Short-Term Preexposure to Novel Enriched Environment Augments Hippocampal Ripples in Urethane-Anesthetized Mice.

Learning and memory are affected by novel enriched environment, a condition where animals play and interact with a variety of toys and conspecifics. Exposure of animals to the novel enriched environments improves memory by altering neural plasticity during natural sleep, a process called memory consolidation. The hippocampus, a pivotal brain region for learning and memory, generates high-frequency oscillations called ripples during sleep, which is required for memory consolidation. Naturally occurring sleep shares characteristics in common with general anesthesia in terms of extracellular oscillations, guaranteeing anesthetized animals suitable to examine neural activity in a sleep-like state. However, it is poorly understood whether the preexposure of animals to the novel enriched environment modulates neural activity in the hippocampus under subsequent anesthesia. To ask this question, we allowed mice to freely explore the novel enriched environment or their standard environment, anesthetized them, and recorded local field potentials in the hippocampal CA1 area. We then compared the characteristics of hippocampal ripples between the two groups and found that the amplitude of ripples and the number of successive ripples were larger in the novel enriched environment group than in the standard environment group, suggesting that the afferent synaptic input from the CA3 area to the CA1 area was higher when the animals underwent the novel enriched environment. These results underscore the importance of prior experience that surpasses subsequent physical states from the neurophysiological point of view.

Automatic detection of foot-strike onsets in a rhythmic forelimb movement.

Rhythmic movement is the fundamental motion dynamics characterized by repetitive patterns. Precisely defining onsets in rhythmic movement is essential for a comprehensive analysis of motor functions. Our study introduces an automated method for detecting rat's forelimb foot-strike onsets using deep learning tools. This method demonstrates high accuracy of onset detection by combining two techniques using joint coordinates and behavioral confidence scale. The analysis extends to neural oscillatory responses in the rat's somatosensory cortex, validating the effectiveness of our combined approach. Our technique streamlines experimentation, demanding only a camera and GPU-accelerated computer. This approach is applicable across various contexts and promotes our understanding of brain functions during rhythmic movements.

Lengthened circadian rhythms in mice with self-controlled ambient light intensity.

Laboratory animals are typically maintained under 12-h light and 12-h dark (12:12 LD) conditions with a daytime light intensity of ~ 200 lx. In this study, we designed an apparatus that allowed mice to self-select the room light intensity by nose poking. We measured the behavioral rhythms of the mice under this self-controlled light regimen. The mice quickly learned the relationship between their nose pokes and the resulting changes in the light intensity. Under these conditions, the mice exhibited free-running circadian behavior with a period of 24.5 ± 0.4 h. This circadian period was ~ 1 h longer than that of the same strain of mice when they were kept in constant darkness (DD) after 12:12 LD entrainment, and the lengthened period lasted for at least 30 days. The rhythm of the light intensity controlled by the mice also exhibited a similar period, but the phase of the illuminance rhythm preceded the phase of the locomotor activity rhythm. Mice that did not have access to the light controller were also entrained to the illuminance cycle produced by the mice that did have access to the light controller, but with a slightly delayed phase. The rhythm was likely controlled by the canonical circadian clock because mice with tau mutations in the circadian clock gene CSNK1E exhibited short periods of circadian rhythm under the same conditions. These results indicate that the free-running period of mice in the wild may differ from what they exhibit if they are attuned by forced light cycles in laboratories because mice in their natural habitats can self-control their exposure to ambient light, similar to our experimental conditions.

Low Atmospheric Oxygen Attenuates Alpha Oscillations in the Primary Motor Cortex of Awake Rats.

Oxygen is pivotal for survival of animals. Their cellular activity and cognitive behavior are impaired when atmospheric oxygen is insufficient, called hypoxia. However, concurrent effects of hypoxia on physiological signals are poorly understood. To address this question, we simultaneously recorded local field potentials in the primary motor cortex, primary somatosensory, and anterior cingulate cortex, electrocardiograms, electroolfactograms, and electromyograms of rats under acute hypoxic conditions (i.e., 5.0% O2). Exposure to acute hypoxia significantly attenuated alpha oscillations alone in the primary motor cortex, while we failed to find any effects of acute hypoxia on the oscillatory power in the somatosensory cortex or anterior cingulate cortex. These area- and frequency-specific effects by hypoxia may be accounted for by neural innervation from the brainstem to each cortical area via thalamic relay nuclei. Moreover, we found that heart rate and respiratory rate were increased during acute hypoxia and high heart rate was maintained even after the oxygen level returned to the baseline. Altogether, our study characterizes a systemic effect of atmospheric hypoxia on neural and peripheral signals from physiological viewpoints, leading to bridging a gap between cellular and behavioral levels.

Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.

[Electrophysiological and Pharmacological Research on Neural Activity in the Neocortex and Hippocampus During Sleep].

Sleep is fundamental for living animals. Although they are not conscious during sleep, their brains are continuously working. This neural activity during sleep can be reflected by neural oscillations closely related to cognitive function. While the relationship between neural activity in sleep and cognition has been extensively investigated, it is not fully understood how neural activity in sleep and relevant memory are modulated by specific receptors. In particular, I focused on melatonin receptors and their agonist, ramelteon. While the effects of ramelteon on sleep have been widely documented, it is still poorly understood how ramelteon affects learning and memory as well as neural activity in sleep. To address this question, I first recorded neural oscillations in the neocortex of rats treated with ramelteon and found that ramelteon promoted non-rapid eye movement (NREM) sleep and increased fast gamma power in the primary motor cortex during NREM sleep. I then evaluated the behavioral performance of ramelteon-treated mice using the novel object recognition task and the spontaneous alternation task, demonstrating that ramelteon enhanced object recognition memory and spatial working memory. These results shed light on new aspects of the functions of melatonin receptors.

Postnatal Maturation of Membrane Potential Dynamics during in Vivo Hippocampal Ripples.

Sharp-wave ripples (SWRs) are transient high-frequency oscillations of local field potentials (LFPs) in the hippocampus and play a critical role in memory consolidation. During SWRs, CA1 pyramidal cells exhibit rapid spike sequences that often replay the sequential activity that occurred during behavior. This temporally organized firing activity gradually emerges during 2 weeks after the eye opening; however, it remains unclear how the organized spikes during SWRs mature at the intracellular membrane potential (Vm) level. Here, we recorded Vm of CA1 pyramidal cells simultaneously with hippocampal LFPs from anesthetized immature mice of either sex after the developmental emergence of SWRs. On postnatal days 16 and 17, Vm dynamics around SWRs were premature, characterized by prolonged depolarizations without either pre- or post-SWR hyperpolarizations. The biphasic hyperpolarizations, features typical of adult SWR-relevant Vm, formed by approximately postnatal day 30. This Vm maturation was associated with an increase in SWR-associated inhibitory inputs to pyramidal cells. Thus, the development of SWR-relevant inhibition restricts the temporal windows for spikes of pyramidal cells and allows CA1 pyramidal cells to organize their spike sequences during SWRs.SIGNIFICANCE STATEMENT Sharp-wave ripples (SWRs) are prominent hippocampal oscillations and play a critical role in memory consolidation. During SWRs, hippocampal neurons synchronously emit spikes with organized temporal patterns. This temporal structure of spikes during SWRs develops during the third and fourth postnatal weeks, but the underlying mechanisms are not well understood. Here, we recorded in vivo membrane potentials from hippocampal neurons in premature mice and suggest that the maturation of SWR-associated inhibition enables hippocampal neurons to produce precisely controlled spike times during SWRs.

Machine learning-based segmentation of the rodent hippocampal CA2 area from Nissl-stained sections.

The hippocampus is a center of learning, memory, and spatial navigation. This region is divided into the CA1, CA2, and CA3 areas, which are anatomically different from each other. Among these divisions, the CA2 area is unique in terms of functional relevance to sociality. The CA2 area is often manually detected based on the size, shape, and density of neurons in the hippocampal pyramidal cell layer, but this manual segmentation relying on cytoarchitecture is impractical to apply to a large number of samples and dependent on experimenters' proficiency. Moreover, the CA2 area has been defined based on expression pattern of molecular marker proteins, but it generally takes days to complete immunostaining for such proteins. Thus, we asked whether the CA2 area can be systematically segmented based on cytoarchitecture alone. Since the expression pattern of regulator of G-protein signaling 14 (RGS14) signifies the CA2 area, we visualized the CA2 area in the mouse hippocampus by RGS14-immunostaining and Nissl-counterstaining and manually delineated the CA2 area. We then established "CAseg," a machine learning-based automated algorithm to segment the CA2 area with the F1-score of approximately 0.8 solely from Nissl-counterstained images that visualized cytoarchitecture. CAseg was extended to the segmentation of the prairie vole CA2 area, which raises the possibility that the use of this algorithm can be expanded to other species. Thus, CAseg will be beneficial for investigating unique properties of the hippocampal CA2 area.

Ramelteon administration enhances novel object recognition and spatial working memory in mice.

Ramelteon is used to ameliorate sleep disorders that negatively affect memory performance; however, it remains unknown whether ramelteon strengthens neutral memories, which do not involve reward or punishment. To address this, we monitored behavior of mice treated with vehicle/ramelteon while they performed a novel object recognition task and a spontaneous alternation task. Object memory performance in the novel object recognition task was improved only if ramelteon was injected before training, suggesting that ramelteon specifically enhances the acquisition of object recognition memory. Ramelteon also enhanced spatial working memory in the spontaneous alternation task. Altogether, acute ramelteon treatment enhances memory in quasi-natural contexts.

An open-source application to identify the three-dimensional locations of electrodes implanted into the rat brain from computed tomography images.

Electrophysiological recordings using metal electrodes implanted into the brains have been widely utilized to evaluate neuronal circuit dynamics related to behavior and external stimuli. The most common method for identifying implanted electrode tracks in the brain tissue has been histological examination following postmortem slicing and staining of the brain tissue, which consumes time and resources and occasionally fails to identify the tracks because the brain preparations have been damaged during processing. Recent studies have proposed the use of a promising alternative method, consisting of computed tomography (CT) scanning that can directly reconstruct the three-dimensional arrangements of electrodes in the brains of living animals. In this study, we developed an open-source Python-based application that estimates the location of an implanted electrode from CT image sequences in a rat. After the user manually sets reference coordinates and an area from a sequence of CT images, this application automatically overlays an estimated location of an electrode tip on a histological template image; the estimates are highly accurate, with less than 135 µm of error, irrespective of the depth of the brain region. The estimation of an electrode location can be completed within a few minutes. Our simple and user-friendly application extends beyond currently available CT-based electrode localization methods and opens up the possibility of applying this technique to various electrophysiological recording paradigms.

Theta oscillations represent collective dynamics of multineuronal membrane potentials of murine hippocampal pyramidal cells.

Theta (θ) oscillations are one of the characteristic local field potentials (LFPs) in the hippocampus that emerge during spatial navigation, exploratory sniffing, and rapid eye movement sleep. LFPs are thought to summarize multineuronal events, including synaptic currents and action potentials. However, no in vivo study to date has directly interrelated θ oscillations with the membrane potentials (Vm) of multiple neurons, and it remains unclear whether LFPs can be predicted from multineuronal Vms. Here, we simultaneously patch-clamp up to three CA1 pyramidal neurons in awake or anesthetized mice and find that the temporal evolution of the power and frequency of θ oscillations in Vms (θVms) are weakly but significantly correlate with LFP θ oscillations (θLFP) such that a deep neural network could predict the θLFP waveforms based on the θVm traces of three neurons. Therefore, individual neurons are loosely interdependent to ensure freedom of activity, but they partially share information to collectively produce θLFP.

Enhancement of Motor Cortical Gamma Oscillations and Sniffing Activity by Medial Forebrain Bundle Stimulation Precedes Locomotion.

The medial forebrain bundle (MFB) is a white matter pathway that traverses through mesolimbic structures and includes dopaminergic neural fibers ascending from the ventral tegmental area (VTA). Since dopaminergic signals represent hedonic responses, electrical stimulation of the MFB in animals has been used as a neural reward for operant and spatial tasks. MFB stimulation strongly motivates animals to rapidly learn to perform a variety of behavioral tasks to obtain a reward. Although the MFB is known to connect various brain regions and MFB stimulation dynamically modulates animal behavior, how central and peripheral functions are affected by MFB stimulation per se is poorly understood. To address this question, we simultaneously recorded electrocorticograms (ECoGs) in the primary motor cortex (M1), primary somatosensory cortex (S1), and olfactory bulb (OB) of behaving rats while electrically stimulating the MFB. We found that MFB stimulation increased the locomotor activity of rats. Spectral analysis confirmed that immediately after MFB stimulation, sniffing activity was facilitated and the power of gamma oscillations in the M1 was increased. After sniffing activity and motor cortical gamma oscillations were facilitated, animals started to move. These results provide insight into the importance of sniffing activity and cortical gamma oscillations for motor execution and learning facilitated by MFB stimulation.

In Utero Electroporation for Manipulation of Specific Neuronal Populations.

The complexity of brain functions is supported by the heterogeneity of brain tissue and millisecond-scale information processing. Understanding how complex neural circuits control animal behavior requires the precise manipulation of specific neuronal subtypes at high spatiotemporal resolution. In utero electroporation, when combined with optogenetics, is a powerful method for precisely controlling the activity of specific neurons. Optogenetics allows for the control of cellular membrane potentials through light-sensitive ion channels artificially expressed in the plasma membrane of neurons. Here, we first review the basic mechanisms and characteristics of in utero electroporation. Then, we discuss recent applications of in utero electroporation combined with optogenetics to investigate the functions and characteristics of specific regions, layers, and cell types. These techniques will pave the way for further advances in understanding the complex neuronal and circuit mechanisms that underlie behavioral outputs.

Delayed reinforcement hinders subsequent extinction.

In operant conditioning, animals associate their own behavior with a reinforcer, and the probability of the behavioral responses is increased. This form of learning is called reinforcement. In contrast, when the previously reinforced responses are no longer paired with a reinforcer, these responses are eventually extinguished. The effectiveness of reinforcement depends primarily on time intervals between reinforcers and responses, but it is not fully understood how the intervals affect subsequent extinction. To address this question, we performed electrical stimulation of the rat medial forebrain bundle (MFB), a part of the brain reward system, and an operant task in which the MFB was electrically stimulated 0.1 s (immediate condition) or 1 s (delayed condition) after the rat's nose was poked. During the first half of the task period (a reinforcement period), nose pokes were associated with MFB stimulation. In contrast, during the second half (an extinction period), we did not stimulate the MFB irrespective of nose pokes. We found that rats exhibited increased nose-poke behaviors during the reinforcement period under both conditions, whereas during the extinction period, nose pokes were more persistent in the delayed condition than in the immediate condition. The persistent responses in the extinction period were independent of responses in the reinforcement period. Therefore, reinforcement and extinction are driven by independent neural mechanisms.

Multiple states in ongoing neural activity in the rat visual cortex.

The brain continuously produces internal activity in the absence of afferently salient sensory input. Spontaneous neural activity is intrinsically defined by circuit structures and associated with the mode of information processing and behavioral responses. However, the spatiotemporal dynamics of spontaneous activity in the visual cortices of behaving animals remain almost elusive. Using a custom-made electrode array, we recorded 32-site electrocorticograms in the primary and secondary visual cortex of freely behaving rats and determined the propagation patterns of spontaneous neural activity. Nonlinear dimensionality reduction and unsupervised clustering revealed multiple discrete states of the activity patterns. The activity remained stable in one state and suddenly jumped to another state. The diversity and dynamics of the internally switching cortical states would imply flexibility of neural responses to various external inputs.

Molecular Characterization of Superficial Layers of the Presubiculum During Development.

The presubiculum, a subarea of the parahippocampal region, plays a critical role in spatial navigation and spatial representation. An outstanding aspect of presubicular spatial codes is head-direction selectivity of the firing of excitatory neurons, called head-direction cells. Head-direction selectivity emerges before eye-opening in rodents and is maintained in adulthood through neurophysiological interactions between excitatory and inhibitory neurons. Although the presubiculum has been physiologically profiled in terms of spatial representation during development, the histological characteristics of the developing presubiculum are poorly understood. We found that the expression of vesicular glutamate transporter 2 (VGluT2) could be used to delimit the superficial layers of the presubiculum, which was identified using an anterograde tracer injected into the anterior thalamic nucleus (ATN). Thus, we immunostained slices from mice ranging in age from neonates to adults using an antibody against VGluT2 to evaluate the VGluT2-positive area, which was identified as the superficial layers of the presubiculum, during development. We also immunostained the slices using antibodies against parvalbumin (PV) and somatostatin (SOM) and found that in the presubicular superficial layers, PV-positive neurons progressively increased in number during development, whereas SOM-positive neurons exhibited no increasing trend. In addition, we observed repeating patch structures in presubicular layer III from postnatal days 12. The abundant expression of VGluT2 suggests that the presubicular superficial layers are regulated primarily by VGluT2-mediated excitatory neurotransmission. Moreover, developmental changes in the densities of PV- and SOM-positive interneurons and the emergence of the VGluT2-positive patch structures during adolescence may be associated with the functional development of spatial codes in the superficial layers of the presubiculum.

Acute Ramelteon Treatment Maintains the Cardiac Rhythms of Rats during Non-REM Sleep.

Sleep curtailment negatively affects cardiac activities and thus should be ameliorated by pharmacological methods. One of the therapeutic targets is melatonin receptors, which tune circadian rhythms. Ramelteon, a melatonin MT1/MT2 receptor agonist, has recently been developed to modulate sleep-wake rhythms. To date, the sleep-promoting effect of ramelteon has been widely delineated, but whether ramelteon treatment physiologically influences cardiac function is not well understood. To address this question, we recorded electrocardiograms, electromyograms, and electrocorticograms in the frontal cortex and the olfactory bulb of unrestrained rats treated with either ramelteon or vehicle. We detected vigilance states based on physiological measurements and analyzed cardiac and muscular activities. We found that during non-rapid eye movement (non-REM) sleep, heartrate variability was maintained by ramelteon treatment. Analysis of the electromyograms confirmed that neither microarousal during non-REM sleep nor the occupancy of phasic periods during REM sleep was altered by ramelteon. Our results indicate that ramelteon has a remedial effect on cardiac activity by keeping the heartrate variability and may reduce cardiac dysfunction during sleep.

Deep Learning-Based Classification of GAD67-Positive Neurons Without the Immunosignal.

Excitatory neurons and GABAergic interneurons constitute neural circuits and play important roles in information processing. In certain brain regions, such as the neocortex and the hippocampus, there are fewer interneurons than excitatory neurons. Interneurons have been quantified via immunohistochemistry, for example, for GAD67, an isoform of glutamic acid decarboxylase. Additionally, the expression level of other proteins varies among cell types. For example, NeuN, a commonly used marker protein for postmitotic neurons, is expressed differently across brain regions and cell classes. Thus, we asked whether GAD67-immunopositive neurons can be detected using the immunofluorescence signals of NeuN and the fluorescence signals of Nissl substances. To address this question, we stained neurons in layers 2/3 of the primary somatosensory cortex (S1) and the primary motor cortex (M1) of mice and manually labeled the neurons as either cell type using GAD67 immunosignals. We then sought to detect GAD67-positive neurons without GAD67 immunosignals using a custom-made deep learning-based algorithm. Using this deep learning-based model, we succeeded in the binary classification of the neurons using Nissl and NeuN signals without referring to the GAD67 signals. Furthermore, we confirmed that our deep learning-based method surpassed classic machine-learning methods in terms of binary classification performance. Combined with the visualization of the hidden layer of our deep learning algorithm, our model provides a new platform for identifying unbiased criteria for cell-type classification.

In Vivo Whole-Cell Patch-Clamp Methods: Recent Technical Progress and Future Perspectives.

Brain functions are fundamental for the survival of organisms, and they are supported by neural circuits consisting of a variety of neurons. To investigate the function of neurons at the single-cell level, researchers often use whole-cell patch-clamp recording techniques. These techniques enable us to record membrane potentials (including action potentials) of individual neurons of not only anesthetized but also actively behaving animals. This whole-cell recording method enables us to reveal how neuronal activities support brain function at the single-cell level. In this review, we introduce previous studies using in vivo patch-clamp recording techniques and recent findings primarily regarding neuronal activities in the hippocampus for behavioral function. We further discuss how we can bridge the gap between electrophysiology and biochemistry.

Ramelteon modulates gamma oscillations in the rat primary motor cortex during non-REM sleep.

Sleep disorders adversely affect daily activities and cause physiological and psychiatric problems. The shortcomings of benzodiazepine hypnotics have led to the development of ramelteon, a melatonin MT1 and MT2 agonist. Although the sleep-promoting effects of ramelteon have been documented, few studies have precisely investigated the structure of sleep and neural oscillatory activities. In this study, we recorded electrocorticograms in the primary motor cortex, the primary somatosensory cortex and the olfactory bulb as well as electromyograms in unrestrained rats treated with either ramelteon or vehicle. A neural-oscillation-based algorithm was used to classify the behavior of the rats into three vigilance states (e.g., awake, rapid eye movement (REM) sleep, and non-REM (NREM) sleep). Moreover, we investigated the region-, frequency- and state-specific modulation of extracellular oscillations in the ramelteon-treated rats. We demonstrated that in contrast to benzodiazepine treatment, ramelteon treatment promoted NREM sleep and enhanced fast gamma power in the primary motor cortex during NREM sleep, while REM sleep was unaffected. Gamma oscillations locally coordinate neuronal firing, and thus, ramelteon modulates neural oscillations in sleep states in a unique manner and may contribute to off-line information processing during sleep.