How to optimize communication about animal and animal-free research methods.

There is a long-standing debate about experimental non-human animals and animal-free methods in scientific research. Among the various stakeholders involved in the debate are the scientists. During media broadcasts we, animal researchers and animal-free methods researchers, were positioned as 'opponents'. In this essay we describe our initial rational thoughts and emotions after these events, and how we came together to explore our common ground on animal(-free) experimentation. Realizing that all models have advantages and limitations, our common ground lies in the principles of good scientific research and responsible experimentation. Our communication emanating from the broadcasts has been instrumental in improving communication on animal(-free) experimentation issues by teaming up. We strongly believe that this is essential for making well-informed decisions for the methods we are using now and will be using in the future.

The TD Drive: A Parametric, Open-Source Implant for Multi-Area Electrophysiological Recordings in Behaving and Sleeping Rats.

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and consolidation of memories, are two examples that rely heavily on functional connectivity across the brain. In addition, investigating hemispheric similarities and/or differences goes hand in hand with these multi-area interactions. Electrophysiological studies trying to further elucidate these complex processes thus depend on recording brain activity at multiple locations simultaneously and often in a bilateral fashion. Presented here is a 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously. The open-source design was created employing parametric design principles, allowing prospective users to easily adapt the drive design to their needs by simply adjusting high-level parameters, such as anterior-posterior and mediolateral coordinates of the recording electrode locations. The implant design was validated in n = 20 Lister Hooded rats that performed different tasks. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze using two different commercial recording systems and headstages. Thus, presented here is the adaptable design and assembly of a new electrophysiological implant, facilitating fast preparation and implantation.

Rodent maze studies: from following simple rules to complex map learning.

More than 100 years since the first maze designed for rodent research, researchers now have the choice of a variety of mazes that come in many different shapes and sizes. Still old designs get modified and new designs are introduced to fit new research questions. Yet, which maze is the most optimal to use or which training paradigm should be applied, remains up for debate. In this review, we not only provide a historical overview of maze designs and usages in rodent learning and memory research, but also discuss the possible navigational strategies the animals can use to solve each maze. Furthermore, we summarize the different phases of learning that take place when a maze is used as the experimental task. At last, we delve into how training and maze design can affect what the rodents are actually learning in a spatial task.

Episodic memory development: Bridging animal and human research.

Human episodic memory is not functionally evident until about 2 years of age and continues to develop into the school years. Behavioral studies have elucidated this developmental timeline and its constituent processes. In tandem, lesion and neurophysiological studies in non-human primates and rodents have identified key neural substrates and circuit mechanisms that may underlie episodic memory development. Despite this progress, collaborative efforts between psychologists and neuroscientists remain limited, hindering progress. Here, we seek to bridge human and non-human episodic memory development research by offering a comparative review of studies using humans, non-human primates, and rodents. We highlight critical theoretical and methodological issues that limit cross-fertilization and propose a common research framework, adaptable to different species, that may facilitate cross-species research endeavors.

The pros and cons of using automated sleep scoring in sleep research.

Sleep scoring plays a pivotal role both in sleep research and in clinical practice. Traditionally, this process has relied on manual scoring by human experts, but it is marred by time constraints, and inconsistencies between different scorers. Consequently, the quest for more efficient and reliable approaches has sparked a great interest in the realm of automatic sleep-scoring methods. In this article, we provide an exploration of the merits and drawbacks of automatic sleep scoring, alongside the pressing challenges and critical considerations that demand attention in this evolving field.

CBD lengthens sleep but shortens ripples and leads to intact simple but worse cumulative memory.

Cannabidiol (CBD) is on the rise as over-the-counter medication to treat sleep disturbances, anxiety, pain, and epilepsy due to its action on the excitatory/inhibitory balance in the brain. However, it remains unclear if CBD also leads to adverse effects on memory via changes of sleep macro- and microarchitecture. To investigate the effect of CBD on sleep and memory consolidation, we performed two experiments using the object space task testing for both simple and cumulative memory in rats. We show that oral CBD administration extended the sleep period but changed the properties of rest and non-REM sleep oscillations (delta, spindle, ripples). Specifically, CBD also led to less long (>100 ms) ripples and, consequently, worse cumulative memory consolidation. In contrast, simple memories were not affected. In sum, we can confirm the beneficial effect of CBD on sleep; however, this comes with changes in oscillations that negatively impact memory consolidation.

Increased cortical plasticity leads to memory interference and enhanced hippocampal-cortical interactions.

Our brain is continuously challenged by daily experiences. Thus, how to avoid systematic erasing of previously encoded memories? While it has been proposed that a dual-learning system with 'slow' learning in the cortex and 'fast' learning in the hippocampus could protect previous knowledge from interference, this has never been observed in the living organism. Here, we report that increasing plasticity via the viral-induced overexpression of RGS14414 in the prelimbic cortex leads to better one-trial memory, but that this comes at the price of increased interference in semantic-like memory. Indeed, electrophysiological recordings showed that this manipulation also resulted in shorter NonREM-sleep bouts, smaller delta-waves and decreased neuronal firing rates. In contrast, hippocampal-cortical interactions in form of theta coherence during wake and REM-sleep as well as oscillatory coupling during NonREM-sleep were enhanced. Thus, we provide the first experimental evidence for the long-standing and unproven fundamental idea that high thresholds for plasticity in the cortex protect preexisting memories and modulating these thresholds affects both memory encoding and consolidation mechanisms.

Defensive and offensive behaviours in a Kleefstra syndrome mouse model.

Kleefstra syndrome in humans is characterized by a general delay in development, intellectual disability and autistic features. The mouse model of this disease (Ehmt1±) expresses anxiety, autistic-like traits, and aberrant social interactions with non-cagemates. To investigate how Ehmt1± mice behave with unfamiliar conspecifics, we allowed adult, male animals to freely interact for 10 min in a neutral, novel environment within a host-visitor setting. In trials where the Ehmt1± mice were hosts, there were defensive and offensive behaviors. Our key finding was that Ehmt1± mice displayed defensive postures, attacking and biting; in contrast, wild-type (WT) interacting with other WT did not enact such behaviors. Further, if there was a fight between an Ehmt1± and a WT mouse, the Ehmt1± animal was the most aggressive and always initiated these behaviors.

Memory reactivations during sleep: a neural basis of dream experiences?

Newly encoded memory traces are spontaneously reactivated during sleep. Since their discovery in the 1990s, these memory reactivations have been discussed as a potential neural basis for dream experiences. New results from animal and human research, as well as from the rapidly growing field of sleep and dream engineering, provide essential insights into this question, and reveal both strong parallels and disparities between the two phenomena. We suggest that, although memory reactivations may contribute to subjective experiences across different states of consciousness, they are not likely to be the primary neural basis of dreaming. We identify important limitations in current research paradigms and suggest novel strategies to address this question empirically.

Execution of new trajectories toward a stable goal without a functional hippocampus.

The hippocampus is a critical component of a mammalian spatial navigation system, with the firing sequences of hippocampal place cells during sleep or immobility constituting a "replay" of an animal's past trajectories. A novel spatial navigation task recently revealed that such "replay" sequences of place fields can also prospectively map onto imminent new paths to a goal that occupies a stable location during each session. It was hypothesized that such "prospective replay" sequences may play a causal role in goal-directed navigation. In the present study, we query this putative causal role in finding only minimal effects of muscimol-induced inactivation of the dorsal and intermediate hippocampus on the same spatial navigation task. The concentration of muscimol used demonstrably inhibited hippocampal cell firing in vivo and caused a severe deficit in a hippocampal-dependent "episodic-like" spatial memory task in a watermaze. These findings call into question whether "prospective replay" of an imminent and direct path is actually necessary for its execution in certain navigational tasks.

Sleep scoring in rodents: Criteria, automatic approaches and outstanding issues.

There is nothing we spend as much time on in our lives as we do sleeping, which makes it even more surprising that we currently do not know why we need to sleep. Most of the research addressing this question is performed in rodents to allow for invasive, mechanistic approaches. However, in contrast to human sleep, we currently do not have shared and agreed upon standards on sleep states in rodents. In this article, we present an overview on sleep stages in humans and rodents and a historical perspective on the development of automatic sleep scoring systems in rodents. Further, we highlight specific issues in rodent sleep that also call into question some of the standards used in human sleep research.

Sleep deprivation and hippocampal ripple disruption after one-session learning eliminate memory expression the next day.

Memory reactivation during non-rapid-eye-movement ripples is thought to communicate new information to a systems-wide network and thus can be a key player mediating the positive effect of sleep on memory consolidation. Causal experiments disrupting ripples have only been performed in multiday training paradigms, which decrease but do not eliminate memory performance, and no comparison with sleep deprivation has been made. To enable such investigations, we developed a one-session learning paradigm in a Plusmaze and show that disruption of either sleep with gentle handling or hippocampal ripples with electrical stimulation impaired long-term memory. Furthermore, we detected hippocampal ripples and parietal high-frequency oscillations after different behaviors, and a bimodal frequency distribution in the cortical events was observed. Faster cortical high-frequency oscillations increased after normal learning, a change not seen in the hippocampal ripple-disruption condition, consistent with these having a role in memory consolidation.

Spindle-slow oscillation coupling correlates with memory performance and connectivity changes in a hippocampal network after sleep.

After experiences are encoded, post-encoding reactivations during sleep have been proposed to mediate long-term memory consolidation. Spindle-slow oscillation coupling during NREM sleep is a candidate mechanism through which a hippocampal-cortical dialogue may strengthen a newly formed memory engram. Here, we investigated the role of fast spindle- and slow spindle-slow oscillation coupling in the consolidation of spatial memory in humans with a virtual watermaze task involving allocentric and egocentric learning strategies. Furthermore, we analyzed how resting-state functional connectivity evolved across learning, consolidation, and retrieval of this task using a data-driven approach. Our results show task-related connectivity changes in the executive control network, the default mode network, and the hippocampal network at post-task rest. The hippocampal network could further be divided into two subnetworks of which only one showed modulation by sleep. Decreased functional connectivity in this subnetwork was associated with higher spindle-slow oscillation coupling power, which was also related to better memory performance at test. Overall, this study contributes to a more holistic understanding of the functional resting-state networks and the mechanisms during sleep associated to spatial memory consolidation.

Sleep-spindle frequency: Overnight dynamics, afternoon nap effects, and possible circadian modulation.

Homeostatic and circadian processes play a pivotal role in determining sleep structure, timing, and quality. In sharp contrast with the wide accessibility of the electroencephalogram (EEG) index of sleep homeostasis, an electrophysiological measure of the circadian modulation of sleep is still unavailable. Evidence suggests that sleep-spindle frequencies decelerate during biological night. In order to test the feasibility of measuring this marker in common polysomnographic protocols, the Budapest-Munich database of sleep records (N = 251 healthy subjects, 122 females, age range: 4-69 years), as well as an afternoon nap sleep record database (N = 112 healthy subjects, 30 females, age range: 18-30 years) were analysed by the individual adjustment method of sleep-spindle analysis. Slow and fast sleep-spindle frequencies were characterised by U-shaped overnight dynamics, with highest values in the first and the fourth-to-fifth sleep cycle and the lowest values in the middle of the sleeping period (cycles two to three). Age-related attenuation of sleep-spindle deceleration was evident. Estimated phases of the nadirs in sleep-spindle frequencies were advanced in children as compared to other age groups. Additionally, nap sleep spindles were faster than night sleep spindles (0.57 and 0.39 Hz difference for slow and fast types, respectively). The fine frequency resolution analysis of sleep spindles is a feasible method of measuring the assumed circadian modulation of sleep. Moreover, age-related attenuation of circadian sleep modulation might be measurable by assessing the overnight dynamics in sleep-spindle frequency. Phase of the minimal sleep-spindle frequency is a putative biomarker of chronotype.

Disrupting ripples: Methods, results, and caveats in closed-loop approaches in rodents.

Hippocampal ripple oscillations have been associated with memory reactivations during wake and sleep. These reactivations should contribute to working memory and memory consolidation respectively. In the past decade studies have moved from being observational to actively disrupting ripple-related activity in closed-loop approaches to enable causal investigations into their function. All together these studies have been able to provide evidence that wake, task-related ripple activity is important for working memory and planning but less important for stabilisation of spatial representations. Rest and sleep-related ripple activity, in contrast, is important for long-term memory performance and thus memory consolidation. In this review, we summarise results from different closed-loop approaches in rodents. Further, we highlight differences in detection and stimulation methods as well as controls and discuss how these differences could influence outcomes.

Learning-Induced Shifts in Mice Navigational Strategies Are Unveiled by a Minimal Behavioral Model of Spatial Exploration.

Shifts in spatial patterns produced during the execution of a navigational task can be used to track the effects of the accumulation of knowledge and the acquisition of structured information about the environment. Here, we provide a quantitative analysis of mice behavior while performing a novel goal localization task in a large, modular arena, the HexMaze. To demonstrate the effects of different forms of previous knowledge we first obtain a precise statistical characterization of animals' paths with sub-trial resolution and over different phases of learning. The emergence of a flexible representation of the task is accompanied by a progressive improvement of performance, mediated by multiple, multiplexed time scales. We then use a generative mathematical model of the animal behavior to isolate the specific contributions to the final navigational strategy. We find that animal behavior can be accurately reproduced by the combined effect of a goal-oriented component, becoming stronger with the progression of learning, and of a random walk component, producing choices unrelated to the task and only partially weakened in time.

The HexMaze: A Previous Knowledge Task on Map Learning for Mice.

New information is rarely learned in isolation; instead, most of what we experience can be incorporated into or uses previous knowledge networks in some form. Previous knowledge in form of a cognitive map can facilitate knowledge acquisition and will influence how we learn new spatial information. Here, we developed a new spatial navigation task where food locations are learned in a large, gangway maze to test how mice learn a large spatial map over a longer time period-the HexMaze. Analyzing performance across sessions as well as on specific trials, we can show simple memory effects as well as multiple effects of previous knowledge of the map accelerating both online learning and performance increases over offline periods when incorporating new information. We could identify the following three main phases: (1) learning the initial goal location; (2) faster learning after 2 weeks when learning a new goal location; and then (3) the ability to express one-session learning, leading to long-term memory effect after 12 weeks. Importantly, we are the first to show that buildup of a spatial map is dependent on how much time passes, not how often the animal is trained.

Sleep Leads to Brain-Wide Neural Changes Independent of Allocentric and Egocentric Spatial Training in Humans and Rats.

Sleep is important for memory consolidation and systems consolidation in particular, which is thought to occur during sleep. While there has been a significant amount of research regarding the effect of sleep on behavior and certain mechanisms during sleep, evidence that sleep leads to consolidation across the system has been lacking until now. We investigated the role of sleep in the consolidation of spatial memory in both rats and humans using a watermaze task involving allocentric- and egocentric-based training. Analysis of immediate early gene expression in rodents, combined with functional magnetic resonance imaging in humans, elucidated similar behavioral and neural effects in both species. Sleep had a beneficial effect on behavior in rats and a marginally significant effect in humans. Interestingly, sleep led to changes across multiple brain regions at the time of retrieval in both species and in both training conditions. In rats, sleep led to increased gene expression in the hippocampus, striatum, and prefrontal cortex. In the humans, sleep led to an activity increase in brain regions belonging to the executive control network and a decrease in activity in regions belonging to the default mode network. Thus, we provide cross-species evidence for system-level memory consolidation occurring during sleep.

Shining a light on hippocampal remapping.

In this issue of Neuron, McKenzie et al. (2021) test the degree to which pre-existing biases in hippocampal circuits constrict the encoding of new information via artificial induction of place cell remapping. Their results suggest that the hippocampal spatial map encodes new information via pre-existing latent place fields.

How to Control Behavioral Studies for Rodents-Don't Project Human Thoughts onto Them.

In neuroscience research, we often use behavior as an easy tool and assume a straightforward relationship between memory and behavior. However, many factors are often not accounted for and need to be considered when interpreting a behavioral outcome. This opinion article will discuss factors in rodent studies such as handling and how the animal views the world, that will affect whether memory leads to a certain behavior.