The hippocampus is a brain structure that is known to play a critical role in memory formation (Squire, 1992), but the scope and nature of hippocampal memory processing remains elusive. The discovery of place cells in rodents led to the hypothesis that the hippocampus forms a 'cognitive map' that is essential for spatial learning (O’Keefe and Nadel, 1978). In contrast, human studies have indicated a more general role of the hippocampus in the formation of episodic memories (Scoville and Milner, 1957). In support of the latter view, electrophysiological studies in rodents have found that pyramidal neurons in the dorsal CA1 area of the hippocampus can change their firing in response to arbitrary stimuli, both spatial and non-spatial (Berger et al., 1976; Wood et al., 1999; Moita et al., 2003; Komorowski et al., 2009). However, the functional significance of these observations remains controversial because many non-spatial tasks are studied in restrained animals or under circumstances where spatial and non-spatial influences on hippocampal firing cannot be unequivocally dissociated (O’Keefe, 1999).

To overcome this challenge, we selected a simple learning task—trace eyeblink conditioning—that can be performed independently of spatial location. In this task, a neutral conditioned stimulus (CS, a tone) is followed, after a short delay, by an unconditioned stimulus (US) that evokes an involuntary blink (Figure 1A). With repeated presentations, the subject learns that the CS predicts the US and begins to blink in anticipation (Figure 1C–D). Learning the CS-US association has been shown to be hippocampally dependent in many species, including humans and rodents (Clark and Squire, 1998; Kim et al., 1995; Takehara et al., 2003). Previous electrophysiological studies have found that dorsal CA1 pyramidal neurons change their firing following the CS onset (Berger et al., 1976, 1983; Weiss et al., 1996; Weible et al., 2006), suggesting that these cells encode the non-spatial stimulus. However, these experiments did not characterize the spatial tuning properties of the recorded neurons.

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Here, we disentangle the spatial and non-spatial influences on hippocampal firing by studying trace eyeblink conditioning in freely moving rats. Consistent with previous studies, we find that the CS can evoke significant changes in the firing rate of dorsal CA1 neurons. Additionally, we find that these apparent CS responses are spatially modulated, but surprisingly, they are absent when the animal is moving. These CS-evoked changes occur only when the animal is inactive, produce firing rates that match normal place cell activity, and are accompanied by a cessation of ripples. These observations are more consistent with an arousal-mediated resumption of place cell firing rather than a genuine encoding of the non-spatial stimuli. These results suggest that neurons in dorsal CA1, a major output stage of the hippocampus, transmit exclusively spatial signals even in this hippocampally dependent non-spatial task.

To simultaneously map the spatial and non-spatial influences on hippocampal firing, we trained adult rats in trace eyeblink conditioning while they traversed a linear track (Figure 1B), an environment in which the spatial coding of hippocampal neurons has been well-characterized (O’Keefe, 2007). We presented the eyeblink trials at randomized locations throughout the environment, which enables us to dissociate spatial and non-spatial influences on neuronal activity.

Using chronically implanted tetrode arrays, we recorded from pyramidal cells in the dorsal CA1 region of the hippocampus (an average of 36 cells per training session for a total of 1264). Consistent with previous studies, we found that a subset of cells significantly changed their firing rate following the CS onset (p<0.01 for 10% of cells, two-sided Wilcoxon signed-rank test). However, an analysis of single-trial responses reveals that these cell-level statistics belie a high trial-to-trial variability, and none of the recorded cells respond consistently to the CS onset (Figure 1E–F).

What is the source of this trial-to-trial variability? Does spatial tuning play a role? To address this question, we mapped each cell’s spatial tuning by measuring its average firing rate as a function of the animal’s location while moving around the environment. Cells that were active in the environment had directional place fields distributed throughout the linear track (Figure 2A). The remaining cells had low firing rates everywhere, consistent with place cells that lack a place field in this environment.

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These spatial firing rate maps—which were constructed with eyeblink trials omitted—reveal that a cell’s spatial tuning exerts a strong influence on its response to non-spatial stimuli. For example, the intermittently CS-responsive neuron shown in Figure 1E had a place field in the right endbox, and the trials in which it fired corresponded to the trials presented within this place field (Figure 2B). To visualize the influence of this spatial tuning, we ordered the trials by the place field intensity at the trial delivery location (Figure 2B, bottom panel). Overall, we find that the CS evokes an increase in firing when delivered within a cell’s place field, and silences the cell when delivered outside its place field (Figure 2C–E). This correlation between CS-evoked firing and place field intensity also holds at the single-cell level (Figure 2F). These observations indicate that there are no CA1 neurons that respond consistently to the CS in a space-invariant fashion. Instead, the non-spatial stimulus modulates the firing of place cells.

What is the nature of this modulation? If the CS amplifies ongoing place cell activity, as Figure 2E suggests, then we would expect that CS responses would be most prominent when place cells are actively engaged, such as during running. Surprisingly, we find the opposite: CS-evoked changes in firing rate are absent when the animal is running (Figure 3A) and occur only when the animal is inactive (Figure 3B). Importantly, the lack of CS-evoked changes while running is not accompanied by an impairment of task performance (Figure 3—figure supplement 1). When the animal is sitting, the CS-evoked change in firing actually reflects the fact that the pre-trial firing rates differ from the spatially predicted rates (Figure 3C, grey markers). After the CS onset, firing rates re-align with the place field intensity at the present location (Figure 3C, blue markers). The US evokes a response that briefly exceeds the spatially predicted rates (Figure 3C, red markers), but analysis of this phenomenon is potentially confounded by head movement associated with the unconditioned response.

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Why do pre-trial firing rates in sitting animals differ from the place field intensity? During periods of inactivity, animals may enter a state of quiet wakefulness, in which the firing of place cells is not exclusively governed by the animal’s present location. Instead, the hippocampus exhibits other patterns of firing, such as synchronous bursts (ripples) that may engage neurons with distant place fields (Foster and Wilson, 2006; Csicsvari et al., 2007; Diba and Buzsáki, 2007). We find that when the animal is sitting, ripples spontaneously occur during the pre-trial period, and abruptly cease following the CS onset (Figure 4A). Furthermore, CS-evoked changes in firing rate are more pronounced in trials with detected ripples (Figure 4B) and the firing of these cells outside their place fields is closely associated with the ripple events (Figure 4C). These observations suggest that the animal is indeed in a state of quiet wakefulness during 'sitting' trials, and that the CS triggers an arousal response that leads to a resumption of place cell firing.

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Finally, we investigated the effect of the CS on hippocampal theta oscillations. Previous studies have reported that the CS can reset the theta rhythm, aligning the phase of theta to the CS onset (Moita et al., 2003; Nokia et al., 2010; Darling et al., 2011). To distinguish a genuine phase reset from stimulus-evoked initiation of theta oscillations, it is important to restrict our analysis to trials with ongoing theta activity prior to the CS onset. The prominent theta oscillations during running provide the ideal conditions for this analysis (Figure 5A). Under these conditions, we found that both the CS and US can evoke a sustained alignment of theta phase, but only in one out of three animals (Figure 5D). Even when theta phase alignment did occur, the new phase remained highly correlated with the pre-stimulus phase (Figure 5E), indicating that the observed alignment is due to a subtle phase shift rather than a reset of the theta rhythm. These results confirm that it is possible for the eyeblink stimuli to perturb ongoing theta oscillations, but the absence of this perturbation in two out of three animals shows that it is not necessary to perform the task.

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These findings require a re-evaluation of how dorsal CA1 pyramidal neurons may encode non-spatial information. The simplest form of non-spatial coding would be a purely non-spatial CS cell (Figure 6A) that exhibits a consistent change in firing to the CS independent of the animal’s location. None of the 1264 pyramidal cells responded in this purely non-spatial manner.

Figure 6

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Previous studies that found spatial correlates in hippocampal responses to non-spatial stimuli have hypothesized that these cells encode the conjunction of a specific non-spatial stimulus with a spatial context (Wood et al., 1999; Moita et al., 2003). A conjunction CS-place cell (Figure 6B) would change its firing in a location-dependent manner. At first glance, our data appear to fit this model—the CS responses of place cells are gated by their place fields (Figure 2)—but our key observation is that CS-evoked changes in firing rate disappear when the animal is running (Figure 3). This new finding rejects the conjunctive model because these cells can no longer encode the CS-place conjunction in this behavioral state.

A model that only relies on the known properties of place cell firing provides a more compelling explanation of the experimental observations (Figure 6C). In particular, place cells exhibit place-specific firing only when the animal is moving or alert, and exhibit a different pattern of firing when the animal is in a state of quiet wakefulness. If the CS were able to trigger an arousal response, this would manifest as an increase in firing within a cell’s place field (due to activation of place-specific firing) and a decrease in firing outside the place field (due to cessation of ripple-related firing). Consistent with this hypothesis, we find that in sitting animals, the CS indeed activates place-specific firing, silences out-of-field firing, and abolishes ripples. When the animal is already alert, such as during running, we observe no additional modulation beyond normal place field firing.

Hence the most parsimonious explanation of the experimental data is that dorsal CA1 neurons are all place cells and the apparent responses to the non-spatial stimuli are due to an arousal-mediated resumption of place-specific firing.

These observations clarify the nature of previously-reported patterns of hippocampal activity in eyeblink conditioning. Studies of inactive subjects in a fixed location, such as those with restrained animals (Berger et al., 1983; Weiss et al., 1996; Weible et al., 2006), would find that a subset of cells—those with a place field overlapping the experiment location—consistently increase their firing following the stimulus presentation. Most cells would not have place fields overlapping this location and would be silenced following the stimulus presentation.

The arousal-mediated nature of the hippocampal response can also account for a variety of learning-related phenomena. In particular, any salient stimulus should be able to evoke these hippocampal responses, regardless of whether the task is hippocampally dependent (Berger et al., 1983; Moita et al., 2003; Abe et al., 2014). The salience of the stimulus, and hence its ability to trigger alertness, may change as the subject learns the predictive power of the stimulus, producing a stereotyped evolution of hippocampal activity across learning (McEchron and Disterhoft, 1997). On the other hand, manipulations that decrease the salience of the CS, such as habituation to the unpaired stimuli presented to control animals, would reduce the apparent response (Berger et al., 1976; Weiss et al., 1996).

Previous studies have also reported that the CS can reset the phase of hippocampal theta oscillations (Nokia et al., 2010; Darling et al., 2011). Given the lack of CS-evoked firing rate changes while running, could the CS onset be encoded by this phase reset instead? Analogous to previous studies, we find evidence for an alignment of theta phase to the CS onset. However, a direct comparison of pre- and post-stimulus phase reveals that this effect is due to a slight perturbation of the ongoing theta phase (Figure 5). In particular, this analysis rejects the notion of theta reset to a specific phase. Furthermore, this phase perturbation is present in only one out of three animals, indicating that it is not necessary for performing the task. Moreover, we did not find evidence that theta phase precession was affected by the CS onset (Figure 5—figure supplement 4). Note, however, that this analysis does not rule out the possibility that the CS may modulate the degree of theta phase-locking across brain areas.

Finally, extensive lesion studies have demonstrated the critical importance of the hippocampus in trace eyeblink conditioning (Kim et al., 1995; Takehara et al., 2003). How do our observations fit with this literature? We found that CS-evoked changes in dorsal CA1 firing rates and theta phase are not necessary for task performance—these responses are absent during running, but performance is not impaired—yet dorsally restricted lesions still produce learning deficits (Takehara et al., 2003). Several possible explanations may account for this apparent discrepancy: (1) Dorsal hippocampal lesions may disrupt activity in other hippocampal areas (e.g. ventral CA1), which may contain genuinely CS-responsive neurons. (2) The encoding of the CS may be more subtle than changes in firing rate, but still detectable by postsynaptic targets. (3) The critical engagement of the hippocampus may not occur only during the trial itself. Memory consolidation is hippocampally dependent and proceeds without additional training (Takehara et al., 2003; Takehara-Nishiuchi and McNaughton, 2008), indicating that some eyeblink-related activity occurs well after the stimulus presentation. (4) Even though spatial information may appear irrelevant in this task, the presence of ongoing place-specific firing may still be necessary for learning space-invariant responses. For example, awareness of the context may be necessary for the animal to attend to the appropriate stimuli, or learning a context-dependent association may be a necessary stepping-stone to developing a space-invariant association (Nadel and Willner, 1980).

These findings suggest that the dorsal hippocampus transmits exclusively spatial signals, even in a hippocampally dependent non-spatial task, and constrain the ways in which it may contribute to memory processing. These experiments also highlight the importance of accounting for brain state in studies where alertness changes may confound the observed selectivity to experimental stimuli.

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Author details

1. Kevin Q Shan

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

   Contribution

   KQS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-2621-1274

2. Evgueniy V Lubenov

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

   Contribution

   EVL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-1099-944X

3. Maria Papadopoulou

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

   Contribution

   MP, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Athanassios G Siapas

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

   Contribution

   AGS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   thanos@caltech.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-8837-678X

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