Spatial Learning and Motor Deficits in Vacuolar Protein Sorting-associated Protein 13b (Vps13b) Mutant Mouse

Vacuolar protein sorting-associated protein 13B (VPS13B), also known as COH1, is one of the VPS13 family members which is involved in transmembrane transport, Golgi integrity, and neuritogenesis. Mutations in the VPS13B gene are associated with Cohen syndrome and other cognitive disorders such as intellectual disabilities and autism spectrum disorder (ASD). However, the patho-physiology of VPS13B-associated cognitive deficits is unclear, in part, due to the lack of animal models. Here, we generated a Vps13b exon 2 deletion mutant mouse and analyzed the behavioral phenotypes. We found that Vps13b mutant mice showed reduced activity in open field test and significantly shorter latency to fall in the rotarod test, suggesting that the mutants have motor deficits. In addition, we found that Vps13b mutant mice showed deficits in spatial learning in the hidden platform version of the Morris water maze. The Vps13b mutant mice were normal in other behaviors such as anxiety-like behaviors, working memory and social behaviors. Our results suggest that Vps13b mutant mice may recapitulate key clinical symptoms in Cohen syndrome such as intellectual disability and hypotonia. Vps13b mutant mice may serve as a useful model to investigate the pathophysiology of VPS13B-associated disorders.

Imaging and analysis of genetically encoded calcium indicators linking neural circuits and behaviors

Confirming the direct link between neural circuit activity and animal behavior has been a principal aim of neuroscience. The genetically encoded calcium indicator (GECI), which binds to calcium ions and emits fluorescence visualizing intracellular calcium concentration, enables detection of in vivo neuronal firing activity. Various GECIs have been developed and can be chosen for diverse purposes. These GECI-based signals can be acquired by several tools including two-photon microscopy and microendoscopy for precise or wide imaging at cellular to synaptic levels. In addition, the images from GECI signals can be analyzed with open source codes including constrained non-negative matrix factorization for endoscopy data (CNMF_E) and miniscope 1-photon-based calcium imaging signal extraction pipeline (MIN1PIPE), and considering parameters of the imaged brain regions (e.g., diameter or shape of soma or the resolution of recorded images), the real-time activity of each cell can be acquired and linked with animal behaviors. As a result, GECI signal analysis can be a powerful tool for revealing the functions of neuronal circuits related to specific behaviors.

Circadian Regulation by REV-ERBα Mediates Hippocampal E-LTP in a Time-dependent Manner

Circadian rhythms are driven by circadian oscillators, and these rhythms result in the biological phenomenon of 24-h oscillations. Previous studies suggest that learning and memory are affected by circadian rhythms. One of the genes responsible for generating the circadian rhythm is Rev-erbα. The REV-ERBα protein is a nuclear receptor that acts as a transcriptional repressor, and is a core component of the circadian clock. However, the role of REV-ERBα in neurophysiological processes in the hippocampus has not been characterized yet. In this study, we examined the time-dependent role of REV-ERBα in hippocampal synaptic plasticity using Rev-erbα KO mice. The KO mice lacking REV-ERBα displayed abnormal NMDAR-dependent synaptic potentiation (E-LTP) at CT12~CT14 (subjective night) when compared to their wild-type littermates. However, Rev-erbα KO mice exhibited normal E-LTP at CT0~CT2 (subjective day). We also found that the Rev-erbα KO mice had intact late LTP (L-LTP) at both subjective day and night. Taken together, these results provide evidence that REV-ERBα is critical for hippocampal E-LTP during the dark period.

Inhibition of anterior cingulate cortex excitatory neuronal activity induces conditioned place preference in a mouse model of chronic inflammatory pain

The anterior cingulate cortex (ACC) is known for its role in perception of nociceptive signals and the associated emotional responses. Recent optogenetic studies, involving modulation of neuronal activity in the ACC, show that the ACC can modulate mechanical hyperalgesia. In the present study, we used optogenetic techniques to selectively modulate excitatory pyramidal neurons and inhibitory interneurons in the ACC in a model of chronic inflammatory pain to assess their motivational effect in the conditioned place preference (CPP) test. Selective inhibition of pyramidal neurons induced preference during the CPP test, while activation of parvalbumin (PV)-specific neurons did not. Moreover, chemogenetic inhibition of the excitatory pyramidal neurons alleviated mechanical hyperalgesia, consistent with our previous result. Our results provide evidence for the analgesic effect of inhibition of ACC excitatory pyramidal neurons and a prospective treatment for chronic pain.

Epigenetic regulation and chromatin remodeling in learning and memory

Understanding the underlying mechanisms of memory formation and maintenance has been a major goal in the field of neuroscience. Memory formation and maintenance are tightly controlled complex processes. Among the various processes occurring at different levels, gene expression regulation is especially crucial for proper memory processing, as some genes need to be activated while some genes must be suppressed. Epigenetic regulation of the genome involves processes such as DNA methylation and histone post-translational modifications. These processes edit genomic properties or the interactions between the genome and histone cores. They then induce structural changes in the chromatin and lead to transcriptional changes of different genes. Recent studies have focused on the concept of chromatin remodeling, which consists of 3D structural changes in chromatin in relation to gene regulation, and is an important process in learning and memory. In this review, we will introduce three major epigenetic processes involved in memory regulation: DNA methylation, histone methylation and histone acetylation. We will also discuss general mechanisms of long-term memory storage and relate the epigenetic control of learning and memory to chromatin remodeling. Finally, we will discuss how epigenetic mechanisms can contribute to the pathologies of neurological disorders and cause memory-related symptoms.

Metabotropic glutamate receptor dependent long-term depression in the cortex

Metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD), a type of synaptic plasticity, is characterized by a reduction in the synaptic response, mainly at the excitatory synapses of the neurons. The hippocampus and the cerebellum have been the most extensively studied regions in mGluR-dependent LTD, and Group 1 mGluR has been reported to be mainly involved in this synaptic LTD at excitatory synapses. However, mGluR-dependent LTD in other brain regions may be involved in the specific behaviors or diseases. In this paper, we focus on five cortical regions and review the literature that implicates their contribution to the pathogenesis of several behaviors and specific conditions associated with mGluR-dependent LTD.

Effects of the Female Estrous Cycle on the Sexual Behaviors and Ultrasonic Vocalizations of Male C57BL/6 and Autistic BTBR T+ tf/J Mice

A primary characteristic of autism, which is a neurodevelopmental disorder, is impaired social interaction and communication. Furthermore, patients with autism frequently show abnormal social recognition. In mouse models of autism, social recognition is usually assessed by examining same-sex social behavior using various tests, such as the three-chamber test. However, no studies have examined the ability of male mice with autism to recognize the estrous cycle of female partners. In this study, we investigated the sexual behaviors, especially mounting and ultrasonic vocal communication (USV), of BTBR T+ tf/J (BTBR) mice, which are used as a well-known mouse model of autism, when they encountered estrus or diestrus female mice. As expected, C57BL/6 mice mounted more female mice in the estrus stage compared with the diestrus stage. We found that BTBR mice also mounted more female mice in the estrus stage than female mice in the diestrus stage. Although the USV emission of male mice was not different between estrus and diestrus female mice in both strains, the mounting result implies that BTBR mice distinguish sexual receptivity of females.

Mildly Increased Mechanical Nociceptive Sensitivity in REV-ERBα Knock-out Mice

Nociception is one of the most complex senses that is affected not only by external stimulation but also internal conditions. Previous studies have suggested that circadian rhythm is important in modulating nociception. REV-ERBα knock-out (KO) mice have disrupted circadian rhythm and altered mood-related phenotypes. In this study, we examined the role of REV-ERBα in inflammatory nociception. We found that the nociceptive sensitivity of KO mice was partially enhanced in mechanical nociception. However, this partial alteration was independent of the circadian rhythm. Taken together, deletion of REV-ERBα induced a mild change in mechanical nociceptive sensitivity but this alteration was not dependent on the circadian rhythm.

Which Neurons Will Be the Engram - Activated Neurons and/or More Excitable Neurons?

During past decades, the formation and storage principle of memory have received much attention in the neuroscience field. Although some studies have attempted to demonstrate the nature of the engram, elucidating the memory engram allocation mechanism was not possible because of the limitations of existing methods, which cannot specifically modulate the candidate neuronal population. Recently, the development of new techniques, which offer ways to mark and control specific populations of neurons, may accelerate solving this issue. Here, we review the recent advances, which have provided substantial evidence showing that both candidates (neuronal population that is activated by learning, and that has increased CREB level/excitability at learning) satisfy the criteria of the engram, which are necessary and sufficient for memory expression.

Which Neurons Will Be the Engram - Activated Neurons and/or More Excitable Neurons?

During past decades, the formation and storage principle of memory have received much attention in the neuroscience field. Although some studies have attempted to demonstrate the nature of the engram, elucidating the memory engram allocation mechanism was not possible because of the limitations of existing methods, which cannot specifically modulate the candidate neuronal population. Recently, the development of new techniques, which offer ways to mark and control specific populations of neurons, may accelerate solving this issue. Here, we review the recent advances, which have provided substantial evidence showing that both candidates (neuronal population that is activated by learning, and that has increased CREB level/excitability at learning) satisfy the criteria of the engram, which are necessary and sufficient for memory expression.

Specific Expression of Aplysia Phosphodiesterase 4 in Bag Cells Revealed by in situ Hybridization Analysis

Phosphodiesterases (PDEs) play a key role in the regulation of cyclic adenosine monophosphate (cAMP), which in turn mediates various cellular functions including learning and memory. We previously cloned and characterized three PDE4 isoforms (ApPDE4) from Aplysia kurodai. Using reverse transcription polymerase chain reaction (RT-PCR), we found that ApPDE4 isoforms are primarily expressed in the central nervous system. However, the detailed distribution of ApPDE4 mRNA in Aplysia individual ganglions was not evident. In this study, to determine the distribution of ApPDE4 mRNAs in Aplysia ganglions, we performed in situ hybridization (ISH) using a probe targeting ApPDE4, including the PDE catalytic domain. Interestingly, we found the strongest ISH-positive signals in the symmetrical bag cell clusters of the abdominal ganglion. The R2, R14, L7, L2 and L11 neurons in the abdominal ganglion, LP1 neuron in pleural ganglion, and metacerebral (MCC) neurons were ISH-positive. Mechanosensory neurons of the sensory cluster were also stained on the ventral aspect of the right and left pleural ganglia. Taken together, we found the detailed distribution of ApPDE4 mRNA in Aplysia ganglion and support their roles in serotonin (5-HT)-induced synaptic facilitation of Aplysia mechanosensory neurons.

Mind Bomb-2 Regulates Hippocampus-dependent Memory Formation and Synaptic Plasticity

Notch signaling is a key regulator of neuronal fate during embryonic development, but its function in the adult brain is still largely unknown. Mind bomb-2 (Mib2) is an essential positive regulator of the Notch pathway, which acts in the Notch signal-sending cells. Therefore, genetic deletion of Mib2 in the mouse brain might help understand Notch signaling-mediated cell-cell interactions between neurons and their physiological function. Here we show that deletion of Mib2 in the mouse brain results in impaired hippocampal spatial memory and contextual fear memory. Accordingly, we found impaired hippocampal synaptic plasticity in Mib2 knock-out (KO) mice; however, basal synaptic transmission did not change at the Schaffer collateral-CA1 synapses. Using western blot analysis, we found that the level of cleaved Notch1 was lower in Mib2 KO mice than in wild type (WT) littermates after mild foot shock. Taken together, these data suggest that Mib2 plays a critical role in synaptic plasticity and spatial memory through the Notch signaling pathway.

CTCF as a multifunctional protein in genome regulation and gene expression

CCCTC-binding factor (CTCF) is a highly conserved zinc finger protein and is best known as a transcription factor. It can function as a transcriptional activator, a repressor or an insulator protein, blocking the communication between enhancers and promoters. CTCF can also recruit other transcription factors while bound to chromatin domain boundaries. The three-dimensional organization of the eukaryotic genome dictates its function, and CTCF serves as one of the core architectural proteins that help establish this organization. The mapping of CTCF-binding sites in diverse species has revealed that the genome is covered with CTCF-binding sites. Here we briefly describe the diverse roles of CTCF that contribute to genome organization and gene expression.

Development of a Touch-Screen-Based Paradigm for Assessing Working Memory in the Mouse

Assessing the working memory of the rodent by using a touch-screen system has several advantages (e.g., allowing highly accurate data collection and flexibility in memory task design). However, there is currently no available testing paradigm utilizing touch-screen systems that can assess working memory in the mouse. In this study, we developed a touch-screen testing paradigm in which mice were trained to choose a location that is matched to a sample location after a time delay. Consistent with previous studies, this study showed that mice could not only learn the rule in the delayed matched to position (DMTP), but also could retain a transitory memory of the sample position during delay. This indicates that a touch-screen system can provide a DMTP testing platform to assess working memory in the mouse.

Effect of Food Deprivation on a Delayed Nonmatch-to-place T-maze Task

Food deprivation can affect performance on difficult cognitive task, such as the delayed nonmatch-to-place T-maze task (DNMT). The importance of food deprivation on maintaining high motivation for DNMT task has been emphasized, but not many studies have investigated the optimal conditions for depriving rodents to maximize performance. Establishing appropriate conditions for food deprivation is necessary to maintain DNMT task motivation. We applied different conditions of food deprivation (1-h food restriction vs. 1.5-g food restriction; single caging vs. group caging) and measured body weight and the number of correct choices that 8-week-old C57BL/6J mice made during the DNMT task. The 1.5-g food restriction group maintained 76.0+/-0.6% of their initial body weight, but the final body weight of the 1-h food restriction condition group was reduced to 62.2+/-0.8% of their initial body weight. These results propose that 1.5-g food restriction condition is effective condition for maintaining both body weight and motivation to complete the DNMT task.

Effect of Intensity of Unconditional Stimulus on Reconsolidation of Contextual Fear Memory

Memory reconsolidation is ubiquitous across species and various memory tasks. It is a dynamic process in which memory is modified and/or updated. In experimental conditions, memory reconsolidation is usually characterized by the fact that the consolidated memory is disrupted by a combination of memory reactivation and inhibition of protein synthesis. However, under some experimental conditions, the reactivated memory is not disrupted by inhibition of protein synthesis. This so called "boundary condition" of reconsolidation may be related to memory strength. In Pavlovian fear conditioning, the intensity of unconditional stimulus (US) determines the strength of the fear memory. In this study, we examined the effect of the intensity of US on the reconsolidation of contextual fear memory. Strong contextual fear memory, which is conditioned with strong US, is not disrupted by inhibition of protein synthesis after its reactivation; however, a weak fear memory is often disrupted. This suggests that a US of strong intensity can inhibit reconsolidation of contextual fear memory.

A Computational Model of the Temperature-dependent Changes in Firing Patterns in Aplysia Neurons

We performed experiments using Aplysia neurons to identify the mechanism underlying the changes in the firing patterns in response to temperature changes. When the temperature was gradually increased from 11degrees C to 31degrees C the firing patterns changed sequentially from the silent state to beating, doublets, beating-chaos, bursting-chaos, square-wave bursting, and bursting-oscillation patterns. When the temperature was decreased over the same temperature range, these sequential changes in the firing patterns reappeared in reverse order. To simulate this entire range of spiking patterns we modified nonlinear differential equations that Chay and Lee made using temperature-dependent scaling factors. To refine the equations, we also analyzed the spike pattern changes in the presence of potassium channel blockers. Based on the solutions of these equations and potassium channel blocker experiments, we found that, as temperature increases, the maximum value of the potassium channel relaxation time constant, taun(t) increases, but the maximum value of the probabilities of openings for activation of the potassium channels, n(t) decreases. Accordingly, the voltage-dependent potassium current is likely to play a leading role in the temperature-dependent changes in the firing patterns in Aplysia neurons.

Neuronal RNA granule contains ApCPEB1, a novel cytoplasmic polyadenylation element binding protein, in Aplysia sensory neuron

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3' untranslated regions (3'UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.

Effects of Auditory Environment on Freezing Behavior of Mice in a Novel Context

Freezing behavior is a widely used parameter that represents the level of fear. A number of studies on emotional learning have used this behavior for quantification of fear that results from a cue or a context. Even though the expression of freezing behavior is based on the dynamic interaction of mice with the environment, the effect of environmental stimuli on freezing behavior has not been studied extensively because of its minority compared to the effect of conditioning-stimuli. In this study, we found that the auditory environment of a context affects the freezing behavior of a mouse in it. This effect was not observed when the mouse was exposed to the context for the first time. However, during the second exposure, the level of freezing behavior increased significantly in an intensity-dependent manner, while the type, pitch, and rhythm of additional sounds involved in the context did not induce notable effects. This intensity-dependent effect was unrelated to the level of fear and anxiety, reflecting another aspect of the freezing behavior as a parameter for recognizing the pattern of normal behaviors.

Determination of Behavior and the Stimuli-dependent Transition of the Probability of Behavioral Expression: A Study on the Fluctuation of Behavioral State as Represented by the Level of Body Movement Suppression

The behavior of most animals is extremely complex. Despite accumulating knowledge about the mechanisms of neurons and nervous systems, which regulate these complex behaviors, we have little understanding about how these mechanisms function. In the present study, we analyzed the exploratory behavior of mice repeatedly exposed to a novel context and tracked the changes in the fluctuation patterns of the accumulated level of body movement suppression (BMS). As a result, we found that the fluctuation in BMS can be divided into two phases, which show a pattern of progressive transition from the initial state to the context-dependent and stable equilibrium state. In the former, transition phase, the level of BMS was easily affected by the number of exposures and mental status of mice. However, in the latter, equilibrium phase, the level of BMS was only dependent on the environmental stimuli involved in the context. On the basis of the results, we suggests here a model that explains the determination of complex behavior observed in higher animals by means of the probability of behavioral expression.