Behavior-driven arc expression is reduced in all ventral hippocampal subfields compared to CA1, CA3, and dentate gyrus in rat dorsal hippocampus.

Anatomical connectivity and lesion studies reveal distinct functional heterogeneity along the dorsal-ventral axis of the hippocampus. The immediate early gene Arc is known to be involved in neural plasticity and memory and can be used as a marker for cell activity that occurs, for example, when hippocampal place cells fire. We report here, that Arc is expressed in a greater proportion of cells in dorsal CA1, CA3, and dentate gyrus (DG), following spatial behavioral experiences compared to ventral hippocampal subregions (dorsal CA1 = 33%; ventral CA1 = 13%; dorsal CA3 = 23%; ventral CA3 = 8%; and dorsal DG = 2.5%; ventral DG = 1.2%). The technique used here to obtain estimates of numbers of behavior-driven cells across the dorsal-ventral axis, however, corresponds quite well with samples from available single unit recording studies. Several explanations for the two- to-threefold reduction in spatial behavior-driven cell activity in the ventral hippocampus can be offered. These include anatomical connectivity differences, differential gain of the self-motion signals that appear to alter the scale of place fields and the proportion of active cells, and possibly variations in the neuronal responses to non-spatial information within the hippocampus along its dorso-ventral axis.

Grids from bands, or bands from grids? An examination of the effects of single unit contamination on grid cell firing fields.

Neural recording technology is improving rapidly, allowing for the detection of spikes from hundreds of cells simultaneously. The limiting step in multielectrode electrophysiology continues to be single cell isolation. However, this step is crucial to the interpretation of data from putative single neurons. We present here, in simulation, an illustration of possibly erroneous conclusions that may be reached when poorly isolated single cell data are analyzed. Grid cells are neurons recorded in rodents, and bats, that spike in equally spaced locations in a hexagonal pattern. One theory states that grid firing patterns arise from a combination of band firing patterns. However, we show here that summing the grid firing patterns of two poorly resolved neurons can result in spurious band-like patterns. Thus, evidence of neurons spiking in band patterns must undergo extreme scrutiny before it is accepted. Toward this aim, we discuss single cell isolation methods and metrics.

Nonuniform allocation of hippocampal neurons to place fields across all hippocampal subfields.

The mechanisms governing how the hippocampus selects neurons to exhibit place fields are not well understood. A default assumption in some previous studies was the uniform random draw with replacement (URDWR) model, which, theoretically, maximizes spatial "pattern separation", and predicts a Poisson distribution of the numbers of place fields expressed by a given cell per unit area. The actual distribution of mean firing rates exhibited by a population of hippocampal neurons, however, is approximately exponential or log-normal in a given environment and these rates are somewhat correlated across multiple places, at least under some conditions. The advantage of neural activity-dependent immediate-early gene (IEG) analysis, as a proxy for electrophysiological recording, is the ability to obtain much larger samples of cells, even those whose activity is so sparse that they are overlooked in recording studies. Thus, a more accurate representation of the activation statistics can potentially be achieved. Some previous IEG studies that examined behavior-driven IEG expression in CA1 appear to support URDWR. There was, however, in some of the same studies, an under-recruitment of dentate gyrus granule cells, indicating a highly skewed excitability distribution, which is inconsistent with URDWR. Although it was suggested that this skewness might be related to increased excitability of recently generated granule cells, we show here that CA1, CA3, and subiculum also exhibit cumulative under-recruitment of neurons. Thus, a highly skewed excitability distribution is a general principle common to all major hippocampal subfields. Finally, a more detailed analysis of the frequency distributions of IEG intranuclear transcription foci suggests that a large fraction of hippocampal neurons is virtually silent, even during sleep. Whether the skewing of the excitability distribution is cell-intrinsic or a network phenomenon, and the degree to which this excitability is fixed or possibly time-varying are open questions for future studies. © 2016 Wiley Periodicals, Inc.

An Integrated Platform for in vivo Electrophysiology in Spatial Cognition Experiments.

Spatial cognition research requires behavioral paradigms that can distinguish between different navigational elements, such as allocentric (map-like) navigation and egocentric (e.g., body centered) navigation. To fill this need, we developed a flexible experimental platform that can be quickly modified without the need for significant changes to software and hardware. In this paper, we present this inexpensive and flexible behavioral platform paired with software which we are making freely available. Our behavioral platform serves as the foundation for a range of experiments, and though developed for assessing spatial cognition, it also has applications in the non-spatial domain of behavioral testing. There are two components of the software platform, 'Maze' and 'Stim Trigger'. Both programs can work in conjunction with electrophysiology acquisition systems, allowing for precise time stamping of neural events with behavior. The Maze program includes functionality for automatic reward delivery based on user defined zones. 'Stim Trigger' permits control of brain stimulation via any equipment that can be paired with an Arduino board. We seek to share our software and leverage the potential by expanding functionality in the future to meet the needs of a larger community of researchers.

Hippocampal granule cells opt for early retirement.

Increased excitability and plasticity of adult-generated hippocampal granule cells during a critical period suggests that they may "orthogonalize" memories according to time. One version of this "temporal tag" hypothesis suggests that young granule cells are particularly responsive during a specific time period after their genesis, allowing them to play a significant role in sculpting CA3 representations, after which they become much less responsive to any input. An alternative possibility is that the granule cells active during their window of increased plasticity, and excitability become selectively tuned to events that occurred during that time and participate in later reinstatement of those experiences, to the exclusion of other cells. To discriminate between these possibilities, rats were exposed to different environments at different times over many weeks, and cell activation was subsequently assessed during a single session in which all environments were revisited. Dispersing the initial experiences in time did not lead to the increase in total recruitment at reinstatement time predicted by the selective tuning hypothesis. The data indicate that, during a given time frame, only a very small number of granule cells participate in many experiences, with most not participating significantly in any. Based on these and previous data, the small excitable population of granule cells probably correspond to the most recently generated cells. It appears that, rather than contributing to the recollection of long past events, most granule cells, possibly 90-95%, are effectively "retired." If granule cells indeed sculpt CA3 representations (which remains to be shown), then a possible consequence of having a new set of granule cells participate when old memories are reinstated is that new representations of these experiences might be generated in CA3. Whatever the case, the present data may be interpreted to undermine the standard "orthogonalizer" theory of the role of the dentate gyrus in memory.

Neural ensemble reactivation in rapid eye movement and slow-wave sleep coordinate with muscle activity to promote rapid motor skill learning.

Neural activity patterns of recent experiences are reactivated during sleep in structures critical for memory storage, including hippocampus and neocortex. This reactivation process is thought to aid memory consolidation. Although synaptic rearrangement dynamics following learning involve an interplay between slow-wave sleep (SWS) and rapid eye movement (REM) sleep, most physiological evidence implicates SWS directly following experience as a preferred window for reactivation. Here, we show that reactivation occurs in both REM and SWS and that coordination of REM and SWS activation on the same day is associated with rapid learning of a motor skill. We performed 6 h recordings from cells in rats' motor cortex as they were trained daily on a skilled reaching task. In addition to SWS following training, reactivation occurred in REM, primarily during the pre-task rest period, and REM and SWS reactivation occurred on the same day in rats that acquired the skill rapidly. Both pre-task REM and post-task SWS activation were coordinated with muscle activity during sleep, suggesting a functional role for reactivation in skill learning. Our results provide the first demonstration that reactivation in REM sleep occurs during motor skill learning and that coordinated reactivation in both sleep states on the same day, although at different times, is beneficial for skill learning. This article is part of the Theo Murphy meeting issue 'Memory reactivation: replaying events past, present and future'.

Reactivation of hippocampal ensemble memories during sleep.

Simultaneous recordings were made from large ensembles of hippocampal "place cells" in three rats during spatial behavioral tasks and in slow-wave sleep preceding and following these behaviors. Cells that fired together when the animal occupied particular locations in the environment exhibited an increased tendency to fire together during subsequent sleep, in comparison to sleep episodes preceding the behavioral tasks. Cells that were inactive during behavior, or that were active but had non-overlapping spatial firing, did not show this increase. This effect, which declined gradually during each post-behavior sleep session, may result from synaptic modification during waking experience. Information acquired during active behavior is thus re-expressed in hippocampal circuits during sleep, as postulated by some theories of memory consolidation.

Dynamics of the hippocampal ensemble code for space.

Ensemble recordings of 73 to 148 rat hippocampal neurons were used to predict accurately the animals' movement through their environment, which confirms that the hippocampus transmits an ensemble code for location. In a novel space, the ensemble code was initially less robust but improved rapidly with exploration. During this period, the activity of many inhibitory cells was suppressed, which suggests that new spatial information creates conditions in the hippocampal circuitry that are conducive to the synaptic modification presumed to be involved in learning. Development of a new population code for a novel environment did not substantially alter the code for a familiar one, which suggests that the interference between the two spatial representations was very small. The parallel recording methods outlined here make possible the study of the dynamics of neuronal interactions during unique behavioral events.

Replay of neuronal firing sequences in rat hippocampus during sleep following spatial experience.

The correlated activity of rat hippocampal pyramidal cells during sleep reflects the activity of those cells during earlier spatial exploration. Now the patterns of activity during sleep have also been found to reflect the order in which the cells fired during spatial exploration. This relation was reliably stronger for sleep after the behavioral session than before it; thus, the activity during sleep reflects changes produced by experience. This memory for temporal order of neuronal firing could be produced by an interaction between the temporal integration properties of long-term potentiation and the phase shifting of spike activity with respect to the hippocampal theta rhythm.

The cellular basis of memory.

In the Perspective "Too many rodent carcinogens: Mitogenesis increases mutagenesis" by Bruce N. Ames and Lois Swirsky Gold (31 Aug., p. 970), the last paragraph on page 970 (continuing on page 971) was incorrectly printed. It should have read, "One major group of natural chemicals in the human diet are the chemicals that plants produce to defend themselves, the natural pesticides (4). We calculate that 99.9% (by weight) of the pesticides in our diet are natural. Few natural pesticides have been tested in at least one rodent species, and again about half (27/52) are rodent carcinogens. These 27 occur commonly in plant foods (10). The human diet contains thousands of natural pesticides, and we estimate that the average intake is about 1500 mg per person per day (4). This compares to a total of 0.09 mg per person per day of residues of about 100 synthetic pesticides (4). In addition, of the mold toxins tested at the MTD (induding aflatoxin), 11 out of 16 are rodent carcinogens." Also, in paragraph 3 on page 970, "47,000 8-hydroxydeoxyguanosines per cell" should have been "90,000" per cell.

Spatial selectivity of rat hippocampal neurons: dependence on preparedness for movement.

The mammalian hippocampal formation appears to play a major role in the generation of internal representations of spatial relationships. In rats, this role is reflected in the spatially selective discharge of hippocampal pyramidal cells. The principal metric for coding spatial relationships might be the organism's own movements in space, that is, the spatial relationship between two locations is coded in terms of the movements executed in getting from one to the other. Thus, information from the motor programming systems (or "motor set") may contribute to coding of spatial location by hippocampal neurons. Spatially selective discharge of hippocampal neurons was abolished under conditions of restraint in which the animal had learned that locomotion was impossible. Therefore, hippocampal neuronal activity may reflect the association of movements with their spatial consequences.

Circadian rhythm of synaptic excitability in rat and monkey central nervous system.

Synaptic responses in hippocampal granule cells to stimulation of their afferent fibers from the entorhinal cortex fluctuate with a 24-hour period. The phase of this cycle for rats and monkeys depends on whether the animal is naturally nocturnal or diurnal. In a rat blinded by enucleation, the rhythm persists but drifts out of phase with the rhythm of sighted controls.

NMDA receptor antagonism blocks experience-dependent expansion of hippocampal "place fields".

In agreement with theories of sequence learning, hippocampal place representations expand asymmetrically during repeated route following. This behaviorally induced, experience-dependent expression of neuronal plasticity was blocked by the NMDA(R) antagonist CPP, suggesting that it may result from the temporal asymmetry and associative properties of LTP. NMDA(R) antagonism, however, had no effect on the range of the progressive shift of firing phase of hippocampal cells, relative to the theta rhythm, as the rat traverses the cell's "place field." Thus, when place fields normally expand with experience, the relationship between firing phase and position is altered, as predicted by models that account for "phase precession" on the basis of asymmetry of synaptic connection strengths. These effects of CPP mimic changes that occur during normal aging, suggesting mechanisms by which sequence learning deficits may arise in aged animals.

Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles.

We used fluorescent in-situ hybridization and confocal microscopy to monitor the subcellular distribution of the immediate-early gene Arc. Arc RNA appeared in discrete intranuclear foci within minutes of neuronal activation and subsequently disappeared from the nucleus and accumulated in the cytoplasm by 30 minutes. The time course of nuclear versus cytoplasmic Arc RNA accumulation was distinct, and could therefore be used to infer the activity history of individual neurons at two times. Following sequential exposure of rats to two different environments or to the same environment twice, the proportion of CA1 neurons with cytoplasmic, nuclear or overlapping Arc expression profiles matched predictions derived from ensemble neurophysiological recordings of hippocampal neuronal ensembles. Arc gene induction is thus specifically linked to neural encoding processes.

Computational approaches to hippocampal function.

The results of theoretical work have led researchers to suggest that the hippocampal formation may maximize its memory storage capacity by recoding events into patterns that are as dissimilar to one another, and which use as few neurons per event, as possible.

Imaging neural activity with temporal and cellular resolution using FISH.

Immediate early genes have gained widespread use as neural activity markers in studies of brain function. The recent development of cellular compartment analysis of temporal activity, which combines sensitive fluorescence in situ hybridization and laser scanning confocal microscopy, overcomes the lack of temporal resolution of standard methodologies and allows the tracking of distinct steps in the synthesis and processing of immediate early gene RNAs. Thus, this technique provides information about when individual neurons are activated and allows the visualization, within a single brain, of different neuronal populations engaged by two distinct experiences.

Reactivation of hippocampal cell assemblies: effects of behavioral state, experience, and EEG dynamics.

During slow wave sleep (SWS), traces of neuronal activity patterns from preceding behavior can be observed in rat hippocampus and neocortex. The spontaneous reactivation of these patterns is manifested as the reinstatement of the distribution of pairwise firing-rate correlations within a population of simultaneously recorded neurons. The effects of behavioral state [quiet wakefulness, SWS, and rapid eye movement (REM)], interactions between two successive spatial experiences, and global modulation during 200 Hz electroencephalographic (EEG) "ripples" on pattern reinstatement were studied in CA1 pyramidal cell population recordings. Pairwise firing-rate correlations during often repeated experiences accounted for a significant proportion of the variance in these interactions in subsequent SWS or quiet wakefulness and, to a lesser degree, during SWS before the experience on a given day. The latter effect was absent for novel experiences, suggesting that a persistent memory trace develops with experience. Pattern reinstatement was strongest during sharp wave-ripple oscillations, suggesting that these events may reflect system convergence onto attractor states corresponding to previous experiences. When two different experiences occurred in succession, the statistically independent effects of both were evident in subsequent SWS. Thus, the patterns of neural activity reemerge spontaneously, and in an interleaved manner, and do not necessarily reflect persistence of an active memory (i.e., reverberation). Firing-rate correlations during REM sleep were not related to the preceding familiar experience, possibly as a consequence of trace decay during the intervening SWS. REM episodes also did not detectably influence the correlation structure in subsequent SWS, suggesting a lack of strengthening of memory traces during REM sleep, at least in the case of familiar experiences.

Dentate gyrus and ca1 ensemble activity during spatial reference frame shifts in the presence and absence of visual input.

In rats shuttling between a variably placed landmark of origin and a fixed goal, place fields of hippocampal CA1 cells encode location in two spatial reference frames. On the initial part of the outbound journey, place fields encode location with respect to the origin while on the final segment, place fields are aligned with the goal (Gothard et al., 1996b). An abrupt switch of reference frame can be induced experimentally by shortening the distance between the origin and the goal. Two linked hypotheses concerning this effect were addressed: (1) that the persistent, landmark-referenced firing results from some internal dynamic process (e.g., path integration or "momentum") and is not a result of maintained sensory input from the landmark of origin; and (2) that this hypothetical process is generated by connections either within CA3 or between CA3 and CA1, in which case the effect might be absent from the dentate gyrus. Neuronal ensemble recordings were made simultaneously from CA1 and the dentate gyrus as rats shuttled on a linear track between a variably located box and a goal, under light or dark conditions. The box-referenced firing persisted significantly longer in the dark in both hippocampal subfields, suggesting a competitive interaction between an internal dynamic process and external sensory cues. The similarity between reference frame transitions in the dentate gyrus and the CA1 region suggests that this process probably occurs before CA3, possibly in the entorhinal cortex or subiculum.

Dynamics of hippocampal ensemble activity realignment: time versus space.

Whether hippocampal map realignment is coupled more strongly to position or time was studied in rats trained to shuttle on a linear track. The rats were required to run from a start box and to pause at a goal location at a fixed location relative to stable distal cues (room-aligned coordinate frame). The origin of each lap was varied by shifting the start box and track as a unit (box-aligned coordinate frame) along the direction of travel. As observed by Gothard et al. (1996a), on each lap the hippocampal activity realigned from a representation that was box-aligned to one that was room-aligned. We studied the dynamics of this transition using a measure of how well the moment-by-moment ensemble activity matched the expected activity given the location of the animal in each coordinate frame. The coherency ratio, defined as the ratio of the matches for the two coordinate systems, provides a quantitative measure of the ensemble activity alignment and was used to compare four possible descriptions of the realignment process. The elapsed time since leaving the box provided a better predictor of the occurrence of the transition than any of the three spatial parameters investigated, suggesting that the shift between coordinate systems is at least partially governed by a stochastic, time-dependent process.

The ventral striatum in off-line processing: ensemble reactivation during sleep and modulation by hippocampal ripples.

Previously it has been shown that the hippocampus and neocortex can spontaneously reactivate ensemble activity patterns during post-behavioral sleep and rest periods. Here we examined whether such reactivation also occurs in a subcortical structure, the ventral striatum, which receives a direct input from the hippocampal formation and has been implicated in guidance of consummatory and conditioned behaviors. During a reward-searching task on a T-maze, flanked by sleep and rest periods, parallel recordings were made from ventral striatal ensembles while EEG signals were derived from the hippocampus. Statistical measures indicated a significant amount of reactivation in the ventral striatum. In line with hippocampal data, reactivation was especially prominent during post-behavioral slow-wave sleep, but unlike the hippocampus, no decay in pattern recurrence was visible in the ventral striatum across the first 40 min of post-behavioral rest. We next studied the relationship between ensemble firing patterns in ventral striatum and hippocampal ripples-sharp waves, which have been implicated in pattern replay. Firing rates were significantly modulated in close temporal association with hippocampal ripples in 25% of the units, showing a marked transient enhancement in the average response profile. Strikingly, ripple-modulated neurons in ventral striatum showed a clear reactivation, whereas nonmodulated cells did not. These data suggest, first, the occurrence of pattern replay in a subcortical structure implied in the processing and prediction of reward and, second, a functional linkage between ventral striatal reactivation and a specific type of high-frequency population activity associated with hippocampal replay.