A method to analyze gene expression profiles from hippocampal neurons electrophysiologically recorded in vivo.

Hippocampal pyramidal neurons exhibit diverse spike patterns and gene expression profiles. However, their relationships with single neurons are not fully understood. In this study, we designed an electrophysiology-based experimental procedure to identify gene expression profiles using RNA sequencing of single hippocampal pyramidal neurons whose spike patterns were recorded in living mice. This technique involves a sequence of experiments consisting of in vivo juxtacellular recording and labeling, brain slicing, cell collection, and transcriptome analysis. We demonstrated that the expression levels of a subset of genes in individual hippocampal pyramidal neurons were significantly correlated with their spike burstiness, submillisecond-level spike rise times or spike rates, directly measured by in vivo electrophysiological recordings. Because this methodological approach can be applied across a wide range of brain regions, it is expected to contribute to studies on various neuronal heterogeneities to understand how physiological spike patterns are associated with gene expression profiles.

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.

Hippocampal sharp-wave ripples correlate with periods of naturally occurring self-generated thoughts in humans.

Core features of human cognition highlight the importance of the capacity to focus on information distinct from events in the here and now, such as mind wandering. However, the brain mechanisms that underpin these self-generated states remain unclear. An emerging hypothesis is that self-generated states depend on the process of memory replay, which is linked to sharp-wave ripples (SWRs), which are transient high-frequency oscillations originating in the hippocampus. Local field potentials were recorded from the hippocampus of 10 patients with epilepsy for up to 15 days, and experience sampling was used to describe their association with ongoing thought patterns. The SWR rates were higher during extended periods of time when participants' ongoing thoughts were more vivid, less desirable, had more imaginable properties, and exhibited fewer correlations with an external task. These data suggest a role for SWR in the patterns of ongoing thoughts that humans experience in daily life.

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.

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.

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.

Non-canonical interplay between glutamatergic NMDA and dopamine receptors shapes synaptogenesis.

Direct interactions between receptors at the neuronal surface have long been proposed to tune signaling cascades and neuronal communication in health and disease. Yet, the lack of direct investigation methods to measure, in live neurons, the interaction between different membrane receptors at the single molecule level has raised unanswered questions on the biophysical properties and biological roles of such receptor interactome. Using a multidimensional spectral single molecule-localization microscopy (MS-SMLM) approach, we monitored the interaction between two membrane receptors, i.e. glutamatergic NMDA (NMDAR) and G protein-coupled dopamine D1 (D1R) receptors. The transient interaction was randomly observed along the dendritic tree of hippocampal neurons. It was higher early in development, promoting the formation of NMDAR-D1R complexes in an mGluR5- and CK1-dependent manner, favoring NMDAR clusters and synaptogenesis in a dopamine receptor signaling-independent manner. Preventing the interaction in the neonate, and not adult, brain alters in vivo spontaneous neuronal network activity pattern in male mice. Thus, a weak and transient interaction between NMDAR and D1R plays a structural and functional role in the developing brain.

Stress-induced vagal activity influences anxiety-relevant prefrontal and amygdala neuronal oscillations in male mice.

The vagus nerve crucially affects emotions and psychiatric disorders. However, the detailed neurophysiological dynamics of the vagus nerve in response to emotions and its associated pathological changes remain unclear. In this study, we demonstrated that the spike rates of the cervical vagus nerve change depending on anxiety behavior in an elevated plus maze test, and these changes were eradicated in stress-susceptible male mice. Furthermore, instantaneous spike rates of the vagus nerve were negatively and positively correlated with the power of 2-4 Hz and 20-30 Hz oscillations, respectively, in the prefrontal cortex and amygdala. The oscillations also underwent dynamic changes depending on the behavioral state in the elevated plus maze, and these changes were no longer observed in stress-susceptible and vagotomized mice. Chronic vagus nerve stimulation restored behavior-relevant neuronal oscillations with the recovery of altered behavioral states in stress-susceptible mice. These results suggested that physiological vagal-brain communication underlies anxiety and mood disorders.

Late-spiking retrosplenial cortical neurons are not synchronized with neocortical slow waves in anesthetized mice.

Neocortical slow waves are critical for memory consolidation. The retrosplenial cortex is thought to facilitate the slow wave propagation to regions beyond the neocortex. However, it remains unclear which population is responsible for the slow wave propagation. To address this issue, we performed in vivo whole-cell recordings to identify neurons that were synchronous and asynchronous with slow waves. By quantifying their intrinsic membrane properties, we observed that the former exhibited regular spiking, whereas the latter exhibited late spiking. Thus, these two cell types transmit information in different directions between the neocortex and subcortical regions.

Microglia induce auditory dysfunction after status epilepticus in mice.

Auditory dysfunction and increased neuronal activity in the auditory pathways have been reported in patients with temporal lobe epilepsy, but the cellular mechanisms involved are unknown. Here, we report that microglia play a role in the disinhibition of auditory pathways after status epilepticus in mice. We found that neuronal activity in the auditory pathways, including the primary auditory cortex and the medial geniculate body (MGB), was increased and auditory discrimination was impaired after status epilepticus. We further demonstrated that microglia reduced inhibitory synapses on MGB relay neurons over an 8-week period after status epilepticus, resulting in auditory pathway hyperactivity. In addition, we found that local removal of microglia from the MGB attenuated the increase in c-Fos+ relay neurons and improved auditory discrimination. These findings reveal that thalamic microglia are involved in auditory dysfunction in epilepsy.

[Synaptic phagocytosis by multiple glial cell types].

Neurons in the brain build circuits by synapsing with each other, and glial cells are involved in the formation and elimination of synapses. Glial cells include microglia, astrocytes, and oligodendrocytes, each with distinctive functions supported by different gene expression patterns and morphologies, but all have been shown to regulate the number of synapses in the neuronal circuits through a common function, synaptic phagocytosis. It has also been reported that specific glial cell types phagocytose specific synapses in different brain regions and at different times, and some of the molecular mechanisms involved in each phagocytotic process have been elucidated. For example, microglia, the most frequently reported glial cell type in relation to synaptic phagocytes, are known to recognize various "eat me signals" including complement and phagocytose synapses, contributing to the refinement of neuronal circuits during development. More recently, astrocytes and oligodendrocyte precursor cells have also been shown to be involved in synaptic phagocytosis. Interestingly, there are also reports of different types of glial cells phagocytosing the same types of synapses. And in some cases, it has been suggested that different glial cell types regulate each other's synaptic phagocytosis. In this review, we will discuss the significance of synaptic phagocytosis by multiple types of glial cells by presenting recent studies on synaptic phagocytosis by glial cells.

Effects of theta phase precessing optogenetic intervention on hippocampal neuronal reactivation and spatial maps.

As animals explore environments, hippocampal place cells sequentially fire at progressively earlier phases of theta oscillations in hippocampal local field potentials. In this study, we evaluated the network-level significance of theta phase-entrained neuronal activity in organizing place cell spike patterns. A closed-loop system was developed in which optogenetic stimulation with a temporal pattern replicating theta phase precession is delivered to hippocampal CA1 neurons when rats traversed a particular region on a linear track. Place cells that had place fields during phase precessing stimulation, but not random phase stimulation, showed stronger reactivation during hippocampal sharp-wave ripples in a subsequent rest period. After the rest period, place cells with place fields that emerged during phase precessing stimulation showed more stable place fields. These results imply that neuronal reactivation and stability of spatial maps are mediated by theta phase precession in the hippocampus.

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.

Awake hippocampal synchronous events are incorporated into offline neuronal reactivation.

Learning novel experiences reorganizes hippocampal neuronal circuits, represented as coordinated reactivation patterns in post-experience offline states for memory consolidation. This study examines how awake synchronous events during a novel run are related to post-run reactivation patterns. The disruption of awake sharp-wave ripples inhibited experience-induced increases in the contributions of neurons to post-experience synchronous events. Hippocampal place cells that participate more in awake synchronous events are more strongly reactivated during post-experience synchronous events. Awake synchronous neuronal patterns, in cooperation with place-selective firing patterns, determine cell ensembles that undergo pronounced increases and decreases in their correlated spikes. Taken together, awake synchronous events are fundamental for identifying hippocampal neuronal ensembles to be incorporated into synchronous reactivation during subsequent offline states, thereby facilitating memory consolidation.

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.

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.

Selective serotonin reuptake inhibitors suppress sharp wave ripples in the ventral hippocampus.

Biased memory processing contributes to the development and exacerbation of depression, and thus could represent a potential therapeutic target for stress-induced mental disorders. Synchronized spikes in hippocampal neurons, corresponding to sharp wave ripples (SWRs), may play a crucial role in memory reactivation. In this study, we showed that the frequency of SWRs increased in the ventral hippocampus, but not in the dorsal hippocampus, after stress exposure. Administration of the selective serotonin reuptake inhibitors (SSRIs) fluoxetine and fluvoxamine inhibited the generation of ventral hippocampal SWRs and reduced locomotor activity and local field potential power in the gamma bands. These results suggest that the antidepressant effects of SSRIs may be mediated by the suppression of ventral hippocampal SWRs.

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.