Black box effect: investigating the role of retroactive interference on hippocampal memory mechanisms

Recent studies have shown that decreasing sensory stimulation after learning can enhance memory retention in humans. Amnesic patients and healthy controls expressed significantly better memory for both passages of prose and spatial landmarks when learning was followed by a short period filled with restful wake, rather than an unrelated distractor task (Dewar et al, 2010; Craig et al, 2016). This enhancement was suggested to arise from decreases in memory interference processes. These findings suggest that interference from ongoing sensory stimulation could have a much larger impact on memory and everyday life than previously thought. The aim of this thesis was therefore to investigate the role of retroactive interference in hippocampal-dependent memory consolidation, and to explore the neural mechanisms behind this episodic memory enhancement. To this end, I tested the effects of reducing different types of interference after spatial learning on memory retention in rats. A spatial memory task was used that required no reward, instead using the animal’s natural tendency to detect and explore novelty. To exploit this behaviour experimentally to test memory retention, I used the novel object location (nOL) recognition task. My protocol consisted of a single training trial, during which animals could explore two copies of the same novel object placed in an open field arena. Memory for the object locations was then tested 6h or 24h later, when animals were returned to the arena in which now one of these objects was moved to a novel location. Animals that preferred to explore the object at the novel location expressed memory for the location the objects occupied during the training trial. The role of interference on object location memory was assessed by exposing the animals to different, highly-familiar stimuli (i.e., dark or normally lit holding box, home cage, or cagemate in a holding box) during the 1 h period directly following the training trial. We used gentle handling to prevent rats from falling asleep during this period. I found that animals expressed robust nOL memory when exposed to a dark familiar holding box after learning, but not when they were exposed to their home cage, replicating the memory enhancement effect following reduction of visual stimulation seen in humans. Further experiments sought to isolate what aspects of the dark holding box promoted memory retention as compared to the home cage. To this end, after learning, animals were put into their home cages with their cage mates, which was placed in either an enclosed normally lit (white light) box (WB), or an enclosed dark (red light) box (RB). Neither group expressed memory, suggesting that the black box effect was dependent on animals being socially isolated. Exposure to the WB when alone also prevented the expression of nOL memory. Yet, animals exposed to the RB without cage mates expressed object location memory, establishing that the black holding box effect was dependent on animals being socially isolated and with reduced visual stimulation. These results suggested that interference not only stems from new learning, but can occur simply when exposed to either highly familiar social or visual stimuli. Object location memory is known to depend on the hippocampus. The activity of pyramidal neurons within the hippocampus (place cells) represents the location of an animal within its environment. This activity is context-dependent, and has been shown to be modulated by the manipulation of objects within these environments (Deshmukh et al, 2013; Burke et al, 2011). Therefore, to explore the neural mechanisms underpinning the ‘black box effect’, I recorded place cells in the dorsal CA1 of rats. I first focused on the spatially-selective firing of place cells to study whether post-learning stimulation could affect the spatial stability of place cell firing within a novel environment, thereby causing memory interference. Animals explored a novel environment for 10 min, after which they spent 3 h awake in either the WB or RB. Then, 6 h after the initial exposure, animals explored the same environment again. Analysis of place cell firing indicated that whilst the overall firing and spatial properties of place cells were not different between groups, the stability of place fields between the initial and repeated exposures was significantly enhanced in the dark (RB) box group. Therefore, reducing visual stimulation after learning promoted place field stability, consistent with the behavioural results. To determine whether these changes in place field stability correlated to the strength of object location memory, a third set of experiments investigated the influence of objects on place field expression during a nOL behavioural task. As seen previously, implanted rats expressed object location memory for 6 h when exposed to the RB, but not the WB, after learning. In contrast to these findings, no differences in the firing and spatial properties of place fields both over and between sessions were found between the WB and RB groups. The introduction, movement and removal of objects, however, did affect various measures of place field stability and synchronicity. The apparent object-place field relationship was investigated further, and results suggested that place fields were more likely to be expressed away from objects during the probe trial if the animal had significant memory for the object locations. Overall, the results reported in my thesis show that long-term memory formation, in terms of behavioural as well a subset of electrophysiological measures, benefits from reduced sensory stimulation after learning. These findings highlight that even low levels of sensory stimulation can have a drastic impact on spatial memory and correlated neural activity. This has important implications for experimental design, as well as life outside of the laboratory

The Black Box effect: sensory stimulation after learning interferes with the retention of long-term object location memory in rats

Reducing sensory experiences during the period that immediately follows learning improves long-term memory retention in healthy humans, and even preserves memory in patients with amnesia. To date, it is entirely unclear why this is the case, and identifying the neurobiological mechanisms underpinning this effect requires suitable animal models, which are currently lacking. Here, we describe a straightforward experimental procedure in rats that future studies can use to directly address this issue. Using this method, we replicated the central findings on quiet wakefulness obtained in humans: We show that rats that spent 1 h alone in a familiar dark and quiet chamber (the Black Box) after exploring two objects in an open field expressed long-term memory for the object locations 6 h later, while rats that instead directly went back into their home cage with their cage mates did not. We discovered that both visual stimulation and being together with conspecifics contributed to the memory loss in the home cage, as exposing rats either to light or to a cage mate in the Black Box was sufficient to disrupt memory for object locations. Our results suggest that in both rats and humans, everyday sensory experiences that normally follow learning in natural settings can interfere with processes that promote long-term memory retention, thereby causing forgetting in form of retroactive interference. The processes involved in this effect are not sleep-dependent because we prevented sleep in periods of reduced sensory experience. Our findings, which also have implications for research practices, describe a potentially useful method to study the neurobiological mechanisms that might explain why normal sensory processing after learning impairs memory both in healthy humans and in patients suffering from amnesia

The medial entorhinal cortex is necessary for the stimulus control over hippocampal place fields by distal, but not proximal, landmarks

A fundamental property of place cells in the hippocampus is the anchoring of their firing fields to salient landmarks within the environment. However, it is unclear how such information reaches the hippocampus. In the current experiment, we tested the hypothesis that the stimulus control exerted by distal visual landmarks requires input from the medial entorhinal cortex (MEC). Place cells were recorded from mice with ibotenic acid lesions of the MEC (n = 7) and from sham-lesioned mice (n = 6) following 90° rotations of either distal landmarks or proximal cues in a cue- controlled environment. We found that lesions of the MEC impaired the anchoring of place fields to distal landmarks, but not proximal cues. We also observed that, relative to sham-lesioned mice, place cells in animals with MEC lesions exhibited significantly reduced spatial information and increased sparsity. These results support the view that distal landmark information reaches the hippocampus via the MEC, but that proximal cue information can do so via an alternative neural pathway

Experience-dependent changes in hippocampal spatial activity and hippocampal circuit function are disrupted in a rat model of Fragile X Syndrome

BACKGROUND: Fragile X syndrome (FXS) is a common single gene cause of intellectual disability and autism spectrum disorder. Cognitive inflexibility is one of the hallmarks of FXS with affected individuals showing extreme difficulty adapting to novel or complex situations. To explore the neural correlates of this cognitive inflexibility, we used a rat model of FXS (Fmr1(−/y)). METHODS: We recorded from the CA1 in Fmr1(−/y) and WT littermates over six 10-min exploration sessions in a novel environment—three sessions per day (ITI 10 min). Our recordings yielded 288 and 246 putative pyramidal cells from 7 WT and 7 Fmr1(−/y) rats, respectively. RESULTS: On the first day of exploration of a novel environment, the firing rate and spatial tuning of CA1 pyramidal neurons was similar between wild-type (WT) and Fmr1(−/y) rats. However, while CA1 pyramidal neurons from WT rats showed experience-dependent changes in firing and spatial tuning between the first and second day of exposure to the environment, these changes were decreased or absent in CA1 neurons of Fmr1(−/y) rats. These findings were consistent with increased excitability of Fmr1(−/y) CA1 neurons in ex vivo hippocampal slices, which correlated with reduced synaptic inputs from the medial entorhinal cortex. Lastly, activity patterns of CA1 pyramidal neurons were dis-coordinated with respect to hippocampal oscillatory activity in Fmr1(−/y) rats. LIMITATIONS: It is still unclear how the observed circuit function abnormalities give rise to behavioural deficits in Fmr1(−/y) rats. Future experiments will focus on this connection as well as the contribution of other neuronal cell types in the hippocampal circuit pathophysiology associated with the loss of FMRP. It would also be interesting to see if hippocampal circuit deficits converge with those seen in other rodent models of intellectual disability. CONCLUSIONS: In conclusion, we found that hippocampal place cells from Fmr1(−/y) rats show similar spatial firing properties as those from WT rats but do not show the same experience-dependent increase in spatial specificity or the experience-dependent changes in network coordination. Our findings offer support to a network-level origin of cognitive deficits in FXS. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13229-022-00528-z