Progressive Excitability Changes in the Medial Entorhinal Cortex in the 3xTg Mouse Model of Alzheimer's Disease Pathology.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by memory loss and progressive cognitive impairments. In mouse models of AD pathology, studies have found neuronal and synaptic deficits in hippocampus, but less is known about changes in medial entorhinal cortex (MEC), which is the primary spatial input to the hippocampus and an early site of AD pathology. Here, we measured neuronal intrinsic excitability and synaptic activity in MEC layer II (MECII) stellate cells, MECII pyramidal cells, and MEC layer III (MECIII) excitatory neurons at 3 and 10 months of age in the 3xTg mouse model of AD pathology, using male and female mice. At 3 months of age, before the onset of memory impairments, we found early hyperexcitability in intrinsic properties of MECII stellate and pyramidal cells, but this was balanced by a relative reduction in synaptic excitation (E) compared with inhibition (I; E/I ratio), suggesting intact homeostatic mechanisms regulating MECII activity. Conversely, MECIII neurons had reduced intrinsic excitability at this early time point with no change in synaptic E/I ratio. By 10 months of age, after the onset of memory deficits, neuronal excitability of MECII pyramidal cells and MECIII excitatory neurons was largely normalized in 3xTg mice. However, MECII stellate cells remained hyperexcitable, and this was further exacerbated by an increased synaptic E/I ratio. This observed combination of increased intrinsic and synaptic hyperexcitability suggests a breakdown in homeostatic mechanisms specifically in MECII stellate cells at this postsymptomatic time point, which may contribute to the emergence of memory deficits in AD.SIGNIFICANCE STATEMENT AD causes cognitive deficits, but the specific neural circuits that are damaged to drive changes in memory remain unknown. Using a mouse model of AD pathology that expresses both amyloid and tau transgenes, we found that neurons in the MEC have altered excitability. Before the onset of memory impairments, neurons in layer 2 of MEC had increased intrinsic excitability, but this was balanced by reduced inputs onto the cell. However, after the onset of memory impairments, stellate cells in MEC became further hyperexcitable, with increased excitability exacerbated by increased synaptic inputs. Thus, it appears that MEC stellate cells are uniquely disrupted during the progression of memory deficits and may contribute to cognitive deficits in AD.

A shared neural ensemble links distinct contextual memories encoded close in time.

Recent studies suggest that a shared neural ensemble may link distinct memories encoded close in time. According to the memory allocation hypothesis, learning triggers a temporary increase in neuronal excitability that biases the representation of a subsequent memory to the neuronal ensemble encoding the first memory, such that recall of one memory increases the likelihood of recalling the other memory. Here we show in mice that the overlap between the hippocampal CA1 ensembles activated by two distinct contexts acquired within a day is higher than when they are separated by a week. Several findings indicate that this overlap of neuronal ensembles links two contextual memories. First, fear paired with one context is transferred to a neutral context when the two contexts are acquired within a day but not across a week. Second, the first memory strengthens the second memory within a day but not across a week. Older mice, known to have lower CA1 excitability, do not show the overlap between ensembles, the transfer of fear between contexts, or the strengthening of the second memory. Finally, in aged mice, increasing cellular excitability and activating a common ensemble of CA1 neurons during two distinct context exposures rescued the deficit in linking memories. Taken together, these findings demonstrate that contextual memories encoded close in time are linked by directing storage into overlapping ensembles. Alteration of these processes by ageing could affect the temporal structure of memories, thus impairing efficient recall of related information.

The role of intrinsic excitability in the evolution of memory: Significance in memory allocation, consolidation, and updating.

Memory is a dynamic process that is continuously regulated by both synaptic and intrinsic neural mechanisms. While numerous studies have shown that synaptic plasticity is important in various types and phases of learning and memory, neuronal intrinsic excitability has received relatively less attention, especially regarding the dynamic nature of memory. In this review, we present evidence demonstrating the importance of intrinsic excitability in memory allocation, consolidation, and updating. We also consider the intricate interaction between intrinsic excitability and synaptic plasticity in shaping memory, supporting both memory stability and flexibility.

Psychostimulants and cognition: a continuum of behavioral and cognitive activation.

Psychostimulants such as cocaine have been used as performance enhancers throughout recorded history. Although psychostimulants are commonly prescribed to improve attention and cognition, a great deal of literature has described their ability to induce cognitive deficits, as well as addiction. How can a single drug class be known to produce both cognitive enhancement and impairment? Properties of the particular stimulant drug itself and individual differences between users have both been suggested to dictate the outcome of stimulant use. A more parsimonious alternative, which we endorse, is that dose is the critical determining factor in cognitive effects of stimulant drugs. Herein, we review several popular stimulants (cocaine, amphetamine, methylphenidate, modafinil, and caffeine), outlining their history of use, mechanism of action, and use and abuse today. One common graphic depiction of the cognitive effects of psychostimulants is an inverted U-shaped dose-effect curve. Moderate arousal is beneficial to cognition, whereas too much activation leads to cognitive impairment. In parallel to this schematic, we propose a continuum of psychostimulant activation that covers the transition from one drug effect to another as stimulant intake is increased. Low doses of stimulants effect increased arousal, attention, and cognitive enhancement; moderate doses can lead to feelings of euphoria and power, as well as addiction and cognitive impairment; and very high doses lead to psychosis and circulatory collapse. This continuum helps account for the seemingly disparate effects of stimulant drugs, with the same drug being associated with cognitive enhancement and impairment.

Simultaneous dual-color calcium imaging in freely-behaving mice.

Miniaturized fluorescence microscopes (miniscopes) enable imaging of calcium events from a large population of neurons in freely behaving animals. Traditionally, miniscopes have only been able to record from a single fluorescence wavelength. Here, we present a new open-source dual-channel Miniscope that simultaneously records two wavelengths in freely behaving animals. To enable simultaneous acquisition of two fluorescent wavelengths, we incorporated two CMOS sensors into a single Miniscope. To validate our dual-channel Miniscope, we imaged hippocampal CA1 region that co-expressed a dynamic calcium indicator (GCaMP) and a static nuclear signal (tdTomato) while mice ran on a linear track. Our results suggest that, even when neurons were registered across days using tdTomato signals, hippocampal spatial coding changes over time. In conclusion, our novel dual-channel Miniscope enables imaging of two fluorescence wavelengths with minimal crosstalk between the two channels, opening the doors to a multitude of new experimental possibilities. Teaser: Novel open-source dual-channel Miniscope that simultaneously records two wavelengths with minimal crosstalk in freely behaving animals.

Dissociable contributions of the amygdala and ventral hippocampus to stress-induced changes in defensive behavior.

Stress can have profound consequences on mental health. While much is known about the neural circuits supporting associative memories of stressful events, our understanding of the circuits underlying the non-associative impacts of stress, such as heightened stress sensitivity and anxiety-related behavior, is limited. Here, we demonstrate that the ventral hippocampus (vHC) and basolateral amygdala (BLA) support distinct non-associative behavioral changes following stress. Inhibiting stress-induced protein synthesis in the BLA blocked subsequent increases in stress sensitivity but not anxiety-related behaviors. Conversely, inhibiting stress-induced protein synthesis in the vHC blocked subsequent increases in anxiety-related behavior but not stress sensitivity. Inhibiting neuronal activity in the BLA and vHC during the assessment of stress sensitivity or anxiety-related behavior recapitulated these structures' dissociable contributions to defensive behavior. Lastly, blocking the associative memory of a stressor had no impact on stress-induced changes in anxiety-related behavior. These findings highlight that multiple memory systems support the long-lasting effects of stress.

Distinct changes to hippocampal and medial entorhinal circuits emerge across the progression of cognitive deficits in epilepsy.

Temporal lobe epilepsy (TLE) causes pervasive and progressive memory impairments, yet the specific circuit changes that drive these deficits remain unclear. To investigate how hippocampal-entorhinal dysfunction contributes to progressive memory deficits in epilepsy, we performed simultaneous in vivo electrophysiology in hippocampus (HPC) and medial entorhinal cortex (MEC) of control and epileptic mice 3 or 8 weeks after pilocarpine-induced status epilepticus (Pilo-SE). We found that HPC synchronization deficits (including reduced theta power, coherence, and altered interneuron spike timing) emerged within 3 weeks of Pilo-SE, aligning with early-onset, relatively subtle memory deficits. In contrast, abnormal synchronization within MEC and between HPC-MEC emerged later, by 8 weeks after Pilo-SE, when spatial memory impairment was more severe. Furthermore, a distinct subpopulation of MEC layer 3 excitatory neurons (active at theta troughs) was specifically impaired in epileptic mice. Together, these findings suggest that hippocampal-entorhinal circuit dysfunction accumulates and shifts as cognitive impairment progresses in TLE.

Ensemble-specific deficit in neuronal intrinsic excitability in aged mice.

With the prevalence of age-related cognitive deficits on the rise, it is essential to identify cellular and circuit alterations that contribute to age-related memory impairment. Increased intrinsic neuronal excitability after learning is important for memory consolidation, and changes to this process could underlie memory impairment in old age. Some studies find age-related deficits in hippocampal neuronal excitability that correlate with memory impairment but others do not, possibly due to selective changes only in activated neural ensembles. Thus, we tagged CA1 neurons activated during learning and recorded their intrinsic excitability 5 hours or 7 days post-training. Adult mice exhibited increased neuronal excitability 5 hours after learning, specifically in ensemble (learning-activated) CA1 neurons. As expected, ensemble excitability returned to baseline 7 days post-training. In aged mice, there was no ensemble-specific excitability increase after learning, which was associated with impaired hippocampal memory performance. These results suggest that CA1 may be susceptible to age-related impairments in post-learning ensemble excitability and underscore the need to selectively measure ensemble-specific changes in the brain.

Manipulating single-unit theta phase-locking with PhaSER: An open-source tool for real-time phase estimation and manipulation.

The precise timing of neuronal spiking relative to the brain's endogenous oscillations (i.e., phase-locking or spike-phase coupling) has long been hypothesized to coordinate cognitive processes and maintain excitatory-inhibitory homeostasis. Indeed, disruptions in theta phase-locking have been described in models of neurological diseases with associated cognitive deficits and seizures, such as Alzheimer's disease, temporal lobe epilepsy, and autism spectrum disorders. However, due to technical limitations, determining if phase-locking causally contributes to these disease phenotypes has not been possible until recently. To fill this gap and allow for the flexible manipulation of single-unit phase-locking to on-going endogenous oscillations, we developed PhaSER, an open-source tool that allows for phase-specific manipulations. PhaSER can deliver optogenetic stimulation at defined phases of theta in order to shift the preferred firing phase of neurons relative to theta in real-time. Here, we describe and validate this tool in a subpopulation of inhibitory neurons that express somatostatin (SOM) in the CA1 and dentate gyrus (DG) regions of the dorsal hippocampus. We show that PhaSER is able to accurately deliver a photo-manipulation that activates opsin+ SOM neurons at specified phases of theta in real-time in awake, behaving mice. Further, we show that this manipulation is sufficient to alter the preferred firing phase of opsin+ SOM neurons without altering the referenced theta power or phase. All software and hardware requirements to implement real-time phase manipulations during behavior are available online (https://github.com/ShumanLab/PhaSER).

Progressive excitability changes in the medial entorhinal cortex in the 3xTg mouse model of Alzheimer's disease pathology.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is characterized by memory loss and progressive cognitive impairments. In mouse models of AD pathology, studies have found neuronal and synaptic deficits in the hippocampus, but less is known about what happens in the medial entorhinal cortex (MEC), which is the primary spatial input to the hippocampus and an early site of AD pathology. Here, we measured the neuronal intrinsic excitability and synaptic activity in MEC layer II (MECII) stellate cells, MECII pyramidal cells, and MEC layer III (MECIII) excitatory neurons at early (3 months) and late (10 months) time points in the 3xTg mouse model of AD pathology. At 3 months of age, prior to the onset of memory impairments, we found early hyperexcitability in MECII stellate and pyramidal cells' intrinsic properties, but this was balanced by a relative reduction in synaptic excitation (E) compared to inhibition (I), suggesting intact homeostatic mechanisms regulating activity in MECII. Conversely, MECIII neurons had reduced intrinsic excitability at this early time point with no change in the synaptic E/I ratio. By 10 months of age, after the onset of memory deficits, neuronal excitability of MECII pyramidal cells and MECIII excitatory neurons was largely normalized in 3xTg mice. However, MECII stellate cells remained hyperexcitable and this was further exacerbated by an increased synaptic E/I ratio. This observed combination of increased intrinsically and synaptically generated excitability suggests a breakdown in homeostatic mechanisms specifically in MECII stellate cells at this post-symptomatic time point. Together, these data suggest that the breakdown in homeostatic excitability mechanisms in MECII stellate cells may contribute to the emergence of memory deficits in AD.

Dissociable contributions of the amygdala and ventral hippocampus to stress-induced changes in defensive behavior.

Severe stress can produce multiple persistent changes in defensive behavior. While much is known about the circuits supporting stress-induced associative fear responses, how circuit plasticity supports the broader changes in defensive behavior observed after severe stress remains unclear. Here, we find that stress-induced plasticity in the ventral hippocampus (vHC) and basolateral amygdala (BLA) support doubly dissociable defensive behavioral changes. Stress-induced protein synthesis in the BLA was found to support lasting enhancements in stress sensitivity but not enhancements in exploratory anxiety-related behaviors, whereas protein synthesis in the vHC was found to support enhancements in anxiety-related behavior but not enhancements in stress sensitivity. Like protein synthesis, neuronal activity of the BLA and vHC were found to differentially support the expression of these same defensive behaviors. Lastly, blockade of associative fear had no impact on stress-induced changes in anxiety-related behavior. These findings highlight that multiple memory-systems support stress-induced defensive behavior changes.

Chronotate: An open-source tool for manual timestamping and quantification of animal behavior.

A core necessity to behavioral neuroscience research is the ability to accurately measure performance on behavioral assays, such as the novel object location and novel object recognition tasks. These tasks are widely used in neuroscience research and measure a rodent's instinct for investigating novel features as a proxy to test their memory of a previous experience. Automated tools for scoring behavioral videos can be cost prohibitive and often have difficulty distinguishing between active investigation of an object and simply being in close proximity to an object. As such, many experimenters continue to rely on hand scoring interactions using stopwatches, which makes it difficult to review scoring after-the-fact and results in the loss of temporal information. Here, we introduce Chronotate, a free, open-source tool to aid in manually scoring novel object behavior videos. The software consists of an interactive video player with keyboard integration for marking timestamps of behavioral events during video playback, making it simple to quickly score and review bouts of rodent-object interaction. In addition, Chronotate outputs detailed interaction bout data, allowing for nuanced behavioral performance analyses. Using this detailed temporal information, we demonstrate that novel object location performance peaks within the first 3 s of interaction time and preference for the novel location becomes reduced across the test session. Thus, Chronotate can be used to determine the temporal structure of interactions on this task and can provide new insight into the memory processes that drive this behavior. Chronotate is available for download at: https://github.com/ShumanLab/Chronotate.

Aversive experience drives offline ensemble reactivation to link memories across days.

Memories are encoded in neural ensembles during learning and stabilized by post-learning reactivation. Integrating recent experiences into existing memories ensures that memories contain the most recently available information, but how the brain accomplishes this critical process remains unknown. Here we show that in mice, a strong aversive experience drives the offline ensemble reactivation of not only the recent aversive memory but also a neutral memory formed two days prior, linking the fear from the recent aversive memory to the previous neutral memory. We find that fear specifically links retrospectively, but not prospectively, to neutral memories across days. Consistent with prior studies, we find reactivation of the recent aversive memory ensemble during the offline period following learning. However, a strong aversive experience also increases co-reactivation of the aversive and neutral memory ensembles during the offline period. Finally, the expression of fear in the neutral context is associated with reactivation of the shared ensemble between the aversive and neutral memories. Taken together, these results demonstrate that strong aversive experience can drive retrospective memory-linking through the offline co-reactivation of recent memory ensembles with memory ensembles formed days prior, providing a neural mechanism by which memories can be integrated across days.

Minian, an open-source miniscope analysis pipeline.

Miniature microscopes have gained considerable traction for in vivo calcium imaging in freely behaving animals. However, extracting calcium signals from raw videos is a computationally complex problem and remains a bottleneck for many researchers utilizing single-photon in vivo calcium imaging. Despite the existence of many powerful analysis packages designed to detect and extract calcium dynamics, most have either key parameters that are hard-coded or insufficient step-by-step guidance and validations to help the users choose the best parameters. This makes it difficult to know whether the output is reliable and meets the assumptions necessary for proper analysis. Moreover, large memory demand is often a constraint for setting up these pipelines since it limits the choice of hardware to specialized computers. Given these difficulties, there is a need for a low memory demand, user-friendly tool offering interactive visualizations of how altering parameters at each step of the analysis affects data output. Our open-source analysis pipeline, Minian (miniscope analysis), facilitates the transparency and accessibility of single-photon calcium imaging analysis, permitting users with little computational experience to extract the location of cells and their corresponding calcium traces and deconvolved neural activities. Minian contains interactive visualization tools for every step of the analysis, as well as detailed documentation and tips on parameter exploration. Furthermore, Minian has relatively small memory demands and can be run on a laptop, making it available to labs that do not have access to specialized computational hardware. Minian has been validated to reliably and robustly extract calcium events across different brain regions and from different cell types. In practice, Minian provides an open-source calcium imaging analysis pipeline with user-friendly interactive visualizations to explore parameters and validate results.

ezTrack-A Step-by-Step Guide to Behavior Tracking.

Tracking animal behavior by video is one of the most common tasks in neuroscience. Previously, we have validated ezTrack, a free, flexible, and easy-to-use software for the analysis of animal behavior. ezTrack's Location Tracking Module can be used for the positional analysis of an individual animal and is applicable to a wide range of behavioral tasks. Separately, ezTrack's Freeze Analysis Module is designed for the analysis of defensive freezing behavior. ezTrack supports a range of desirable tools, including options for cropping and masking portions of the field of view, defining regions of interest, producing summary data for specified portions of time, algorithms to remove the influence of electrophysiology cables and other tethers, batch processing of multiple videos, and video down-sampling. Moreover, ezTrack produces a range of interactive plots and visualizations to promote users' confidence in their results. In this protocols paper, we provide step-by-step instructions for the use of ezTrack, from tips for recording behavior to instructions for using the software for video analysis. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Software environment installation Basic Protocol 2: Using the Location Tracking Module Basic Protocol 3: Using the Freeze Analysis Module.

Sleep deprivation and Pavlovian fear conditioning.

Sleep has been suggested to play a role in memory consolidation. Prior rodent studies have used sleep deprivation to examine this relationship. First, we reexamined the effects of sleep deprivation on Pavlovian fear conditioning. We found that the deprivation method itself (i.e., gentle handling) induced deficits independent of sleep. Second, we examined an alternative method of sleep deprivation using amphetamine and found that this method failed to induce amnesia. These data indicate that sleep deprivation is a problematic way to examine the role of sleep in memory consolidation, and an alternative paradigm is proposed.

Breakdown of spatial coding and interneuron synchronization in epileptic mice.

Temporal lobe epilepsy causes severe cognitive deficits, but the circuit mechanisms remain unknown. Interneuron death and reorganization during epileptogenesis may disrupt the synchrony of hippocampal inhibition. To test this, we simultaneously recorded from the CA1 and dentate gyrus in pilocarpine-treated epileptic mice with silicon probes during head-fixed virtual navigation. We found desynchronized interneuron firing between the CA1 and dentate gyrus in epileptic mice. Since hippocampal interneurons control information processing, we tested whether CA1 spatial coding was altered in this desynchronized circuit, using a novel wire-free miniscope. We found that CA1 place cells in epileptic mice were unstable and completely remapped across a week. This spatial instability emerged around 6 weeks after status epilepticus, well after the onset of chronic seizures and interneuron death. Finally, CA1 network modeling showed that desynchronized inputs can impair the precision and stability of CA1 place cells. Together, these results demonstrate that temporally precise intrahippocampal communication is critical for spatial processing.

Modafinil and memory: effects of modafinil on Morris water maze learning and Pavlovian fear conditioning.

Modafinil has been shown to promote wakefulness and some studies suggest the drug can improve cognitive function. Because of many similarities, the mechanism of action may be comparable to classical psychostimulants, although the exact mechanisms of modafinil's actions in wakefulness and cognitive enhancement are unknown. The current study aims to further examine the effects of modafinil as a cognitive enhancer on hippocampus-dependent memory in mice. A high dose of modafinil (75 mg/kg ip) given before training improved acquisition on a Morris water maze. When given only before testing, modafinil did not affect water maze performance. We also examined modafinil (0.075 to 75 mg/kg) on Pavlovian fear conditioning. A low dose of pretraining modafinil (0.75 mg/kg) enhanced memory of contextual fear conditioning (tested off-drug 1 week later) whereas a high dose (75 mg/kg) disrupted memory. Pretraining modafinil did not affect cued conditioning at any dose tested, and immediate posttraining modafinil had no effect on either cued or contextual fear. These results suggest that modafinil's effects of memory are more selective than amphetamine or cocaine and specific to hippocampus-dependent memory.

ezTrack: An open-source video analysis pipeline for the investigation of animal behavior.

Tracking animal behavior by video is one of the most common tasks in the life sciences. Although commercial software exists for executing this task, they often present enormous cost to the researcher and can entail purchasing hardware that is expensive and lacks adaptability. Additionally, the underlying code is often proprietary. Alternatively, available open-source options frequently require model training and can be challenging for those inexperienced with programming. Here we present an open-source and platform independent set of behavior analysis pipelines using interactive Python that researchers with no prior programming experience can use. Two modules are described. One module can be used for the positional analysis of an individual animal, amenable to a wide range of behavioral tasks. A second module is described for the analysis of freezing behavior. For both modules, a range of interactive plots and visualizations are available to confirm that chosen parameters produce the anticipated results. Moreover, batch processing tools for the fast analysis of multiple videos is provided, and frame-by-frame output makes alignment with biological recording data simple. Lastly, options for cropping video frames to mitigate the influence of fiberoptic/electrophysiology cables, analyzing specified portions of time, and defining regions of interest, are readily implemented.