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.

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'.

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.

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.

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.

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.

Declarative memory consolidation in humans: a prospective functional magnetic resonance imaging study.

Retrieval of recently acquired declarative memories depends on the hippocampus, but with time, retrieval is increasingly sustainable by neocortical representations alone. This process has been conceptualized as system-level consolidation. Using functional magnetic resonance imaging, we assessed over the course of three months how consolidation affects the neural correlates of memory retrieval. The duration of slow-wave sleep during a nap/rest period after the initial study session and before the first scan session on day 1 correlated positively with recognition memory performance for items studied before the nap and negatively with hippocampal activity associated with correct confident recognition. Over the course of the entire study, hippocampal activity for correct confident recognition continued to decrease, whereas activity in a ventral medial prefrontal region increased. These findings, together with data obtained in rodents, may prompt a revision of classical consolidation theory, incorporating a transfer of putative linking nodes from hippocampal to prelimbic prefrontal areas.

Sparse, environmentally selective expression of Arc RNA in the upper blade of the rodent fascia dentata by brief spatial experience.

After a spatial behavioral experience, hippocampal CA1 pyramidal cells express the activity-regulated, immediate early gene Arc in an environment-specific manner, and in similar proportions ( 40%) to cells exhibiting electrophysiologically recorded place fields under similar conditions. Theoretical accounts of the function of the fascia dentata suggest that it plays a role in pattern separation during encoding. The hypothesis that the dentate gyrus (DG) uses a sparse, and thus more orthogonal, coding scheme has been supported by the observation that, while granule cells do exhibit place fields, most are silent in a given environment. To quantify the degree of sparsity of DG coding and its corresponding ability to generate distinct environmental representations, behaviorally induced Arc expression was assessed using in situ hybridization coupled with confocal microscopy. The proportion of Arc(+) cells in the "upper blade" of the fascia dentata (i.e., the portion that abuts CA1) increased in an environment-specific fashion, approximately 4-fold above cage-control activity, after behavioral exploration. Surprisingly, cells in the lower blade of the fascia dentata, which are capable of expressing Arc following electrical stimulation, exhibited virtually no behaviorally-induced Arc expression. This difference was confirmed using "line scan" analyses, which also revealed no patterns or gradients of activity along the upper blade of the DG. The expression of Arc in the upper blade was quantitatively similar after exploring familiar or novel environments. When animals explored two different environments, separated by 20 min, a new group of cells responded to the second environment, whereas two separated experiences in the same environment did not activate a new set of granular cells. Thus, granule cells generate distinct codes for different environments. These findings suggest differential contribution of upper and lower blade neurons to plastic networks and confirm the hypothesis that the DG uses sparse coding that may facilitate orthogonalization of information.

A test of the reverberatory activity hypothesis for hippocampal 'place' cells.

One of several tenable hypotheses that can be proposed to explain the complex dynamics of spatially selective hippocampal neural activity postulates that the region of space over which a given cell receives its external input is actually much smaller than the classical 'place field.' According to this notion, the later portions of the field reflect some form of network hysteresis resulting from 'reverberatory' activity within reentrant, synaptically coupled cell assemblies within the hippocampus. This hypothesis predicts that transient, global inhibition, induced after the onset of firing, might truncate the remainder of the place field. To test this hypothesis, principal afferents to the hippocampus were stimulated bilaterally in rats running on a circular track, evoking widespread inhibition throughout the hippocampus, and abolishing all spike activity from simultaneously recorded populations of CA1 pyramidal cells for periods of 150-300 ms. Stimulation at any point within the place field of a given cell suppressed firing only for such brief intervals, followed by an immediate resumption for the remainder of the field. These results suggest that without additional cellular and/or synaptic mechanisms, reverberatory activity alone within the hippocampus does not account for the shape and spatial extent of place fields.

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.

Coordinated reactivation of distributed memory traces in primate neocortex.

Conversion of new memories into a lasting form may involve the gradual refinement and linking together of neural representations stored widely throughout neocortex. This consolidation process may require coordinated reactivation of distributed components of memory traces while the cortex is "offline," i.e., not engaged in processing external stimuli. Simultaneous neural ensemble recordings from four sites in the macaque neocortex revealed such coordinated reactivation. In motor, somatosensory, and parietal cortex (but not prefrontal cortex), the behaviorally induced correlation structure and temporal patterning of neural ensembles within and between regions were preserved, confirming a major tenet of the trace-reactivation theory of memory consolidation.

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.

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.

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.

Independence of firing correlates of anatomically proximate hippocampal pyramidal cells.

In neocortex, neighboring neurons frequently exhibit correlated encoding properties. There is conflicting evidence whether a similar phenomenon occurs in hippocampus. To assess this quantitatively, a comparison was made of the spatial and temporal firing correlations within and between local groups of hippocampal cells, spaced 350-1400 microm apart. No evidence of clustering was found in a sample of >3000 neurons. Moreover, cells active in two environments were uniformly interspersed at a scale of <100 microm, as assessed by the activity-induced gene Arc. Independence of encoding characteristics implies uncorrelated inputs, which could enhance the capacity of the hippocampus to store arbitrary associations.

Hippocampal place-cell firing during movement in three-dimensional space.

"Place" cells of the rat hippocampus are coupled to "head direction" cells of the thalamus and limbic cortex. Head direction cells are sensitive to head direction in the horizontal plane only, which leads to the question of whether place cells similarly encode locations in the horizontal plane only, ignoring the z axis, or whether they encode locations in three dimensions. This question was addressed by recording from ensembles of CA1 pyramidal cells while rats traversed a rectangular track that could be tilted and rotated to different three-dimensional orientations. Cells were analyzed to determine whether their firing was bound to the external, three-dimensional cues of the environment, to the two-dimensional rectangular surface, or to some combination of these cues. Tilting the track 45 degrees generally provoked a partial remapping of the rectangular surface in that some cells maintained their place fields, whereas other cells either gained new place fields, lost existing fields, or changed their firing locations arbitrarily. When the tilted track was rotated relative to the distal landmarks, most place fields remapped, but a number of cells maintained the same place field relative to the x-y coordinate frame of the laboratory, ignoring the z axis. No more cells were bound to the local reference frame of the recording apparatus than would be predicted by chance. The partial remapping demonstrated that the place cell system was sensitive to the three-dimensional manipulations of the recording apparatus. Nonetheless the results were not consistent with an explicit three-dimensional tuning of individual hippocampal neurons nor were they consistent with a model in which different sets of cells are tightly coupled to different sets of environmental cues. The results are most consistent with the statement that hippocampal neurons can change their "tuning functions" in arbitrary ways when features of the sensory input or behavioral context are altered. Understanding the rules that govern the remapping phenomenon holds promise for deciphering the neural circuitry underlying hippocampal function.

Hippocampal synaptic plasticity is modulated by theta rhythm in the fascia dentata of adult and aged freely behaving rats.

A modulatory role for the hippocampal theta rhythm in synaptic plasticity is suggested by the observations that theta occurs during exploratory behaviors, spatial learning is impaired when the theta rhythm is disrupted, and excitation of hippocampal principal cells is phase-coupled to the theta wave. The theta phase affects the nature of the plasticity induced in urethane-anesthetized rats and in the carbachol-treated in vitro slice preparation, but these oscillations are phenomenologically different from natural theta, and the effects of theta phase on plasticity under natural conditions have not been reported. We therefore examined the effects of theta phase on the magnitude of long-term potentiation (LTP) in awake rats running on a linear track for a food reward. Twelve adult and 10 aged F344 male rats were implanted with a stimulating electrode in the perforant path and a recording electrode in the hilus of the fascia dentata. Stimuli were delivered at the peak or trough of the hilar theta rhythm. In both adult and aged, memory-impaired rats, LTP lasting at least 48 h was induced when stimuli were delivered at the positive theta peak, whereas LTP was not induced when stimuli were delivered at the negative troughs. Consistent with the finding that the threshold for LTP induction is increased at this synapse in old rats, the magnitude of LTP induced at the peak of theta rhythm was significantly lower in old animals. These data confirm that LTP can be modulated by locomotion-induced theta, and that this modulation is at least qualitatively preserved across age.

Reactivation of hippocampal ensemble activity patterns in the aging rat.

In young rats, the pattern of neuronal ensemble activity correlations expressed among hippocampal pyramidal cells during behavior persists during subsequent quiet wakefulness and slow-wave sleep, a process that may facilitate the consolidation of episodic memories. The present study explored the hypothesis that age-related changes in this process might contribute to memory impairments observed during normal aging. Neuronal activity was recorded from CA1 pyramidal cells, and in both young and old rats, there was a strong similarity between the resting epoch activity patterns and those from the preceding behavior epoch. This similarity was strongest during sharp-wave events. There were no detectable differences in the reactivation process or the decay rate between the young and old age groups. Thus, age differences in spatial memory do not appear to be explainable by differences in the spontaneous reactivation of familiar patterns within the hippocampus during the immediate postbehavior period.