neuron ensembles showed stronger selectivity these features during the CS-US interval. Specifically, during the early
for the identity of the CS during the early phase of the trace interval phase of the CS-US interval, phasic-firing neurons showed greater se-
than persistent-firing neuron ensembles while the selectivity for the CS- lectivity for the identity of the CS than the upcoming US while their
US relationship was comparable between the two groups of neurons. selectivity for the upcoming US became stronger towards the end of
trace interval. Although a similar shift in the selectivity was reported in
4.3. Discussion persistent-firing patterns in the lateral prefrontal cortex in monkeys
during a delayed paired associate task (Rainer et al., 1999), persistent-
By applying two different screening criteria to the group of pre- firing neurons in our dataset did not show this coding property. The two
frontal neurons, we identified two largely non-overlapping populations studies differ in many aspects, including the difference in the duration
of neurons, one with persistent-firing responses and the other with of the time interval between the task used (0.5 msec versus 1 sec), the
phasic-firing responses during the intervals between the CS and US brain region, and species. Therefore, future studies are needed to fur-
(trace intervals) in trace eyeblink conditioning. Similar to time cells in ther examine the time-dependent shift in the selectivity of neuron ac-
the hippocampus (MacDonald et al., 2013, 2011; Modi et al., 2014; tivity during inter-event intervals.
Pastalkova et al., 2008; Salz et al., 2016), phasic-firing cells reached the Collectively, these results suggest that neurons in the medial pre-
maximum firing rate at various time points during the trace interval, frontal cortex, regardless of their firing profiles, play a role in all three
thereby forming a sequential firing pattern that covered the entire critical processes underlying the association formation between sti-
duration of the CS-US interval. These neurons formed a different se- mulus and punishment over a temporal gap. Phasic-firing patterns,
quential firing pattern when the same stimulus was presented by itself however, showed a better selectivity for the task features important for
and when a different stimulus was paired with the same punishment. trace holding and explicit timing, raising the possibility that for the
These findings suggest that in parallel to neurons showing the well- encoding of short time intervals (less than one second), population
established persistent firing patterns (Hattori et al., 2014; Siegel & coding in phasic-firing neuron ensembles may have a computational
Mauk, 2013; Siegel et al., 2012; Takehara-Nishiuchi & McNaughton, advantage over rate coding in individual persistent-firing neurons.
2008), other neurons in the mPFC show phasic firings selective for a
specific moment during the interval between temporally discontiguous 5. Concluding remarks
To compare the contribution of phasic- and persistent-firing neurons Memories of daily experiences consist of complex spatial and tem-
to trace holding, temporal expectation, and explicit timing, we em- poral sequences of events. Compared to well-documented neural re-
ployed SVM classification analysis to quantify their selectivity for the presentations of the spatial information (Hartley et al., 2014; Moser
identity of the CS, the CS-US relationship, and the temporal position et al., 2008), our understanding of neural representations of the tem-
within the trace interval. The strong selectivity for these features was poral information is still developing. The present review aimed to
found in both persistent- and phasic-firing neurons, suggesting that provide a concise summary of single neuron firing patterns observed
both of these neuron types contribute to trace holding, temporal ex- while rodents or non-human primates associate stimuli, outcome, and
pectation, and explicit timing. Phasic-firing neurons showed stronger responses across a temporal gap. Our survey included careful con-
selectivity for the temporal position than persistent-firing neurons, sideration of the experimental design used in each study which allowed
which was expected due to the criteria used for selecting phasic-firing for deciphering the selectivity of each neuron firing pattern for the
neurons (i.e., significant firing rate variations across five time bins content of preceding events (trace holding), the content of following
during the CS-US interval). This observation is also consistent with the events (temporal expectation), and the temporal position within inter-
successful decoding of the elapsed time from sequential firing patterns event intervals (explicit timing). Across the various behavioral

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

Table 2
Neuron firing patterns and brain regions supporting trace holding, temporal expectation and explicit timing in time-linked memory tasks. Task refers to a particular
paradigm used in each study. Duration refers to the delay between two stimuli or stimulus-response used in each task.
Pattern Task Duration (sec) Species Region Citation

Trace holding Persistent Visuomotor learning 0.5 Monkey Lateral prefrontal Asaad et al. (1998)
Phasic Paired associate 10 Rat Hippocampus MacDonald et al. (2011)
Persistent Trace eyeblink 0.5 Rat Medial prefrontal Morrissey et al. (2017)
Persistent Paired associate 2 Monkey Area 36 Naya et al. (2001)
Persistent Paired associate 2 Monkey Area TE Naya et al. (2001)
Persistent Paired associate 2 Monkey Area 36 Naya et al. (2003)
Persistent Paired associate 2 Monkey Area TE Naya et al. (2003)
Persistent Paired associate 1 Monkey Lateral prefrontal Rainer et al. (1999)
Persistent Paired associate 4 Monkey Inferior temporal Sakai and Miyashita (1991)
Persistent Visuomotor learning 0.7 Monkey Hippocampus Wirth et al. (2003)
Persistent Visuomotor learning 0.7 Monkey Hippocampus Yanike et al. (2004)
Persistent Visuomotor learning 0.7 Monkey Perirhinal Yanike et al. (2009)

Temporal expectation Persistent Visuomotor learning 0.5 Monkey Lateral prefrontal Asaad et al. (1998)
Persistent Trace conditioning 1.5 Mouse Striatum Bakhurin et al. (2016)
Persistent Trace eyeblink 0.5 Rabbit Striatum Flores and Disterhoft (2009)
Persistent Trace eyeblink 0.5 Rabbit Striatum Flores and Disterhoft (2013)
Persistent Trace fear 20 Rat Medial prefrontal Gilmartin and McEchron (2005)
Persistent Trace eyeblink 0.5 Rabbit Medial prefrontal Hattori et al. (2014)
Phasic Paired associate 10 Rat Hippocampus MacDonald et al. (2011)
Persistent Trace eyeblink 0.5 Rabbit Hippocampus McEchron and Disterhoft (1997)
Persistent Trace eyeblink 0.5 Rabbit Hippocampus McEchron et al. (2001)
Phasic Trace eyeblink 0.25 Mouse Hippocampus Modi et al. (2014)
Persistent Trace eyeblink 0.5 Rat Medial prefrontal Morrissey et al. (2017)
Persistent Paired associate 2 Monkey Area 36 Naya et al. (2001)
Persistent Paired associate 2 Monkey Area TE Naya et al. (2001)
Persistent Paired associate 2 Monkey Area 36 Naya et al. (2003)
Persistent Paired associate 2 Monkey Area TE Naya et al. (2003)
Persistent Visuomotor learning 1 Monkey Dorsolateral prefrontal Pasupathy and Miller (2005)
Persistent Visuomotor learning 1 Monkey Striatum Pasupathy and Miller (2005)
Persistent Paired associate 1 Monkey Lateral prefrontal Rainer et al. (1999)
Persistent Paired associate 4 Monkey Inferior temporal Sakai and Miyashita (1991)
Persistent Trace eyeblink 0.5 Rat Medial prefrontal Takehara-Nishiuchi and McNaughton (2008)

Explicit timing Phasic Interval timing 1.5 Mouse Striatum Bakhurin et al. (2017
Phasic Interval timing 1.5 Mouse Orbitofrontal Bakhurin et al. (2017)
Persistent Interval timing 3, 12 Rat Medial prefrontal Emmons et al. (2017)
Persistent Interval timing 3, 12 Rat Dorsal striatum Emmons et al. (2017)
Phasic Paired associate 10 Rat Hippocampus MacDonald et al. (2011)
Persistent Interval timing 10, 40 Rat Striatum Matell et al. (2003)
Persistent Interval timing 10, 40 Rat Medial prefrontal Matell et al. (2003)
Phasic Interval timing 12, 24, 36, 48, 60 Mouse Striatum Mello et al. (2015)
Phasic Trace eyeblink 0.25 Mouse Hippocampus Modi et al. (2014)
Persistent Visuomotor learning 0.7 Monkey Hippocampus Sakon et al. (2014)
Persistent Interval timing 1.5, 2.5 Rat Medial prefrontal Xu et al. (2014)

paradigms used in neurophysiological studies, two distinct firing pat- The survey highlights remarkable similarity among neural firing
terns, tonic and phasic firings, were observed during intervals separ- patterns observed during the inter-event interval whose duration ranges
ating two events (Table 2). Tonic firing patterns in the prefrontal, in- from milliseconds to seconds. In parallel, it also illuminates some dif-
ferior temporal cortices, and hippocampus are selective for the content ferences in the selectivity for trace holding, temporal expectation, and
of preceding and following events, suggesting their role in representa- explicit timing between firing patterns within each brain region as well
tions of trace holding and temporal expectation. This coding property is as between brain regions. Below we list several unresolved points that
observed in paradigms in which the duration of the interval ranging need to be addressed in future studies.
from 0.5 sec to a few seconds. In parallel, tonic firings in the primate
hippocampus, rodent medial prefrontal cortex, and striatum signal ex- 1. Potential functional differences between tonic- and phasic-firing
plicit timing by scaling the slope of firing rate changes over inter-event patterns need further investigation. Our discussion regarding this
intervals depending on their duration. The coding property was ob- point is limited because most studies only report neurons with either
served during the interval ranging from 1.5 to 12 sec. of these firing patterns. This is likely due to the typical step of neural
In parallel, a group of neurons with phasic firings form a sequential activity analysis, in which investigators construct a screening cri-
firing pattern spanning the entire interval ranging from 0.25 to 10 sec. terion for neurons with a specific firing pattern of interest. Given the
Sequential firing patterns in the hippocampus form orthogonal patterns co-existence of tonic- and phasic-firing neurons in some brain re-
selective for the identity of preceding and following events as well as gions (see, Sections 3 and 4), it is important to search for neurons
the duration of the interval. In contrast, sequential firing patterns in the with various firing patterns by applying several screening criteria to
striatum encode the different duration of intervals by scaling each a single dataset. This allows for directly comparing the selectivity
neuron’s firing duration while maintaining the ordinal position of its for various task features between neurons with different firing
firing timing within the intervals. In the medial prefrontal cortex, the profiles, leading to a richer description of neural representations of
sequential firing pattern is observed in parallel to persistent firings, and the temporal information (see Diehl, Hon, Leutgeb, & Leutgeb, 2017
they outperform persistent firings in the selectivity for explicit timing for successful application of this approach to spatial representations
and trace holding, but not for temporal expectation. in the medial entorhinal cortex).

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

2. Another point that requires further investigation is the influence of authors thank Dr. Mark Morrissey for collecting single neurons firing
motor output on neuron firing patterns. In trace conditioning, ani- patterns used for the data analysis in Section 4.
mals initiate behavioral responses in the middle of intervals between
the stimulus and outcome. In fact, in some cases, the temporal References
profile of firing patterns appears to model the temporal pattern of
behavior (e.g., Fig. 1D). Even in conditional visuomotor learning and Asaad, W. F., Rainer, G., & Miller, E. K. (1998). Neural activity in the primate prefrontal
interval timing tasks, in which animals are trained to withhold any cortex during associative learning. Neuron, 21, 1399–1407.
Baeg, E. H., Kim, Y. B., Jang, J., Kim, H. T., Mook-Jung, I., & Jung, M. W. (2001). Fast
behavioral responses during the interval, they may internally plan spiking and regular spiking neural correlates of fear conditioning in the medial
the action (Namboodiri & Shuler, 2014) and even change their body prefrontal cortex of the rat. Cerebral Cortex, 11, 441–451.
position to a very small degree which cannot be detected without Bakhurin, K. I., Goudar, V., Shobe, J. L., Claar, L. D., Buonomano, D. V., & Masmanidis, S.
C. (2017). Differential encoding of time by prefrontal and striatal network dynamics.
close monitoring (Cowen & McNaughton, 2007). Uncovering whe- Journal of Neuroscience, 37(4), 854–870.
ther observed neural responses are cause or consequence of beha- Bakhurin, K. I., Mac, V., Golshani, P., & Masmanidis, S. C. (2016). Temporal correlations
vioral manifestation of memory is notoriously difficult: neuron among functionally specialized striatal neural ensembles in reward-conditioned mice.
Journal of Neurophysiology, 115, 1521–1532.
firing patterns selective for memory should be correlated with
Bangasser, D. A., Waxler, D. E., Santollo, J., & Shors, T. J. (2006). Trace conditioning and
movement reporting memory expression. One case that successfully the hippocampus: The importance of contiguity. Journal of Neuroscience, 26,
tackled this challenge is an elegant study by Siegel and Mauk 8702–8706.
Berger, T. W., Alger, B., & Thompson, R. F. (1976). Neuronal substrate of classical con-
(2013). By recording single neuron firings from the medial pre-
ditioning in the hippocampus. Science, 192, 483–485.
frontal cortex while rabbits received trace eyeblink conditioning, Berger, T. W., Laham, R. I., & Thompson, R. F. (1980). Hippocampal unit-behavior cor-
they found that persistent firing patterns in some, but not all, pre- relations during classical conditioning. Brain Research, 193, 229–248.
frontal neurons are abolished with the inactivation of the cere- Berger, T. W., & Orr, W. B. (1983). Hippocampectomy selectively disrupts discrimination
reversal conditioning of the rabbit nictitating membrane response. Behavioural Brain
bellum which supresses the motor output (i.e., conditioned eyelid Research, 8, 49–68.
responses). Thus, although some persistent firing patterns are an Berger, T. W., & Thompson, R. F. (1978). Neuronal plasticity in the limbic system during
efferent copy of eyelid movement, others are likely related to tem- classical conditioning of the rabbit nictitating membrane response. I. The hippo-
campus. Brain Research, 145, 323–346.
poral stimulus association formed as a result of the conditioning. Beylin, A. V., Gandhi, C. C., Wood, G. E., Talk, A. C., Matzel, L. D., & Shors, T. J. (2001).
Applying a similar approach to the other paradigms holds the pro- The role of the hippocampus in trace conditioning: Temporal discontinuity or task
mise of disentangling memory-related firing patterns from motor- difficulty? Neurobiology of Learning and Memory, 76, 447–461.
Brasted, P. J., & Wise, S. P. (2004). Comparison of learning-related neuronal activity in
related firing patterns. the dorsal premotor cortex and striatum. European Journal of Neuroscience, 19,
3. Lastly, potential differences in the roles that each brain region plays 721–740.
for time-linked memory call for further investigations. As reviewed Bunsey, M., & Eichenbaum, H. (1993). Critical role of the parahippocampal region for
paired-associate learning in rats. Behavioral Neuroscience, 107, 740.
in Section 2, all behavioral paradigms assessing time-linked memory
Bunsey, M., & Eichenbaum, H. (1996). Conservation of hippocampal memory function in
depend on an interconnected network including many brain regions. rats and humans. Nature, 379, 255–257.
By comparing neuron firing patterns between these brain regions Burman, M. A., Starr, M. J., & Gewirtz, J. C. (2006). Dissociable effects of hippocampus
lesions on expression of fear and trace fear conditioning memories in rats.
during the same memory paradigm would provide valuable insight
Hippocampus, 16, 103–113.
into the difference in the selectivity for task features between the Chang, C.-C., & Lin, C.-J. (2011). LIBSVM: A Library for Support Vector Machines. ACM
brain regions (e.g., Bakhurin et al., 2017; Emmons et al., 2017; Transactions on Intelligent Systems and Technology, 2 27:1–27:27.
Hattori et al., 2014; Yanike et al., 2009). Moreover, the combination Chowdhury, N., Quinn, J. J., & Fanselow, M. S. (2005). Dorsal hippocampus involvement
in trace fear conditioning with long, but not short, trace intervals in mice. Behavioral
of electrophysiological recordings from one brain region with ma- Neuroscience, 119, 1396.
nipulations of another brain region is essential for uncovering the Cortes, C., & Vapnik, V. (1995). Support-vector networks. Machine Learning, 20, 273–297.
influence that the latter brain region has on neural firing patterns in Coull, J. T., & Nobre, A. C. (2008). Dissociating explicit timing from temporal expectation
with fMRI. Current Opinion in Neurobiology, 18, 137–144.
the former brain region. Reversible inactivation (e.g., Emmons et al., Cowen, S. L., & McNaughton, B. L. (2007). Selective delay activity in the medial pre-
2017) or optogenetic inhibition during a specific phase of the task frontal cortex of the rat: Contribution of sensorimotor information and contingency.
(e.g., Robinson et al., 2017) would effectively identify the source of Journal of Neurophysiology, 98, 303–316.
Diehl, G. W., Hon, O. J., Leutgeb, S., & Leutgeb, J. K. (2017). Grid and nongrid cells in
specific information represented in the neural firing patterns. medial entorhinal cortex represent spatial location and environmental features with
complementary coding schemes. Neuron, 94, 83–92.
In summary, convergent evidence from behavioral and neurophy- Eacott, M. J., & Gaffan, D. (1992). Inferotemporal-frontal disconnection: The uncinate
fascicle and visual associative learning in monkeys. European Journal of Neuroscience,
siological studies points to distributed neural representations of tem- 4, 1320–1332.
poral organization of events that we encounter during everyday life. In Emmons, E. B., De Corte, B. J., Kim, Y., Parker, K. L., Matell, M. S., & Narayanan, N. S.
various brain regions involved in this important aspect of everyday (2017). Rodent medial frontal control of temporal processing in the dorsomedial
striatum. Journal of Neuroscience, 37, 8718–8733.
memory, neurons show tonic and phasic firing patterns during time
Erickson, C. A., & Desimone, R. (1999). Responses of macaque perirhinal neurons during
intervals separating two events. Depending on the brain regions, these and after visual stimulus association learning. Journal of Neuroscience, 19,
firing patterns are selective for the prior event, expectation, or timing of 10404–10416.
the coming event with different strength. Future studies are necessary Esclassan, F., Coutureau, E., Di Scala, G., & Marchand, A. R. (2009). A cholinergic-de-
pendent role for the entorhinal cortex in trace fear conditioning. Journal of
to further characterize the difference in the selectivity between two Neuroscience, 29, 8087–8093.
firing patterns within each brain region as well as between brain re- Finnerty, G. T., Shadlen, M. N., Jazayeri, M., Nobre, A. C., & Buonomano, D. V. (2015).
gions. These investigations, along with those directed at neural re- Time in cortical circuits. Journal of Neuroscience, 35, 13912–13916.
Flores, L. C., & Disterhoft, J. F. (2009). Caudate nucleus is critically involved in trace
presentations of time in more flexible, dynamic settings, such as social eyeblink conditioning. Journal of Neuroscience, 29, 14511–14520.
interaction, communication, and language (Finnerty, Shadlen, Jazayeri, Flores, L. C., & Disterhoft, J. F. (2013). Caudate nucleus in retrieval of trace eyeblink
Nobre, & Buonomano, 2015; Goel & Buonomano, 2014), would further conditioning after consolidation. Journal of Neuroscience, 33, 2828–2836.
Fujimichi, R., Naya, Y., Koyano, K. W., Takeda, M., Takeuchi, D., & Miyashita, Y. (2010).
expand our understanding of how the brain processes time. Unitized representation of paired objects in area 35 of the macaque perirhinal cortex.
European Journal of Neuroscience, 32, 659–667.
Acknowledgements Fuster, J. M., & Alexander, G. E. (1971). Neuron activity related to short-term memory.
Science, 173, 652–654.
Gaffan, D., & Harrison, S. (1988). Inferotemporal-frontal disconnection and fornix
This work was supported by NSERC Discovery Grant (RGPIN-2015- transection in visuomotor conditional learning by monkeys. Behavioural Brain
05458), CIHR Operating Grant (MOP-133693), Ontario Research Fund Research, 31, 149–163.
Gilbert, P. E., & Kesner, R. P. (2003). Localization of function within the dorsal hippo-
Early Researcher Award (ER 13-09-01), CFI Leaders Opportunity Fund
campus: The role of the CA3 subregion in paired-associate learning. Behavioral
(25026) to KT, NSERC graduate fellowship (396157093) to MP. The

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

Neuroscience, 117, 1385. hippocampus of rabbits during acquisition of trace eyeblink conditioned responses.
Gilmartin, M. R., & Helmstetter, F. J. (2010). Trace and contextual fear conditioning Journal of Neurophysiology, 78, 1030–1044.
require neural activity and NMDA receptor-dependent transmission in the medial McEchron, M. D., Weible, A. P., & Disterhoft, J. F. (2001). Aging and learning-specific
prefrontal cortex. Learning and Memory, 17, 289–296. changes in single-neuron activity in CA1 hippocampus during rabbit trace eyeblink
Gilmartin, M. R., Kwapis, J. L., & Helmstetter, F. J. (2012). Trace and contextual fear conditioning. Journal of Neurophysiology, 86, 1839–1857.
conditioning are impaired following unilateral microinjection of muscimol in the Meck, W. H. (2006). Neuroanatomical localization of an internal clock: A functional link
ventral hippocampus or amygdala, but not the medial prefrontal cortex. Neurobiology between mesolimbic, nigrostriatal, and mesocortical dopaminergic systems. Brain
of Learning and Memory, 97, 452–464. Research, 1109, 93–107.
Gilmartin, M. R., & McEchron, M. D. (2005). Single neurons in the medial prefrontal Mello, G. B., Soares, S., & Paton, J. J. (2015). A scalable population code for time in the
cortex of the rat exhibit tonic and phasic coding during trace fear conditioning. striatum. Current Biology, 25, 1113–1122.
Behavioral Neuroscience, 119, 1496–1510. Misane, I., Tovote, P., Meyer, M., Spiess, J., Ögren, S. O., & Stiedl, O. (2005). Time-
Gilmartin, M. R., Miyawaki, H., Helmstetter, F. J., & Diba, K. (2013). Prefrontal activity dependent involvement of the dorsal hippocampus in trace fear conditioning in mice.
links nonoverlapping events in memory. Journal of Neuroscience, 33, 10910–10914. Hippocampus, 15, 418–426.
Goel, A., & Buonomano, D. V. (2014). Timing as an intrinsic property of neural networks: Modi, M. N., Dhawale, A. K., & Bhalla, U. S. (2014). CA1 cell activity sequences emerge
Evidence from in vivo and in vitro experiments. Philosophical Transactions of the Royal after reorganization of network correlation structure during associative learning.
Society B, 369, 20120460. Elife, 3, e01982.
Graves, C. A., & Solomon, P. R. (1985). Age-related disruption of trace but not delay Morrissey, M. D., Insel, N., & Takehara-Nishiuchi, K. (2017). Generalizable knowledge
classical conditioning of the rabbit’s nictitating membrane response. Behavioral outweighs incidental details in prefrontal ensemble code over time. Elife, 6, e22177.
Neuroscience, 99, 88. Morrissey, M. D., Maal-Bared, G., Brady, S., & Takehara-Nishiuchi, K. (2012). Functional
Han, C. J., O’Tuathaigh, C. M., van Trigt, L., Quinn, J. J., Fanselow, M. S., Mongeau, R., ... dissociation within the entorhinal cortex for memory retrieval of an association be-
Anderson, D. J. (2003). Trace but not delay fear conditioning requires attention and tween temporally discontiguous stimuli. Journal of Neuroscience, 32(16), 5356–5361.
the anterior cingulate cortex. Proceedings of the National Academy of Sciences, 100, Moser, E. I., Kropff, E., & Moser, M.-B. (2008). Place cells, grid cells, and the brain’s
13087–13092. spatial representation system. Annual Review of Neuroscience, 31, 69–89.
Hartley, T., Lever, C., Burgess, N., & O’Keefe, J. (2014). Space in the brain: How the Moyer, J. R., Jr., Deyo, R. A., & Disterhoft, J. F. (1990). Hippocampectomy disrupts trace
hippocampal formation supports spatial cognition. Philosophical Transactions of the eye-blink conditioning in rabbits. Behavioral Neuroscience, 104, 243–252.
Royal Society B, 369, 20120510. Munera, A., Gruart, A., Munoz, M. D., Fernandez-Mas, R., & Delgado-Garcia, J. M. (2001).
Hattori, S., Chen, L., Weiss, C., & Disterhoft, J. F. (2015). Robust hippocampal re- Hippocampal pyramidal cell activity encodes conditioned stimulus predictive value
sponsivity during retrieval of consolidated associative memory: Hippocampal during classical conditioning in alert cats. Journal of Neurophysiology, 86, 2571–2582.
Involvement after Memory Consolidation. Hippocampus, 25, 655–669. Murray, E. A., Gaffan, D., & Mishkin, M. (1993). Neural substrates of visual stimulus-
Hattori, S., Yoon, T., Disterhoft, J. F., & Weiss, C. (2014). Functional reorganization of a stimulus association in rhesus monkeys. Journal of Neuroscience, 13, 4549–4561.
prefrontal cortical network mediating consolidation of trace eyeblink conditioning. Murray, E. A., & Wise, S. P. (1996). Role of the hippocampus plus subjacent cortex but not
Journal of Neuroscience, 34, 1432–1445. amygdala in visuomotor conditional learning in rhesus monkeys. Behavioral
Higuchi, S.-I., & Miyashita, Y. (1996). Formation of mnemonic neuronal responses to Neuroscience, 110, 1261.
visual paired associates in inferotemporal cortex is impaired by perirhinal and en- Namboodiri, V. M. K., & Shuler, M. G. H. (2014). Report of interval timing or action?
torhinal lesions. Proceedings of the National Academy of Sciences, 93, 739–743. Proceedings of the National Academy of Sciences, 111 E2239–E2239.
Hoehler, F. K., & Thompson, R. F. (1980). Effect of the interstimulus (CS–UCS) interval on Narayanan, N. S., Horst, N. K., & Laubach, M. (2006). Reversible inactivations of rat
hippocampal unit activity during classical conditioning of the nictitating membrane medial prefrontal cortex impair the ability to wait for a stimulus. Neuroscience, 139,
response of the rabbit (Oryctolagus cuniculus). Journal of Comparative and 865–876.
Physiological Psychology, 94, 201. Narayanan, N. S., Land, B. B., Solder, J. E., Deisseroth, K., & DiLeone, R. J. (2012).
Hu, B., Chen, H., Feng, H., Zeng, Y., Yang, L., Fan, Z. L., ... Sui, J. F. (2010). Disrupted Prefrontal D1 dopamine signaling is required for temporal control. Proceedings of the
topography of the acquired trace-conditioned eyeblink responses in guinea pigs after National Academy of Sciences, 109, 20726–20731.
suppression of cerebellar cortical inhibition to the interpositus nucleus. Brain Naya, Y., & Suzuki, W. A. (2011). Integrating what and when across the primate medial
Research, 1337, 41–55. temporal lobe. Science, 333, 773–776.
Ivry, R. B., & Spencer, R. M. (2004). The neural representation of time. Current Opinion in Naya, Y., Yoshida, M., & Miyashita, Y. (2001). Backward spreading of memory-retrieval
Neurobiology, 14, 225–232. signal in the primate temporal cortex. Science, 291, 661–664.
Kamin, L. J., & Schaub, R. E. (1963). Effects of conditioned stimulus intensity on the Naya, Y., Yoshida, M., & Miyashita, Y. (2003). Forward processing of long-term asso-
conditioned emotional response. Journal of Comparative and Physiological Psychology, ciative memory in monkey inferotemporal cortex. Journal of Neuroscience, 23,
56, 502. 2861–2871.
Kesner, R. P., Hunsaker, M. R., & Gilbert, P. E. (2005). The role of CA1 in the acquisition Pakaprot, N., Kim, S., & Thompson, R. F. (2009). The role of the cerebellar interpositus
of an object-trace-odor paired associate task. Behavioral Neuroscience, 119, 781. nucleus in short and long term memory for trace eyeblink conditioning. Behavioral
Kholodar-Smith, D. B., Boguszewski, P., & Brown, T. H. (2008). Auditory trace fear Neuroscience, 123, 54–61.
conditioning requires perirhinal cortex. Neurobiology of Learning and Memory, 90, Parker, A., & Gaffan, D. (1998). Memory after frontal/temporal disconnection in mon-
537–543. keys: Conditional and non-conditional tasks, unilateral and bilateral frontal lesions.
Kishimoto, Y., Kawahara, S., Mori, H., Mishina, M., & Kirino, Y. (2001). Long-trace in- Neuropsychologia, 36, 259–271.
terval eyeblink conditioning is impaired in mutant mice lacking the NMDA receptor Parker, K. L., Chen, K.-H., Kingyon, J. R., Cavanagh, J. F., & Narayanan, N. S. (2014). D1-
subunit ε1: Eyeblink conditioning in GluRε1 mutant mice. European Journal of dependent 4 Hz oscillations and ramping activity in rodent medial frontal cortex
Neuroscience, 13, 1221–1227. during interval timing. Journal of Neuroscience, 34, 16774–16783.
Kitamura, T., Pignatelli, M., Suh, J., Kohara, K., Yoshiki, A., Abe, K., & Tonegawa, S. Parker, K. L., Ruggiero, R. N., & Narayanan, N. S. (2015). Infusion of D1 dopamine re-
(2014). Island cells control temporal association memory. Science, 343, 896–901. ceptor agonist into medial frontal cortex disrupts neural correlates of interval timing.
Kraus, B. J., Robinson, R. J., White, J. A., Eichenbaum, H., & Hasselmo, M. E. (2013). Frontiers in Behavioral Neuroscience, 9.
Hippocampal “time cells”: Time versus path integration. Neuron, 78, 1090–1101. Pastalkova, E., Itskov, V., Amarasingham, A., & Buzsáki, G. (2008). Internally generated
Kronforst-Collins, M. A., & Disterhoft, J. F. (1998). Lesions of the caudal area of rabbit cell assembly sequences in the rat hippocampus. Science, 321, 1322–1327.
medial prefrontal cortex impair trace eyeblink conditioning. Neurobiology of Learning Pasupathy, A., & Miller, E. K. (2005). Different time courses of learning-related activity in
and Memory, 69, 147–162. the prefrontal cortex and striatum. Nature, 433, 873–876.
Kwapis, J. L., Jarome, T. J., Schiff, J. C., & Helmstetter, F. J. (2011). Memory con- Pavlov, I. P. (1927). Conditional reflexes: An investigation of the physiological activity of
solidation in both trace and delay fear conditioning is disrupted by intra-amygdala the cerebral cortex.
infusion of the protein synthesis inhibitor anisomycin. Learning and Memory, 18, Petrides, M. (1982). Motor conditional associative-learning after selective prefrontal le-
728–732. sions in the monkey. Behavioural Brain Research, 5, 407–413.
LeDoux, J. E., Iwata, J., Cicchetti, P., & Reis, D. J. (1988). Different projections of the Petter, E. A., Lusk, N. A., Hesslow, G., & Meck, W. H. (2016). Interactive roles of the
central amygdaloid nucleus mediate autonomic and behavioral correlates of condi- cerebellum and striatum in sub-second and supra-second timing: Support for an in-
tioned fear. Journal of Neuroscience, 8, 2517–2529. itiation, continuation, adjustment, and termination (ICAT) model of temporal pro-
MacDonald, C. J., Carrow, S., Place, R., & Eichenbaum, H. (2013). Distinct hippocampal cessing. Neuroscience & Biobehavioral Reviews, 71, 739–755.
time cell sequences represent odor memories in immobilized rats. Journal of Powell, D. A., & Churchwell, J. (2002). Mediodorsal thalamic lesions impair trace eye-
Neuroscience, 33, 14607–14616. blink conditioning in the rabbit. Learning and Memory, 9, 10–17.
MacDonald, C. J., Lepage, K. Q., Eden, U. T., & Eichenbaum, H. (2011). Hippocampal Rainer, G., Rao, S. C., & Miller, E. K. (1999). Prospective coding for objects in primate
“Time Cells” bridge the gap in memory for discontiguous events. Neuron, 71, prefrontal cortex. Journal of Neuroscience, 19, 5493–5505.
737–749. Raybuck, J. D., & Lattal, K. M. (2011). Double dissociation of amygdala and hippocampal
Marlin, N. A. (1981). Contextual associations in trace conditioning. Animal Learning & contributions to trace and delay fear conditioning. PloS One, 6, e15982.
Behavior, 9, 519–523. Robinson, N. T., Priestley, J. B., Rueckemann, J. W., Garcia, A. D., Smeglin, V. A., Marino,
Matell, M. S., Meck, W. H., & Nicolelis, M. A. (2003). Interval timing and the encoding of F. A., & Eichenbaum, H. (2017). Medial entorhinal cortex selectively supports tem-
signal duration by ensembles of cortical and striatal neurons. Behavioral Neuroscience, poral coding by hippocampal neurons. Neuron, 94, 677–688.
117, 760. Rolls, E. T. (2000). Functions of the primate temporal lobe cortical visual areas in in-
McEchron, M. D., Bouwmeester, H., Tseng, W., Weiss, C., & Disterhoft, J. F. (1998). variant visual object and face recognition. Neuron, 27, 205–218.
Hippocampectomy disrupts auditory trace fear conditioning and contextual fear Runyan, J. D., Moore, A. N., & Dash, P. K. (2004). A role for prefrontal cortex in memory
conditioning in the rat. Hippocampus, 8, 638–646. storage for trace fear conditioning. Journal of Neuroscience, 24, 1288–1295.
McEchron, M. D., & Disterhoft, J. F. (1997). Sequence of single neuron changes in CA1 Ryou, J. W., Cho, S. Y., & Kim, H. T. (2001). Lesions of the entorhinal cortex impair

M. Pilkiw, K. Takehara-Nishiuchi Neurobiology of Learning and Memory xxx (xxxx) xxx–xxx

acquisition of hippocampal-dependent trace conditioning. Neurobiology of Learning Takehara-Nishiuchi, K., & McNaughton, B. L. (2008). Spontaneous changes of neocortical
and Memory, 75, 121–127. code for associative memory during consolidation. Science, 322, 960–963.
Sakai, K., & Miyashita, Y. (1991). Neural organization for the long-term memory of paired Tam, S. K., & Bonardi, C. (2012). Dorsal hippocampal involvement in appetitive trace
associates. Nature, 354, 152–155. conditioning and interval timing. Behavioral Neuroscience, 126, 258.
Sakon, J. J., Naya, Y., Wirth, S., & Suzuki, W. A. (2014). Context-dependent incremental Tanaka, K. (1996). Inferotemporal cortex and object vision. Annual Review of
timing cells in the primate hippocampus. Proceedings of the National Academy of Neuroscience, 19, 109–139.
Sciences, 111, 18351–18356. Tanninen, S. E., Yu, X., Giritharan, T., Tran, L., Bakir, R., Volle, J., ... Takehara-Nishiuchi,
Salz, D. M., Tiganj, Z., Khasnabish, S., Kohley, A., Sheehan, D., Howard, M. W., & K. (2015). Cholinergic, but not NMDA, receptors in the lateral entorhinal cortex
Eichenbaum, H. (2016). Time cells in hippocampal area CA3. Journal of Neuroscience, mediate acquisition in trace eyeblink conditioning. Hippocampus, 25, 1456–1464.
36, 7476–7484. Tremblay, L., Hollerman, J. R., & Schultz, W. (1998). Modifications of reward expecta-
Schmaltz, L. W., & Theios, J. (1972). Acquisition and extinction of a classically condi- tion-related neuronal activity during learning in primate striatum. Journal of
tioned response in hippocampectomized rabbits (Oryctolagus cuniculus). Journal of Neurophysiology, 80, 964–977.
Comparative and Physiological Psychology, 79(2), 328–333. Tseng, W., Guan, R., Disterhoft, J. F., & Weiss, C. (2004). Trace eyeblink conditioning is
Selden, N. R. W., Everitt, B. J., Jarrard, L. E., & Robbins, T. W. (1991). Complementary hippocampally dependent in mice. Hippocampus, 14, 58–65.
roles for the amygdala and hippocampus in aversive conditioning to explicit and Volle, J., Yu, X., Sun, H., Tanninen, S. E., Insel, N., & Takehara-Nishiuchi, K. (2016).
contextual cues. Neuroscience, 42, 335–350. Enhancing prefrontal neuron activity enables associative learning of temporally
Siegel, J. J. (2014). Modification of persistent responses in medial prefrontal cortex disparate events. Cell Reports, 15, 2400–2410.
during learning in trace eyeblink conditioning. Journal of Neurophysiology, 112, Walker, A. G., & Steinmetz, J. E. (2008). Hippocampal lesions in rats differentially affect
2123–2137. long- and short-trace eyeblink conditioning. Physiology & Behavior, 93, 570–578.
Siegel, J. J. (2016). Prefrontal single-neuron responses after changes in task contingencies Weible, A. P., McEchron, M. D., & Disterhoft, J. F. (2000). Cortical involvement in ac-
during trace eyeblink conditioning in rabbits. Eneuro, 3 ENEURO–0057. quisition and extinction of trace eyeblink conditioning. Behavioral Neuroscience, 114,
Siegel, J. J., Kalmbach, B., Chitwood, R. A., & Mauk, M. D. (2012). Persistent activity in a 1058–1067.
cortical-to-subcortical circuit: Bridging the temporal gap in trace eyelid conditioning. Weiss, C., Knuttinen, M.-G., Power, J. M., Patel, R. I., O’connor, M. S., & Disterhoft, J. F.
Journal of Neurophysiology, 107, 50–64. (1999). Trace eyeblink conditioning in the freely moving rat: Optimizing the con-
Siegel, J. J., & Mauk, M. D. (2013). Persistent activity in prefrontal cortex during trace ditioning parameters. Behavioral Neuroscience, 113, 1100.
eyelid conditioning: dissociating responses that reflect cerebellar output from those Wirth, S., Yanike, M., Frank, L. M., Smith, A. C., Brown, E. N., & Suzuki, W. A. (2003).
that do not. Journal of Neuroscience, 33, 15272–15284. Single neurons in the monkey hippocampus and learning of new associations. Science,
Siegel, J. J., Taylor, W., Gray, R., Kalmbach, B., Zemelman, B. V., Desai, N. S., ... 300, 1578–1581.
Chitwood, R. A. (2015). Trace eyeblink conditioning in mice is dependent upon the Woodruff-Pak, D. S., Lavond, D. G., & Thompson, R. F. (1985). Trace conditioning:
dorsal medial prefrontal cortex, cerebellum, and amygdala: behavioral character- Abolished by cerebellar nuclear lesions but not lateral cerebellar cortex aspirations.
ization and functional circuitry. Eneuro, 2 ENEURO-0051. Brain Research, 348, 249–260.
Solomon, P. R., Vander Schaaf, E. R., Thompson, R. F., & Weisz, D. J. (1986). Xu, M., Zhang, S., Dan, Y., & Poo, M. (2014). Representation of interval timing by tem-
Hippocampus and trace conditioning of the rabbit’s classically conditioned nictitating porally scalable firing patterns in rat prefrontal cortex. Proceedings of the National
membrane response. Behavioral Neuroscience, 100, 729. Academy of Sciences, 111, 480–485.
Suter, E. E., Weiss, C., & Disterhoft, J. F. (2013). Perirhinal and postrhinal, but not lateral Yanike, M., Wirth, S., Smith, A. C., Brown, E. N., & Suzuki, W. A. (2009). Comparison of
entorhinal, cortices are essential for acquisition of trace eyeblink conditioning. associative learning-related signals in the macaque perirhinal cortex and hippo-
Learning and Memory, 20, 80–84. campus. Cerebral Cortex, 19(5), 1064–1078.
Takeda, M., Naya, Y., Fujimichi, R., Takeuchi, D., & Miyashita, Y. (2005). Active main- Yanike, M., Wirth, S., & Suzuki, W. A. (2004). Representation of well-learned information
tenance of associative mnemonic signal in monkey inferior temporal cortex. Neuron, in the monkey hippocampus. Neuron, 42, 477–487.
48, 839–848. Yin, B., & Meck, W. H. (2014). Comparison of interval timing behaviour in mice following
Takehara, K., Kawahara, S., Takatsuki, K., & Kirino, Y. (2002). Time-limited role of the dorsal or ventral hippocampal lesions with mice having δ-opioid receptor gene de-
hippocampus in the memory for trace eyeblink conditioning in mice. Brain Research, letion. Philosophical Transactions of the Royal Society of London. Series B, Biological
951, 183–190. sciences, 369, 20120466.
Takehara-Nishiuchi, K., Kawahara, S., & Kirino, Y. (2005). NMDA receptor-dependent Yoon, T., & Otto, T. (2007). Differential contributions of dorsal vs. ventral hippocampus
processes in the medial prefrontal cortex are important for acquisition and the early to auditory trace fear conditioning. Neurobiology of Learning and Memory, 87,
stage of consolidation during trace, but not delay eyeblink conditioning. Learning and 464–475.
Memory, 12, 606–614.