Medial entorhinal cortex mediates learning of context-dependent interval timing behavior.

Episodic memory requires encoding the temporal structure of experience and relies on brain circuits in the medial temporal lobe, including the medial entorhinal cortex (MEC). Recent studies have identified MEC 'time cells', which fire at specific moments during interval timing tasks, collectively tiling the entire timing period. It has been hypothesized that MEC time cells could provide temporal information necessary for episodic memories, yet it remains unknown whether they display learning dynamics required for encoding different temporal contexts. To explore this, we developed a new behavioral paradigm requiring mice to distinguish temporal contexts. Combined with methods for cellular resolution calcium imaging, we found that MEC time cells display context-dependent neural activity that emerges with task learning. Through chemogenetic inactivation we found that MEC activity is necessary for learning of context-dependent interval timing behavior. Finally, we found evidence of a common circuit mechanism that could drive sequential activity of both time cells and spatially selective neurons in MEC. Our work suggests that the clock-like firing of MEC time cells can be modulated by learning, allowing the tracking of various temporal structures that emerge through experience.

Medial entorhinal cortex plays a specialized role in learning of flexible, context-dependent interval timing behavior.

Episodic memory requires encoding the temporal structure of experience and relies on brain circuits in the medial temporal lobe, including the medial entorhinal cortex (MEC). Recent studies have identified MEC 'time cells', which fire at specific moments during interval timing tasks, collectively tiling the entire timing period. It has been hypothesized that MEC time cells could provide temporal information necessary for episodic memories, yet it remains unknown whether MEC time cells display learning dynamics required for encoding different temporal contexts. To explore this, we developed a novel behavioral paradigm that requires distinguishing temporal contexts. Combined with methods for cellular resolution calcium imaging, we find that MEC time cells display context-dependent neural activity that emerges with task learning. Through chemogenetic inactivation we find that MEC activity is necessary for learning of context-dependent interval timing behavior. Finally, we find evidence of a common circuit mechanism that could drive sequential activity of both time cells and spatially selective neurons in MEC. Our work suggests that the clock-like firing of MEC time cells can be modulated by learning, allowing the tracking of various temporal structures that emerge through experience.

Medial entorhinal cortex plays a specialized role in learning of flexible, context-dependent interval timing behavior.

In order to survive and adapt in a dynamic environment, animals must perceive and remember the temporal structure of events and actions across a wide range of timescales, including so-called interval timing on the scale of seconds to minutes1,2. Episodic memory (i.e. the ability to remember specific, personal events that occur in spatial and temporal context) requires accurate temporal processing and is known to require neural circuits in the medial temporal lobe (MTL), including medial entorhinal cortex (MEC)3-5. Recently, it has been discovered that neurons in MEC termed time cells, fire regularly at brief moments as animals engage in interval timing behavior, and as a population, display sequential neural activity that tiles the entire timed epoch6. It has been hypothesized that MEC time cell activity could provide temporal information necessary for episodic memories, yet it remains unknown whether the neural dynamics of MEC time cells display a critical feature necessary for encoding experience. That is, whether MEC time cells display context-dependent activity. To address this question, we developed a novel behavioral paradigm that requires learning complex temporal contingencies. Applying this novel interval timing task in mice, in concert with methods for manipulating neural activity and methods for large-scale cellular resolution neurophysiological recording, we have uncovered a specific role for MEC in flexible, context-dependent learning of interval timing behavior. Further, we find evidence for a common circuit mechanism that could drive both sequential activity of time cells and spatially selective neurons in MEC.

Inactivation of the Medial Entorhinal Cortex Selectively Disrupts Learning of Interval Timing.

The entorhinal-hippocampal circuit can encode features of elapsed time, but nearly all previous research focused on neural encoding of "implicit time." Recent research has revealed encoding of "explicit time" in the medial entorhinal cortex (MEC) as mice are actively engaged in an interval timing task. However, it is unclear whether the MEC is required for temporal perception and/or learning during such explicit timing tasks. We therefore optogenetically inactivated the MEC as mice learned an interval timing "door stop" task that engaged mice in immobile interval timing behavior and locomotion-dependent navigation behavior. We find that the MEC is critically involved in learning of interval timing but not necessary for estimating temporal duration after learning. Together with our previous research, these results suggest that activity of a subcircuit in the MEC that encodes elapsed time during immobility is necessary for learning interval timing behaviors.

Navigating Through Time: A Spatial Navigation Perspective on How the Brain May Encode Time.

Interval timing, which operates on timescales of seconds to minutes, is distributed across multiple brain regions and may use distinct circuit mechanisms as compared to millisecond timing and circadian rhythms. However, its study has proven difficult, as timing on this scale is deeply entangled with other behaviors. Several circuit and cellular mechanisms could generate sequential or ramping activity patterns that carry timing information. Here we propose that a productive approach is to draw parallels between interval timing and spatial navigation, where direct analogies can be made between the variables of interest and the mathematical operations necessitated. Along with designing experiments that isolate or disambiguate timing behavior from other variables, new techniques will facilitate studies that directly address the neural mechanisms that are responsible for interval timing.

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

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.

Physiological Properties of Neurons in Bat Entorhinal Cortex Exhibit an Inverse Gradient along the Dorsal-Ventral Axis Compared to Entorhinal Neurons in Rat.

Medial entorhinal cortex (MEC) grid cells exhibit firing fields spread across the environment on the vertices of a regular tessellating triangular grid. In rodents, the size of the firing fields and the spacing between the firing fields are topographically organized such that grid cells located more ventrally in MEC exhibit larger grid fields and larger grid-field spacing compared with grid cells located more dorsally. Previous experiments in brain slices from rodents have shown that several intrinsic cellular electrophysiological properties of stellate cells in layer II of MEC change systematically in neurons positioned along the dorsal-ventral axis of MEC, suggesting that these intrinsic cellular properties might control grid-field spacing. In the bat, grid cells in MEC display a functional topography in terms of grid-field spacing, similar to what has been reported in rodents. However, it is unclear whether neurons in bat MEC exhibit similar gradients of cellular physiological properties, which may serve as a conserved mechanism underlying grid-field spacing in mammals. To test whether entorhinal cortex (EC) neurons in rats and bats exhibit similar electrophysiological gradients, we performed whole-cell patch recordings along the dorsal-ventral axis of EC in bats. Surprisingly, our data demonstrate that the sag response properties and the resonance properties recorded in layer II neurons of entorhinal cortex in the Egyptian fruit bat demonstrate an inverse relationship along the dorsal-ventral axis compared with the rat. SIGNIFICANCE STATEMENT: As animals navigate, neurons in medial entorhinal cortex (MEC), termed grid cells, discharge at regular spatial intervals. In bats and rats, the spacing between the firing fields of grid cells changes systematically along the dorsal-ventral axis of MEC. It has been proposed that these changes could be generated by systematic differences in the intrinsic cellular physiology of neurons distributed along the dorsal-ventral axis of MEC. The results from our study show that key intrinsic physiological properties of neurons in entorhinal cortex of the bat and rat change in the opposite direction along the dorsal-ventral axis of entorhinal cortex, suggesting that these intrinsic physiological properties cannot account in the same way across species for the change in grid-field spacing shown along the dorsal-ventral axis.

The functional micro-organization of grid cells revealed by cellular-resolution imaging.

Establishing how grid cells are anatomically arranged, on a microscopic scale, in relation to their firing patterns in the environment would facilitate a greater microcircuit-level understanding of the brain's representation of space. However, all previous grid cell recordings used electrode techniques that provide limited descriptions of fine-scale organization. We therefore developed a technique for cellular-resolution functional imaging of medial entorhinal cortex (MEC) neurons in mice navigating a virtual linear track, enabling a new experimental approach to study MEC. Using these methods, we show that grid cells are physically clustered in MEC compared to nongrid cells. Additionally, we demonstrate that grid cells are functionally micro-organized: the similarity between the environment firing locations of grid cell pairs varies as a function of the distance between them according to a "Mexican hat"-shaped profile. This suggests that, on average, nearby grid cells have more similar spatial firing phases than those further apart.

Bat and rat neurons differ in theta-frequency resonance despite similar coding of space.

Both bats and rats exhibit grid cells in medial entorhinal cortex that fire as they visit a regular array of spatial locations. In rats, grid-cell firing field properties correlate with theta-frequency rhythmicity of spiking and membrane-potential resonance; however, bat grid cells do not exhibit theta rhythmic spiking, generating controversy over the role of theta rhythm. To test whether this discrepancy reflects differences in rhythmicity at a cellular level, we performed whole-cell patch recordings from entorhinal neurons in both species to record theta-frequency resonance. Bat neurons showed no theta-frequency resonance, suggesting grid-cell coding via different mechanisms in bats and rats or lack of theta rhythmic contributions to grid-cell firing in either species.

Effects of acetylcholine on neuronal properties in entorhinal cortex.

The entorhinal cortex (EC) receives prominent cholinergic innervation from the medial septum and the vertical limb of the diagonal band of Broca (MSDB). To understand how cholinergic neurotransmission can modulate behavior, research has been directed toward identification of the specific cellular mechanisms in EC that can be modulated through cholinergic activity. This review focuses on intrinsic cellular properties of neurons in EC that may underlie functions such as working memory, spatial processing, and episodic memory. In particular, the study of stellate cells (SCs) in medial entorhinal has resulted in discovery of correlations between physiological properties of these neurons and properties of the unique spatial representation that is demonstrated through unit recordings of neurons in medial entorhinal cortex (mEC) from awake-behaving animals. A separate line of investigation has demonstrated persistent firing behavior among neurons in EC that is enhanced by cholinergic activity and could underlie working memory. There is also evidence that acetylcholine plays a role in modulation of synaptic transmission that could also enhance mnemonic function in EC. Finally, the local circuits of EC demonstrate a variety of interneuron physiology, which is also subject to cholinergic modulation. Together these effects alter the dynamics of EC to underlie the functional role of acetylcholine in memory.

Neuromodulation of I(h) in layer II medial entorhinal cortex stellate cells: a voltage-clamp study.

Stellate cells in layer II of medial entorhinal cortex (mEC) are endowed with a large hyperpolarization-activated cation current [h current (I(h))]. Recent work using in vivo recordings from awake behaving rodents demonstrate that I(h) plays a significant role in regulating the characteristic spatial periodicity of "grid cells" in mEC. A separate, yet related, line of research demonstrates that grid field spacing changes as a function of behavioral context. To understand the neural mechanism or mechanisms that could be underlying these changes in grid spacing, we have conducted voltage-clamp recordings of I(h) in layer II stellate cells. In particular, we have studied I(h) under the influence of several neuromodulators. The results demonstrate that I(h) amplitude can be both upregulated and downregulated through activation of distinct neuromodulators in mEC. Activation of muscarinic acetylcholine receptors produces a significant decrease in the I(h) tail current and a hyperpolarizing shift in the activation, whereas upregulation of cAMP through application of forskolin produces a significant increase in the I(h) amplitude and a depolarizing shift in I(h) activation curve. In addition, there was evidence of differential modulation of I(h) along the dorsal-ventral axis of mEC. Voltage-clamp protocols were also used to determine whether M current is present in stellate cells. In contrast to CA1 pyramidal neurons, which express M current, the data demonstrate that M current is not present in stellate cells. The results from this study provide key insights into a potential mechanism that could be underlying changes seen in grid field spacing during distinct behavioral contexts.

Possible role of acetylcholine in regulating spatial novelty effects on theta rhythm and grid cells.

Existing pharmacological and lesion data indicate that acetylcholine plays an important role in memory formation. For example, increased levels of acetylcholine in the hippocampal formation are known to be associated with successful encoding while disruption of the cholinergic system leads to impairments on a range of mnemonic tasks. However, cholinergic signaling from the medial septum also plays a central role in generating and pacing theta-band oscillations throughout the hippocampal formation. Recent experimental results suggest a potential link between these distinct phenomena. Environmental novelty, a condition associated with strong cholinergic drive, has been shown to induce an expansion in the firing pattern of entorhinal grid cells and a reduction in the frequency of theta measured from the LFP. Computational modeling suggests the spatial activity of grid cells is produced by interference between neuronal oscillators; scale being determined by theta-band oscillations impinging on entorhinal stellate cells, the frequency of which is modulated by acetylcholine. Here we propose that increased cholinergic signaling in response to environmental novelty triggers grid expansion by reducing the frequency of the oscillations. Furthermore, we argue that cholinergic induced grid expansion may enhance, or even induce, encoding by producing a mismatch between expanded grid cells and other spatial inputs to the hippocampus, such as boundary vector cells. Indeed, a further source of mismatch is likely to occur between grid cells of different native scales which may expand by different relative amounts.

Cholinergic modulation of the resonance properties of stellate cells in layer II of medial entorhinal cortex.

In vitro whole cell patch-clamp recordings of stellate cells in layer II of medial entorhinal cortex show a subthreshold membrane potential resonance in response to a sinusoidal current injection of varying frequency. Physiological recordings from awake behaving animals show that neurons in layer II medial entorhinal cortex, termed "grid cells," fire in a spatially selective manner such that each cell's multiple firing fields form a hexagonal grid. Both the spatial periodicity of the grid fields and the resonance frequency change systematically in neurons along the dorsal to ventral axis of medial entorhinal cortex. Previous work has also shown that grid field spacing and acetylcholine levels change as a function of the novelty to a particular environment. Using in vitro whole cell patch-clamp recordings, our study shows that both resonance frequency and resonance strength vary as a function of cholinergic modulation. Furthermore, our data suggest that these changes in resonance properties are mediated through modulation of h-current and m-current.

A phase code for memory could arise from circuit mechanisms in entorhinal cortex.

Neurophysiological data reveals intrinsic cellular properties that suggest how entorhinal cortical neurons could code memory by the phase of their firing. Potential cellular mechanisms for this phase coding in models of entorhinal function are reviewed. This mechanism for phase coding provides a substrate for modeling the responses of entorhinal grid cells, as well as the replay of neural spiking activity during waking and sleep. Efforts to implement these abstract models in more detailed biophysical compartmental simulations raise specific issues that could be addressed in larger scale population models incorporating mechanisms of inhibition.