Evidence for a subcircuit in medial entorhinal cortex representing elapsed time during immobility

Abstract

The medial entorhinal cortex (MEC) is known to contain spatial encoding neurons that likely contribute to encoding spatial aspects of episodic memories. However, little is known about the role MEC plays in encoding temporal aspects of episodic memories, particularly during immobility. Here using a virtual ‘Door Stop’ task for mice, we show that MEC contains a representation of elapsed time during immobility, with individual time-encoding neurons activated at a specific moment during the immobile interval. This representation consisted of a sequential activation of time-encoding neurons and displayed variations in progression speed that correlated with variations in mouse timing behavior. Time- and space-encoding neurons were preferentially active during immobile and locomotion periods, respectively, were anatomically clustered with respect to each other, and preferentially encoded the same variable across tasks or environments. These results suggest the existence of largely non-overlapping subcircuits in MEC encoding time during immobility or space during locomotion.

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Articles
https://doi.org/10.1038/s41593-018-0252-8
Department of Neurobiology, Northwestern University, Evanston Illinois, Evanston, IL, USA. *e-mail: d-dombeck@northwestern.edu
Over the past 50 years, research from humans and animal
models have implicated the medial temporal lobe, which
includes the hippocampus and MEC, in the formation
of personal memories of events that occur at specific places and
involve specific time intervals1,2. While a vast amount of research
has uncovered cellular substrates in the hippocampus and MEC
that likely make up the spatial representation required for these epi-
sodic memories3–8, our understanding of the temporal representa-
tion is substantially less advanced and has focused mostly on the
hippocampus9–11. Time-related neurons were first demonstrated in
the hippocampus using studies in which rodents were moving to
some degree, either in a running wheel12, on a treadmill13, or in a
small box14. Notably, one study found hippocampal time-related
activity during immobility15. These so-called hippocampal ‘time
cells’ fire briefly and consistently at specific times during the task,
such that behavioral time periods are tiled by a sequence of brief
neuronal activations. Strikingly, specialized circuitry representing
spatial information during immobility has also been demonstrated
in the hippocampus16,17. This suggests that separate circuitry within
the medial temporal lobe might be used to encode behaviorally rel-
evant variables between mobile and immobile periods, though it is
unclear from these studies whether the representation of elapsed
time maps onto a particular circuit(s).
In MEC, one study18 found that MEC grid cells can provide timing-
related information during treadmill running, and a separate study
found MEC neurons that were more active at low running speeds
rather than high speeds during locomotion19. Inactivation of MEC
during such mobile periods was found to produce deficits in encod-
ing memories across trace periods20,21, produce deficits in a temporal
memory task, and cause instability in downstream hippocampal time
cells22. These studies suggest that a code for elapsed time may exist
in MEC during locomotion, but it is currently unknown whether the
neural circuitry in MEC forms a representation of elapsed time during
immobility, when sensory cues may not change in a temporally infor-
mative manner. Furthermore, if such a representation exists in MEC, it
is unknown how the neural circuitry might be organized to generate it.
Results
To explore these ideas, we used our previously developed functional
two-photon imaging methods23 to optically record from popula-
tions of layer II MEC neurons (Fig. 1a and Supplementary Fig. 1)
during mouse navigation in a novel virtual Door Stop task. The
Door Stop task combines both a locomotion-dependent virtual
navigation phase and an explicit instrumental timing phase that
was separated in time and location from reward delivery (Fig. 1b
and Supplementary Fig. 2a). Mice were trained to run down a lin-
ear track to a specific location where they encountered an invisible
door, which they could not run past, though they could still run
on the treadmill. At the door location, the mice were required to
stop and wait for at least 6 s (an auditory click signaled the start of
the 6-s interval once the treadmill velocity fell below a threshold;
see Methods); if the mice began running on the treadmill before
the expiration of the 6 s interval, the mice could not progress past
the closed door and the trial would start over (signaled by another
click). After the 6-s interval, the door would open and the mice
could run down the remaining length of the track to the reward
zone. After 6–8 weeks of training, mice ran to the invisible door and
stopped on their first attempt for the full 6-s wait period on 55.1%
of trials (Fig. 1c), referred to as ‘correct trials’. To easily compare
neural activity during immobile timing periods and neural activity
during locomotion periods, we excluded a transition zone between
these periods and excluded the reward zone when behavior was
more ambiguous (Fig. 1e, Supplementary Fig. 2a, and see Methods).
During the wait periods, mice mostly sat immobile with essentially
0 velocity with small jerky movements occurring during 12.9% of
the wait period to maintain balance on the treadmill (velocity over
wait periods = 0.33 ± 1.00 cm/s (mean ± s.d.); Fig. 1d,e). All of the
data presented in Figs. 2–4 using the (invisible door) Door Stop task
come only from these correct trials (see Supplementary Fig. 2b–f
for velocity on all trials). Since the mice could not see the invisible
door opening at the end of the 6-s interval, this Door Stop task
therefore required an internal temporal representation for efficient
completion.
Evidence for a subcircuit in medial entorhinal
cortex representing elapsed time during
immobility
JamesG.Heys and DanielA.Dombeck *
The medial entorhinal cortex (MEC) is known to contain spatial encoding neurons that likely contribute to encoding spatial
aspects of episodic memories. However, little is known about the role MEC plays in encoding temporal aspects of episodic
memories, particularly during immobility. Here using a virtual ‘Door Stop’ task for mice, we show that MEC contains a repre-
sentation of elapsed time during immobility, with individual time-encoding neurons activated at a specific moment during the
immobile interval. This representation consisted of a sequential activation of time-encoding neurons and displayed variations
in progression speed that correlated with variations in mouse timing behavior. Time- and space-encoding neurons were prefer-
entially active during immobile and locomotion periods, respectively, were anatomically clustered with respect to each other,
and preferentially encoded the same variable across tasks or environments. These results suggest the existence of largely non-
overlapping subcircuits in MEC encoding time during immobility or space during locomotion.
NATURE NEUROSCIENCE | VOL 21 | NOVEMBER 2018 | 1574–1582 | www.nature.com/natureneuroscience
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