Early-life sleep disruption impairs subtle social behaviours in prairie voles: a pose-estimation study.

Early-life sleep disruption (ELSD) has been shown to have long-lasting effects on social behaviour in adult prairie voles (Microtus ochrogaster), including impaired expression of pair bonding during partner preference testing. However, due to the limitations of manual behaviour tracking, the effects of ELSD across the time course of pair bonding have not yet been described, hindering our ability to trace mechanisms. Here, we used pose estimation to track prairie voles during opposite-sex cohabitation, the process leading to pair bonding. Male-female pairs were allowed to interact through a mesh divider in the home cage for 72 h, providing variables of body direction, distance-to-divider and locomotion speed. We found that control males displayed periodic patterns of body orientation towards females during cohabitation. In contrast, ELSD males showed reduced duration and ultradian periodicity of these body orientation behaviours towards females. Furthermore, in both sexes, ELSD altered spatial and temporal patterns of locomotion across the light/dark cycles of the 72 h recordings. This study allows a comprehensive behavioural assessment of the effects of ELSD on later life sociality and highlights subtle prairie vole behaviours. Our findings may shed light on neurodevelopmental disorders featuring sleep disruption and social deficits, such as autism spectrum disorders.

LabGym: Quantification of user-defined animal behaviors using learning-based holistic assessment.

Quantifying animal behavior is important for biological research. Identifying behaviors is the prerequisite of quantifying them. Current computational tools for behavioral quantification typically use high-level properties such as body poses to identify the behaviors, which constrains the information available for a holistic assessment. Here we report LabGym, an open-source computational tool for quantifying animal behaviors without this constraint. In LabGym, we introduce "pattern image" to represent the animal's motion pattern, in addition to "animation" that shows all spatiotemporal details of a behavior. These two pieces of information are assessed holistically by customizable deep neural networks for accurate behavior identifications. The quantitative measurements of each behavior are then calculated. LabGym is applicable for experiments involving multiple animals, requires little programming knowledge to use, and provides visualizations of behavioral datasets. We demonstrate its efficacy in capturing subtle behavioral changes in diverse animal species.

The temporal structure of REM sleep shows minute-scale fluctuations across brain and body in mice and humans.

Rapid eye movement sleep (REM) is believed to have a binary temporal structure with "phasic" and "tonic" microstates, characterized by motoric activity versus quiescence, respectively. However, we observed in mice that the frequency of theta activity (a marker of rodent REM) fluctuates in a nonbinary fashion, with the extremes of that fluctuation correlating with phasic-type and tonic-type facial motricity. Thus, phasic and tonic REM may instead represent ends of a continuum. These cycles of brain physiology and facial movement occurred at 0.01 to 0.06 Hz, or infraslow frequencies, and affected cross-frequency coupling and neuronal activity in the neocortex, suggesting network functional impact. We then analyzed human data and observed that humans also demonstrate nonbinary phasic/tonic microstates, with continuous 0.01 to 0.04-Hz respiratory rate cycles matching the incidence of eye movements. These fundamental properties of REM can yield insights into our understanding of sleep health.

Sleep Alterations in a Mouse Model of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder showing progressive neuronal loss in several brain areas and a broad spectrum of motor and non-motor symptoms, including ataxia and altered sleep. While sleep disturbances are known to play pathophysiologic roles in other neurodegenerative disorders, their impact on SCA3 is unknown. Using spectrographic measurements, we sought to quantitatively characterize sleep electroencephalography (EEG) in SCA3 transgenic mice with confirmed disease phenotype. We first measured motor phenotypes in 18-31-week-old homozygous SCA3 YACMJD84.2 mice and non-transgenic wild-type littermate mice during lights-on and lights-off periods. We next implanted electrodes to obtain 12-h (zeitgeber time 0-12) EEG recordings for three consecutive days when the mice were 26-36 weeks old. EEG-based spectroscopy showed that compared to wild-type littermates, SCA3 homozygous mice display: (i) increased duration of rapid-eye movement sleep (REM) and fragmentation in all sleep and wake states; (ii) higher beta power oscillations during REM and non-REM (NREM); and (iii) additional spectral power band alterations during REM and wake. Our data show that sleep architecture and EEG spectral power are dysregulated in homozygous SCA3 mice, indicating that common sleep-related etiologic factors may underlie mouse and human SCA3 phenotypes.

3D Printed Skull Cap and Benchtop Fabricated Microwire-Based Microelectrode Array for Custom Rat Brain Recordings.

Microwire microelectrode arrays (MEAs) have been a popular low-cost tool for chronic electrophysiological recordings and are an inexpensive means to record the electrical dynamics crucial to brain function. However, both the fabrication and implantation procedures for multi-MEAs on a single rodent are time-consuming and the accuracy and quality are highly manual skill-dependent. To address the fabrication and implantation challenges for microwire MEAs, (1) a computer-aided designed and 3D printed skull cap for the pre-determined implantation locations of each MEA and (2) a benchtop fabrication approach for low-cost custom microwire MEAs were developed. A proof-of-concept design of a 32-channel 4-MEA (8-wire each) recording system was prototyped and tested through Sprague Dawley rat recordings. The skull cap design, based on the CT-scan of a single rat conforms well with multiple Sprague Dawley rats of various sizes, ages, and weight with a minimal bregma alignment error (A/P axis standard error of the mean = 0.25 mm, M/L axis standard error of the mean = 0.07 mm, n = 6). The prototyped 32-channel system was able to record the spiking activities over five months. The developed benchtop fabrication method and the 3D printed skull cap implantation platform would enable neuroscience groups to conduct in-house design, fabrication, and implantation of customizable microwire MEAs at a lower cost than the current commercial options and experience a shorter lead time for the design modifications and iterations.

Developing an IV Ketamine Clinic for Treatment-Resistant Depression: a Primer.

The efficacy of subanesthetic intravenous ketamine for treatment resistant depression (TRD) has spurred a growth of clinics nationwide that provide this service. Ketamine is an FDA-approved drug as an anesthetic but remains unapproved for psychiatric indications, and this status raises a number of short- and long-term safety and efficacy concerns that need to be addressed when implementing and developing this type of clinic. Using a framework of systems, provider, and patient domains, we provide a review of the key challenges in providing ketamine infusions and suggest potential approaches. Under systems issues, we highlight broad stakeholder engagement involving cross-departmental and multidisciplinary considerations, business case development, and delineation of administrative standard operating procedures. In the provider domain, we highlight specific roles for different treatment team members as well as suggested training requirements. In the patient domain, we identify a variety of standard operating procedures involving initial patient assessment parameters, ketamine dosing and administration guidelines, and safety monitoring procedures. Together, this review provides key considerations for developing a ketamine clinic for depression, in an effort to meet the pressing demand for this novel treatment option while helping to ensure its safe implementation.

Repeated Dosing of Ketamine in the Forced Swim Test: Are Multiple Shots Better Than One?

The anesthetic drug ketamine has been successfully repurposed as an antidepressant in human subjects. This represents a breakthrough for clinical psychopharmacology, because unlike monoaminergic antidepressants, ketamine has rapid onset, including in Major Depressive Disorder (MDD) that is resistant to conventional pharmacotherapy. This rapid therapeutic onset suggests a unique mechanism of action, which continues to be investigated in reverse translational studies in rodents. A large fraction of rodent and human studies of ketamine have focused on the effects of only a single administration of ketamine, which presents a problem because MDD is typically a persistent illness that may require ongoing treatment with this drug to prevent relapse. Here we review behavioral studies in rodents that used repeated dosing of ketamine in the forced swim test (FST), with an eye toward eventual mechanistic studies. A subset of these studies carried out additional experiments with only a single injection of ketamine for comparison, and several studies used chronic psychosocial stress, where stress is a known causative factor in some cases of MDD. We find that repeated ketamine can in some cases paradoxically produce increases in immobility in the FST, especially at high doses such as 50 or 100 mg/kg. Several studies however provide evidence that repeated dosing is more effective than a single dose at decreasing immobility, including behavioral effects that last longer. Collectively, this growing literature suggests that repeated dosing of ketamine has prominent depression-related effects in rodents, and further investigation may help optimize the use of this drug in humans experiencing MDD.

Flexible High-Resolution Force and Dimpling Measurement System for Pia and Dura Penetration During In Vivo Microelectrode Insertion Into Rat Brain.

OBJECTIVE: Understanding the in vivo force and tissue dimpling during micro-electrode implantation into the brain are important for neuro-electrophysiology to minimize damage while enabling accurate placement and stable chronic extracellular electrophysiological recordings. Prior studies were unable to measure the sub-mN forces exerted during in vivo insertion of small electrodes. Here, we have investigated the in vivo force and dimpling depth profiles during brain surface membrane rupture (including dura) in anesthetized rats. METHODS: A µN-resolution cantilever beam-based measurement system was designed, built, and calibrated and adapted for in vivo use. A total of 244 in vivo insertion tests were conducted on 8 anesthetized rats with 121 through pia mater and 123 through dura and pia combined. RESULTS: Both microwire tip sharpening and diameter reduction reduced membrane rupture force (insertion force) and eased brain surface penetration. But dimpling depth and rupture force are not always strongly correlated. Multi-shank silicon probes showed smaller dimpling and rupture force per shank than single shank devices. CONCLUSION: A force measurement system with flexible range and µN-level resolution (up to 0.032 µN) was achieved and proved feasible. For both pia-only and dura-pia penetrations in anesthetized rats, the rupture force and membrane dimpling depth at rupture are linearly related to the microwire diameter. SIGNIFICANCE: We have developed a new system with both µN-level resolution and capacity to be used in vivo for measurement of force profiles of various neural interfaces into the brain. This allows quantification of brain tissue cutting and provides design guidelines for optimal neural interfaces.

Multiple cholinesterase inhibitors have antidepressant-like properties in the mouse forced swim test.

There is high clinical interest in improving the pharmacological treatment of individuals with Major Depressive Disorder (MDD). This neuropsychiatric disorder continues to cause significant morbidity and mortality worldwide, where existing pharmaceutical treatments such as selective serotonin reuptake inhibitors often have limited efficacy. In a recent publication, we demonstrated an antidepressant-like role for the acetylcholinesterase inhibitor (AChEI) donepezil in the C57BL/6J mouse forced swim test (FST). Those data added to a limited literature in rodents and human subjects which suggests AChEIs have antidepressant properties, but added the novel finding that donepezil only showed antidepressant-like properties at lower doses (0.02, 0.2 mg/kg). At a high dose (2.0 mg/kg), donepezil tended to promote depression-like behavior, suggesting a u-shaped dose-response curve for FST immobility. Here we investigate the effects of three other AChEIs with varying molecular structures: galantamine, physostigmine, and rivastigmine, to test whether they also exhibit antidepressant-like effects in the FST. We find that these drugs do exhibit therapeutic-like effects at low but not high doses, albeit at lower doses for physostigmine. Further, we find that their antidepressant-like effects are not mediated by generalized hyperactivity in the novel open field test, and are also not accompanied by anxiolytic-like properties. These data further support the hypothesis that acetylcholine has a u-shaped dose-response relationship with immobility in the C57BL/6J mouse FST, and provide a rationale for more thoroughly investigating whether reversible AChEIs as a class can be repurposed for the treatment of MDD in human subjects.

Defining the latent period of epileptogenesis and epileptogenic zone in a focal cortical dysplasia type II (FCDII) rat model.

OBJECTIVES: Focal cortical dysplasia type II (FCDII) is one of the most common underlying pathologies in patients with drug-resistant epilepsy. However, mechanistic understanding of FCDII fails to keep pace with genetic discoveries, primarily due to the significant challenge in developing a clinically relevant animal model. Conceptually and clinically important questions, such as the unknown latent period of epileptogenesis and the controversial epileptogenic zone, remain unknown in all experimental FCDII animal models, making it even more challenging to investigate the underlying epileptogenic mechanisms. METHODS: In this study, we used continuous video-electroencephalography (EEG) monitoring to detect the earliest interictal and ictal events in a clustered regularly interspaced short palindromic repeats (CRISPR)-in utero electroporation (IUE) FCDII rat model that shares genetic, pathological, and electroclinical signatures with those observed in humans. We then took advantage of in vivo local field potential (LFP) recordings to localize the epileptogenic zone in these animals. RESULTS: To the best of our knowledge, we showed for the first time that epileptiform discharges emerged during the third postnatal week, and that the first seizure occurred as early as during the fourth postnatal week. We also showed that both interictal and ictal discharges are localized within the dysplastic cortex, concordant with human clinical data. SIGNIFICANCE: Together, our work identified the temporal and spatial frame of epileptogenesis in a highly clinically relevant FCDII animal model, paving the way for mechanistic studies at molecular, cellular, and circuitry levels.

Sex- and stress-dependent effects of a single injection of ketamine on open field and forced swim behavior.

Ketamine has emerged as a novel treatment for common psychiatric conditions such as Major Depressive Disorder (MDD) and anxiety disorders, many of which can be initiated and exacerbated by psychological stress. Sex differences in the frequency of both anxiety and depressive disorders are well known and could be due to sex differences in neuroendocrine responses to stress. Ketamine is known to modulate the hormonal response to stress, specifically corticosterone. It is not clear if the acute effect of ketamine on corticosterone differs by sex, or what role this could play in subsequent behavior. Here we test whether a single injection of (R,S)-ketamine (30 mg/kg, i.p.), administered either with or without unpredictable chronic stress (UCS), has different sustained effects on open field test (OFT), elevated zero maze (EZM) or forced swim test (FST) behavior in female versus male C57BL/6J mice. In the OFT (24 h post-injection), ketamine increased center square exploration in males but not females. In contrast, in the FST (72 h post-injection), females showed a trend toward a decrease in immobility after ketamine whereas males were not strongly modulated. These behavioral effects of ketamine were stronger in the presence of UCS than in unstressed animals. UCS animals also showed lower corticosterone after injection than unstressed animals, and in the presence of UCS ketamine increased corticosterone; these effects were similar in both sexes. Corticosterone post-injection did not predict subsequent behavior. These findings complement a growing preclinical literature suggesting both stress-dependency and sex differences in OFT and FST behavioral responses to ketamine.LAY SUMMARYIn humans, it is known that major depression and anxiety disorders, which can be caused or made worse by exposure to psychological stress, occur roughly twice as frequently in women than in men, but the underpinnings of these effects are not well characterized. In the current study, we explored how sex interacts with stress and ketamine (a rapidly acting antidepressant) by assessing both open field and forced swim behavior in mice after chronic mild stress. We report the novel finding that male mice exhibit greater exploration of the aversive center square in the open field after ketamine, whereas females trended toward lower immobility (often interpreted as an antidepressant-like effect) in the forced swim test after this drug, and these effects were amplified by prior stress exposure.

The cholinesterase inhibitor donepezil has antidepressant-like properties in the mouse forced swim test.

Finding new antidepressant agents is of high clinical priority given that many cases of major depressive disorder (MDD) do not respond to conventional monoaminergic antidepressants such as the selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants, and monoamine oxidase inhibitors. Recent findings of effective fast-acting antidepressants indicate that there are biological substrates to be taken advantage of for fast relief of depression and that we may find further treatments in this category. In this vein, the cholinergic system may be a relatively overlooked target for antidepressant medications, given its major role in motivation and attention. Furthermore, the classically engaged monoaminergic neurotransmitter systems in depression treatment-serotonin, norepinephrine, and dopamine-interact directly at times with cholinergic signaling. Here we investigate in greater detail how the cholinergic system may impact depression-related behavior, by administering widely ranging doses of the cholinesterase inhibitor drug, donepezil, to C57BL/6J mice in the forced swim test. First, we confirm prior findings that this drug, which is thought to boost synaptic acetylcholine, promotes depression-like behavior at a high dose (2.0 mg/kg, i.p.). But we also find paradoxically that it has an antidepressant-like effect at lower doses (0.02 and 0.2 mg/kg). Further this antidepressant-like effect is not due to generalized hyperactivity, since we did not observe increased locomotor activity in the open field test. These data support a novel antidepressant-like role for donepezil at lower doses as part of an overall u-shaped dose-response curve. This raises the possibility that donepezil could have antidepressant properties in humans suffering from MDD.

Patterned activation of action potential patterns during offline states in the neocortex: replay and non-replay.

Action potential generation (spiking) in the neocortex is organized into repeating non-random patterns during both awake experiential states and non-engaged states ranging from inattention to sleep to anaesthesia-and even occur in slice preparations. Repeating patterns in a given population of neurons between states may imply a common means by which cortical networks can be engaged despite brain state changes, but super-imposed on this common firing is a variability that is both specific to ongoing inputs and can be re-shaped by experience. This similarity with specifically induced variance may allow for a range of processes including perception, memory consolidation and network homeostasis. Here, we review how patterned activity in neocortical populations has been studied and what it may imply for a cortex that must be both static and plastic. This article is part of the Theo Murphy meeting issue 'Memory reactivation: replaying events past, present and future'.

Repurposing Cholinesterase Inhibitors as Antidepressants? Dose and Stress-Sensitivity May Be Critical to Opening Possibilities.

When stress becomes chronic it can trigger lasting brain and behavioral changes including Major Depressive Disorder (MDD). There is conflicting evidence regarding whether acetylcholinesterase inhibitors (AChEIs) may have antidepressant properties. In a recent publication, we demonstrated a strong dose-dependency of the effect of AChEIs on antidepressant-related behavior in the mouse forced swim test: whereas the AChEI donepezil indeed promotes depression-like behavior at a high dose, it has antidepressant-like properties at lower doses in the same experiment. Our data therefore suggest a Janus-faced dose-response curve for donepezil in depression-related behavior. In this review, we investigate the mood-related properties of AChEIs in greater detail, focusing on both human and rodent studies. In fact, while there have been many studies showing pro-depressant activity by AChEIs and this is a major concept in the field, a variety of other studies in both humans and rodents show antidepressant effects. Our study was one of the first to systematically vary dose to include very low concentrations while measuring behavioral effects, potentially explaining the apparent disparate findings in the field. The possibility of antidepressant roles for AChEIs in rodents may provide hope for new depression treatments. Importantly, MDD is a psychosocial stress-linked disorder, and in rodents, stress is a major experimental manipulation for studying depression mechanisms, so an important future direction will be to determine the extent to which these depression-related effects are stress-sensitive. In sum, gaining a greater understanding of the potentially therapeutic mood-related effects of low dose AChEIs, both in rodent models and in human subjects, should be a prioritized topic in ongoing translational research.

Author Correction: Neuronal firing rates diverge during REM and homogenize during non-REM.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rodent ketamine depression-related research: Finding patterns in a literature of variability.

Discovering that the anesthetic drug ketamine has rapidly acting antidepressant effects in many individuals with major depression is one of the most important findings in clinical psychopharmacology in recent decades. The initial report of these effects in human subjects was based on a foundation of rodent preclinical studies carried out in the 1990s, and subsequent investigation has included both further studies in individuals with depression, as well as reverse translational experiments in animal models, especially rodents. While there is general agreement in the rodent literature that ketamine has rapidly-acting, and generally sustained, antidepressant-like properties, there are also points of contention across studies, including the precise mechanism of action of this drug. In this review, we briefly summarize prominent yet variable findings regarding the mechanism of action. We also discuss a combination of similarities and variances in the rodent literature in the antidepressant-like effects of ketamine as a function of dose, species and strain, test, stressor, and presumably sex of the experimenter. We then present previously unpublished mouse strain comparison data suggesting that subanesthetic ketamine does not have robust antidepressant-like properties in unstressed animals, and may actually promote depression-like behavior, in contrast to widely reported findings. We conclude that the data best support the notion of ketamine action principally via NMDA receptor antagonism, transiently boosting glutamatergic (and possibly other) signaling in diverse brain circuits. We also suggest that future studies should address in greater detail the extent to which antidepressant-like properties of this drug are stress-sensitive, in an effort to better model major depression present in humans.

In vivo electrophysiological recordings of the effects of antidepressant drugs.

Antidepressant drugs are a standard biological treatment for various neuropsychiatric disorders, yet relatively little is known about their electrophysiologic and synaptic effects on mood systems that set moment-to-moment emotional tone. In vivo electrical recording of local field potentials (LFPs) and single neuron spiking has been crucial for elucidating important details of neural processing and control in many other systems, and yet electrical approaches have not been broadly applied to the actions of antidepressants on mood-related circuits. Here we review the literature encompassing electrophysiologic effects of antidepressants in animals, including studies that examine older drugs, and extending to more recently synthesized novel compounds, as well as rapidly acting antidepressants. The existing studies on neuromodulator-based drugs have focused on recording in the brainstem nuclei, with much less known about their effects on prefrontal or sensory cortex. Studies on neuromodulatory drugs have moreover focused on single unit firing patterns with less emphasis on LFPs, whereas the rapidly acting antidepressant literature shows the opposite trend. In a synthesis of this information, we hypothesize that all classes of antidepressants could have common final effects on limbic circuitry. Whereas NMDA receptor blockade may induce a high powered gamma oscillatory state via direct and fast alteration of glutamatergic systems in mood-related circuits, neuromodulatory antidepressants may induce similar effects over slower timescales, corresponding with the timecourse of response in patients, while resetting synaptic excitatory versus inhibitory signaling to a normal level. Thus, gamma signaling may provide a biomarker (or "neural readout") of the therapeutic effects of all classes of antidepressants.

Stress-sensitive antidepressant-like effects of ketamine in the mouse forced swim test.

Major depression is a stress-linked disease with significant morbidity and the anesthetic drug ketamine is of growing interest in the treatment of depression, since in responsive individuals a single dose has rapid (within hours) antidepressant effects that can be sustained for over a week in some instances. This combination of fast action and a therapeutic effect that lasts far beyond the drug's half-life points to a unique mechanism of action. In this reverse translational study, we investigate the degree to which ketamine counteracts stress-related depression-like behavioral responses by determining whether it affects unstressed animals similarly to stressed mice. To test this, male C57BL/6J mice were given a single injection of vehicle (0.9% saline; i.p.), 10 mg/kg ketamine, or 30 mg/kg ketamine, and were tested in the forced swim test (FST) 24 hours and 7 days later, as well as in the open field test on the eighth day. Unstressed mice had normal group housing, environmental enrichment, and experimenter pre-handling (5 days), whereas stressed animals were subjected to chronic mild stress (single housing, reduced enrichment and minimal handling), where some mice also had daily two-week unpredictable chronic stress (UCS). We find that ketamine (24 hours post-injection) decreases immobility and increases mobile (swimming) behavior (antidepressant-like effects) in UCS animals but does the opposite in unstressed mice, similar to recent human findings. In summary, these data suggest that chronic psychological stress interacts with ketamine treatment to modulate its effects in the C57BL/6J mouse FST, which reinforces the relevance of this test, and this strain of mice, to human, stress-induced depression.

Neuronal firing rates diverge during REM and homogenize during non-REM.

Neurons fire at highly variable intrinsic rates and recent evidence suggests that low- and high-firing rate neurons display different plasticity and dynamics. Furthermore, recent publications imply possibly differing rate-dependent effects in hippocampus versus neocortex, but those analyses were carried out separately and with potentially important differences. To more effectively synthesize these questions, we analyzed the firing rate dynamics of populations of neurons in both hippocampal CA1 and frontal cortex under one framework that avoids the pitfalls of previous analyses and accounts for regression to the mean (RTM). We observed several consistent effects across these regions. While rapid eye movement (REM) sleep was marked by decreased hippocampal firing and increased neocortical firing, in both regions firing rate distributions widened during REM due to differential changes in high- versus low-firing rate cells in parallel with increased interneuron activity. In contrast, upon non-REM (NREM) sleep, firing rate distributions narrowed while interneuron firing decreased. Interestingly, hippocampal interneuron activity closely followed the patterns observed in neocortical principal cells rather than the hippocampal principal cells, suggestive of long-range interactions. Following these undulations in variance, the net effect of sleep was a decrease in firing rates. These decreases were greater in lower-firing hippocampal neurons but also higher-firing frontal cortical neurons, suggestive of greater plasticity in these cell groups. Our results across two different regions, and with statistical corrections, indicate that the hippocampus and neocortex show a mixture of differences and similarities as they cycle between sleep states with a unifying characteristic of homogenization of firing during NREM and diversification during REM.