Hippocampal Engrams Generate Variable Behavioral Responses and Brain-Wide Network States.

Freezing is a defensive behavior commonly examined during hippocampal-mediated fear engram reactivation. How these cellular populations engage the brain and modulate freezing across varying environmental demands is unclear. To address this, we optogenetically reactivated a fear engram in the dentate gyrus subregion of the hippocampus across three distinct contexts in male mice. We found that there were differential amounts of light-induced freezing depending on the size of the context in which reactivation occurred: mice demonstrated robust light-induced freezing in the most spatially restricted of the three contexts but not in the largest. We then utilized graph theoretical analyses to identify brain-wide alterations in cFos expression during engram reactivation across the smallest and largest contexts. Our manipulations induced positive interregional cFos correlations that were not observed in control conditions. Additionally, regions spanning putative "fear" and "defense" systems were recruited as hub regions in engram reactivation networks. Lastly, we compared the network generated from engram reactivation in the small context with a natural fear memory retrieval network. Here, we found shared characteristics such as modular composition and hub regions. By identifying and manipulating the circuits supporting memory function, as well as their corresponding brain-wide activity patterns, it is thereby possible to resolve systems-level biological mechanisms mediating memory's capacity to modulate behavioral states.

Rhythmic coordination and ensemble dynamics in the hippocampal-prefrontal network during odor-place associative memory and decision making.

Memory-guided decision making involves long-range coordination across sensory and cognitive brain networks, with key roles for the hippocampus and prefrontal cortex (PFC). In order to investigate the mechanisms of such coordination, we monitored activity in hippocampus (CA1), PFC, and olfactory bulb (OB) in rats performing an odor-place associative memory guided decision task on a T-maze. During odor sampling, the beta (20-30 Hz) and respiratory (7-8 Hz) rhythms (RR) were prominent across the three regions, with beta and RR coherence between all pairs of regions enhanced during the odor-cued decision making period. Beta phase modulation of phase-locked CA1 and PFC neurons during this period was linked to accurate decisions, with a key role of CA1 interneurons in temporal coordination. Single neurons and ensembles in both CA1 and PFC encoded and predicted animals' upcoming choices, with different cell ensembles engaged during decision-making and decision execution on the maze. Our findings indicate that rhythmic coordination within the hippocampal-prefrontal-olfactory bulb network supports utilization of odor cues for memory-guided decision making.

Internally generated time in the rodent hippocampus is logarithmically compressed.

The Weber-Fechner law proposes that our perceived sensory input increases with physical input on a logarithmic scale. Hippocampal 'time cells' carry a record of recent experience by firing sequentially during a circumscribed period of time after a triggering stimulus. Different cells have 'time fields' at different delays up to at least tens of seconds. Past studies suggest that time cells represent a compressed timeline by demonstrating that fewer time cells fire late in the delay and their time fields are wider. This paper asks whether the compression of time cells obeys the Weber-Fechner Law. Time cells were studied with a hierarchical Bayesian model that simultaneously accounts for the firing pattern at the trial level, cell level, and population level. This procedure allows separate estimates of the within-trial receptive field width and the across-trial variability. After isolating across-trial variability, time field width increased linearly with delay. Further, the time cell population was distributed evenly along a logarithmic time axis. These findings provide strong quantitative evidence that the neural temporal representation in rodent hippocampus is logarithmically compressed and obeys a neural Weber-Fechner Law.

Reactivating hippocampal-mediated memories during reconsolidation to disrupt fear.

Memories are stored in the brain as cellular ensembles activated during learning and reactivated during retrieval. Using the Tet-tag system in mice, we label dorsal dentate gyrus neurons activated by positive, neutral or negative experiences with channelrhodopsin-2. Following fear-conditioning, these cells are artificially reactivated during fear memory recall. Optical stimulation of a competing positive memory is sufficient to update the memory during reconsolidation, thereby reducing conditioned fear acutely and enduringly. Moreover, mice demonstrate operant responding for reactivation of a positive memory, confirming its rewarding properties. These results show that interference from a rewarding experience can counteract negative affective states. While memory-updating, induced by memory reactivation, involves a relatively small set of neurons, we also find that activating a large population of randomly labeled dorsal dentate gyrus neurons is effective in promoting reconsolidation. Importantly, memory-updating is specific to the fear memory. These findings implicate the dorsal dentate gyrus as a potential therapeutic node for modulating memories to suppress fear.

Complementary representations of time in the prefrontal cortex and hippocampus.

Episodic memory binds the spatial and temporal relationships between the elements of experience. The hippocampus encodes space through place cells that fire at specific spatial locations. Similarly, time cells fire sequentially at specific time points within a temporally organized experience. Recent studies in rodents, monkeys, and humans have identified time cells with discrete firing fields and cells with monotonically changing activity in supporting the temporal organization of events across multiple timescales. Using in vivo electrophysiological tetrode recordings, we simultaneously recorded neurons from the prefrontal cortex and dorsal CA1 of the hippocampus while rats performed a delayed match to sample task. During the treadmill mnemonic delay, hippocampal time cells exhibited sparser firing fields with decreasing resolution over time, consistent with previous results. In comparison, temporally modulated cells in the prefrontal cortex showed more monotonically changing firing rates, ramping up or decaying with the passage of time, and exhibited greater temporal precision for Bayesian decoding of time at long time lags. These time cells show exquisite temporal resolution both in their firing fields and in the fine timing of spikes relative to the phase of theta oscillations. Here, we report evidence of theta phase precession in both the prefrontal cortex and hippocampus during the temporal delay, however, hippocampal cells exhibited steeper phase precession slopes and more punctate time fields. To disentangle whether time cell activity reflects elapsed time or distance traveled, we varied the treadmill running speed on each trial. While many neurons contained multiplexed representations of time and distance, both regions were more strongly influenced by time than distance. Overall, these results demonstrate the flexible integration of spatiotemporal dimensions and reveal complementary representations of time in the prefrontal cortex and hippocampus in supporting memory-guided behavior.

In a Temporally Segmented Experience Hippocampal Neurons Represent Temporally Drifting Context But Not Discrete Segments.

There is widespread agreement that episodic memory is organized into a timeline of past experiences. Recent work suggests that the hippocampus may parse the flow of experience into discrete episodes separated by event boundaries. A complementary body of work suggests that context changes gradually as experience unfolds. We recorded from hippocampal neurons as male Long-Evans rats performed 6 blocks of an object discrimination task in sets of 15 trials. Each block was separated by removal from the testing chamber for a delay to enable segmentation. The reward contingency reversed from one block to the next to incentivize segmentation. We expected animals to hold two distinct, recurring representations of context to match the two distinct rule contingencies. Instead, we found that overtrained rats began each block neither above nor below chance but by guessing randomly. While many units had clear firing fields selective to the conjunction of objects in places, a significant population also reflected a continuously drifting code both within block and across blocks. Despite clear boundaries between blocks, we saw no neural evidence for event segmentation in this experiment. Rather, the hippocampal ensemble drifted continuously across time. This continuous drift in the neural representation was consistent with the lack of segmentation observed in behavior.SIGNIFICANCE STATEMENT The neuroscience literature yet to reach consensus on how the hippocampus supports the organization of events across time in episodic memory. Initial studies reported stable hippocampal maps segmented by remapping events. However, it remains unclear whether segmentation is an artifact of cue responsivity. Recently, research has shown that the hippocampal code exhibits continuous drift. Drift may represent a continually evolving context; however, it is unclear whether this is an artifact of changing experiences. We recorded dCA1 in rats performing an object discrimination task designed to segment time. Overtrained rats could not anticipate upcoming context switches but used context boundaries to their advantage. Hippocampal ensembles showed neither evidence of alternating between stable contexts nor sensitivity to boundaries, but showed robust temporal drift.

Complementary Functional Organization of Neuronal Activity Patterns in the Perirhinal, Lateral Entorhinal, and Medial Entorhinal Cortices.

It is commonly conceived that the cortical areas of the hippocampal region are functionally divided into the perirhinal cortex (PRC) and the lateral entorhinal cortex (LEC), which selectively process object information; and the medial entorhinal cortex (MEC), which selectively processes spatial information. Contrary to this notion, in rats performing a task that demands both object and spatial information processing, single neurons in PRC, LEC, and MEC, including those in both superficial and deep cortical areas and in grid, border, and head direction cells of MEC, have a highly similar range of selectivity to object and spatial dimensions of the task. By contrast, representational similarity analysis of population activity reveals a key distinction in the organization of information in these areas, such that PRC and LEC populations prioritize object over location information, whereas MEC populations prioritize location over object information. These findings bring to the hippocampal system a growing emphasis on population analyses as a powerful tool for characterizing neural representations supporting cognition and memory. SIGNIFICANCE STATEMENT: Contrary to the common view that brain regions in the "what" and "where" streams distinctly process object and spatial cues, respectively, we found that both streams encode both object and spatial information but distinctly organize memories for objects and space. Specifically, perirhinal cortex and lateral entorhinal cortex represent objects and, within the object-specific representations, the locations where they occur. Conversely, medial entorhinal cortex represents relevant locations and, within those spatial representations, the objects that occupy them. Furthermore, these findings reach beyond simple notions of perirhinal cortex and lateral entorhinal cortex neurons as object detectors and MEC neurons as position detectors, and point to a more complex organization of memory representations within the medial temporal lobe system.

Representation of memories in the cortical-hippocampal system: Results from the application of population similarity analyses.

Here we consider the value of neural population analysis as an approach to understanding how information is represented in the hippocampus and cortical areas and how these areas might interact as a brain system to support memory. We argue that models based on sparse coding of different individual features by single neurons in these areas (e.g., place cells, grid cells) are inadequate to capture the complexity of experience represented within this system. By contrast, population analyses of neurons with denser coding and mixed selectivity reveal new and important insights into the organization of memories. Furthermore, comparisons of the organization of information in interconnected areas suggest a model of hippocampal-cortical interactions that mediates the fundamental features of memory.

A photosensitive surface capable of inducing electrophysiological changes in NG108-15 neurons.

Retinal prostheses promise to be a viable therapy for many forms of blindness. Direct stimulation of neurons using an organic light-sensitive, self-assembled monolayer surface offers a simple alternative to conventional semiconductor technology. For this purpose we have derivatized an indium tin oxide (ITO) substrate with the photosensitive dye, NK5962, using 3-(aminopropyl)trimethoxysilane (APTMS) as cross-linker. The surface was characterized through contact angle goniometry, electrochemical impedance spectroscopy, grazing angle infrared and ultraviolet-visible spectrophotometry. NG108-15 neurons were grown on the ITO-APTMS-NK5962 surface and neural responses from electrical stimulation vs. photostimulation through the ITO-APTMS-NK5962 surface were measured using patch clamp electrophysiology. Under these conditions, photostimulation of depolarized cells caused an approximate 2-fold increase in voltage-gated sodium (Na(+)) current amplitude at a membrane potential of -30mV. Our results demonstrate the feasibility of stimulating neurons, grown on light-sensitive surfaces, with light impulses, which ultimately may facilitate the fabrication of a simple, passive retinal prosthetic.

Extracorporeal photopheresis normalizes some lymphocyte subsets (including T regulatory cells) in chronic graft-versus-host-disease.

Extracorporeal photopheresis (ECP) has been demonstrated to be clinically effective for the treatment of steroid-refractory graft-versus-host-disease (GvHD). Recently, a murine model suggested that ECP may modulate the allo-reactivity, seen in GvHD, by enhancement of T regulatory cells. T regulatory cells are an important component of immune tolerance and reduced levels of them have been observed in chronic GvHD. Samples were taken from eight chronic GvHD patients prior to receiving any ECP therapy and after three months of ECP. Samples were also obtained from eight age- and sex-matched normal controls. Each sample was evaluated for absolute levels of total lymphocytes and CD4(+) and CD8(+) T cells. T regulatory cell numbers were also assessed accordingly: CD4(+)CD25(high), CD4(+)CD25(high)CD69(-), CD4(+)CD25(high)CD152(+) and CD4(+)CD25(high)FOXP3(+). Following three months of ECP, increases in the absolute counts of total lymphocytes, CD4(+) and CD8(+) T cells and T regulatory cells were observed; however, no selective statistical increase in the percentage of T regulatory cell numbers was observed within the CD4(+) T cell compartment. ECP induces an increase in T regulatory cell numbers; however, this is not specific as there is also an enhancement of both total lymphocyte and CD4(+) T cell numbers. The positive effect of ECP may therefore depend on a more generic re-adjustment of immune homeostasis.

The down-regulation of IL1alpha and IL6, in monocytes exposed to extracorporeal photopheresis (ECP)-treated lymphocytes, is not dependent on lymphocyte phosphatidylserine externalization.

Extracorporeal photopheresis (ECP) has been successfully used to treat some inflammatory conditions. Following ECP, lymphocytes become apoptotic and untreated monocytes, exposed to post-ECP lymphocytes, reduce proinflammatory cytokine secretion. This study attempted to establish if this monocyte immunosuppression was linked to phosphatidylserine externalization (detected using Annexin V) on the apoptotic lymphocytes. Using density gradient and magnetic separation, lymphocytes were isolated from three cutaneous T-cell lymphoma and nine chronic graft versus host disease (cGvHD) patients pre-ECP and prior to re-infusion (post-ECP). The collected lymphocytes were cultured overnight and Annexin V levels determined. Peripheral blood was taken from the same patient 20 h later and the monocytes were isolated. The 'fresh' monocytes were introduced to each 20 h pre- and post-ECP lymphocyte culture, stimulated with lipopolysaccharide (LPS) and Brefeldin A and subsequently tested for intracellular tumour necrosis factor alpha, interleukin 1 alpha (IL1alpha), IL1beta, IL6 and IL8. For cGvHD patients, the relative levels of IL1alpha and IL6 were reduced in the untreated, LPS-stimulated monocytes exposed to post-ECP lymphocytes. However, the down-regulation of IL1alpha and IL6 did not correlate to levels of Annexin V-positive lymphocytes. ECP-treated lymphocytes can reduce the ability of LPS-stimulated monocytes to produce some proinflammatory cytokines; however, this effect is not dependent on phosphatidylserine externalization.

Lymphocytes treated by extracorporeal photopheresis can down-regulate cytokine production in untreated monocytes.

BACKGROUND: Pro-inflammatory cytokines are actively involved in graft-versus host-disease (GvHD) aetiology. Treatment of GvHD, using extracorporeal photopheresis (ECP), has demonstrated clinical efficacy. ECP rapidly reduces the number of T cells that produce tumour necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma) and interleukin (IL)2. ECP-treated cells are re-infused immediately after completion of treatment. This study attempted to determine the influence that ECP-treated cells would have on untreated cells following re-infusion. METHODS: Heparinized samples were taken from 10 chronic GvHD patients, pre-ECP and immediately prior to re-infusion (post-ECP). Lymphocytes and monocytes were isolated using magnetic separation. The post-ECP lymphocytes were mixed with pre-ECP monocytes, while the post-ECP monocytes and pre-ECP lymphocytes were combined. After suitable stimulation, the T cells were tested for intracellular TNFalpha, IFNgamma and IL2, while the monocytes were evaluated for TNFalpha, IL1alpha, IL1beta, IL6 and IL8. RESULTS: Although cytokine secretion is decreased in T cells exposed to ECP, pre-ECP T cells were unaffected by post-ECP monocytes. Post-ECP monocytes demonstrated a reduction in cytokine secretion. Furthermore, untreated monocytes down-regulated cytokine production following exposure to ECP-treated lymphocytes. CONCLUSION: ECP has both a direct and indirect immunosuppressive action, both of which may be beneficial in the treatment of GvHD.

Photopheresis up-regulates CD36 on monocytes and reduces CD25(+) and CD28(+) T cell numbers.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective treatment for some malignant T cell proliferations and disorders associated with pathogenic T cell activation. ECP induces apoptosis in lymphocytes and up-regulates the antigen-processing ability of monocytes. Recently, it has been proposed that the anti-tumour response, generated by ECP, can be enhanced by prolonging the co-culture of the ECP-treated lymphocytes and monocytes prior to re-infusion. However, early markers of lymphoid apoptosis and changes in cytokine secretion have been observed immediately following ECP. This study aimed to determine the rapid effects of ECP on significant markers associated with antigen presentation. METHODS: Pre- and post-ECP samples, from 11 chronic graft versus host disease (cGvHD) and 3 cutaneous T cell lymphoma (CTCL) patients, were tested. The monocytes were evaluated for CD36 and CD91 expression, whilst the number of T cells positive for CD25, CD28, CD62L, CD152 and CD154 were determined. The pre- and post-ECP samples were compared statistically to determine change and both pre- and post-samples were compared to age/sex-matched controls. RESULTS: Monocyte expression of CD36 increased to normal immediately post-ECP in both patient groups, whilst the percentage of CD25(+) and CD28(+) T cells fell significantly in the cGvHD cohort following ECP. CONCLUSION: CD36 is an important receptor for the uptake of apoptotic material and its up-regulation would be beneficial in the antigen-processing mechanism proposed for ECP. No enhancement in markers of T cell activation would indicate that ECP-treated lymphocytes play no direct role in the proposed cytotoxic response. However, reduction in CD25 and CD28 may represent a mechanism beneficial in disease states with excess T cell activation.

Extracorporeal photopheresis differentially regulates the expression of phosphorylated STAT-1 and STAT-5 in treated monocytes and T cells, respectively.

BACKGROUND: Extracorporeal photopheresis (ECP) is effective in conditions with opposing immune etiology. ECP induces an immunomodulatory response through simultaneous monocyte activation and apoptosis induction of lymphocytes. However, ECP is also immunosuppressive, downregulating proinflammatory cytokines. Signal transducers and activators of transcription (STATs) are important mediators of cell-signaling systems, including cytokines. Ultraviolet (UV) immunosuppression is linked to reductions in cytokine-induced STAT phosphorylation. OBJECTIVE: The aim of this study was to find whether ECP downregulates STAT phosphorylation. METHODS: Pre- and post-ECP mononuclear cells from cutaneous T-cell lymphoma and chronic graft-versus-host disease patients were stimulated with either IFNbeta, IL2, or IL15. The percentage of IFNbeta-stimulated monocytes positive for phosphorylated STAT-1 (pSTAT-1) and the number of IL2- and IL15-stimulated T cells expressing phosphorylated STAT-5 (pSTAT-5) were determined at 0 and 24 h. RESULTS: Post-ECP, pSTAT-1 levels in monocytes remained unchanged; however, at 24 h post-ECP the number of T cells expressing pSTAT-5 was reduced. CONCLUSION: Following ECP, monocytes retain their ability to phosphorylate STAT-1, while pSTAT-5 expression is lost in lymphocytes. This differential effect of ECP may account for the diverse population of diseases that benefit.

Extracorporeal photopheresis reduces the number of mononuclear cells that produce pro-inflammatory cytokines, when tested ex-vivo.

Extracorporeal photopheresis (ECP) has been shown to be clinically effective in the treatment of many T cell-mediated conditions. ECP's mechanism of action includes the induction of apoptosis and the release of pro-inflammatory cytokines. Recently, we have observed early lymphoid apoptosis, detectable immediately post ECP. We were interested to determine what influence ECP has on pro-inflammatory cytokine secretion at this early pre-infusion stage. Samples from 6 cutaneous T cell lymphoma (CTCL) and 5 graft versus host disease (GvHD) patients were taken pre ECP and immediately post ECP, prior to re-infusion. Following separation, the PBMCs were added to a cell culture medium and stimulated with PMA, Ionomycin, and Brefeldin A for 6 hours. Using flow cytometry, intracellular cytokine expression of IFNgamma and TNFalpha was determined in the T cell population. The monocytes were evaluated for IL6, IFNgamma, IL12, and TNFalpha. For both patient groups, the number of IFNgamma-expressing T cells fell significantly at re-infusion, whilst both T cell- and monocyte-expressing TNFalpha levels were reduced at re-infusion. All other cytokines tested showed no significant change post ECP. For GvHD, pro-inflammatory cytokines have a pathological role. Their down-regulation may have a direct clinical benefit. However, the reduction in the number of IFNgamma- and TNFalpha-expressing mononuclear cells means, at this early stage, it is unlikely that these cytokines assist in the removal of the malignant Th2 cells present in CTCL.