Activation of prefrontal parvalbumin interneurons ameliorates working memory deficit even under clinically comparable antipsychotic treatment in a mouse model of schizophrenia.

One of the critical unmet medical needs in schizophrenia is the treatment for cognitive deficits. However, the neural circuit mechanisms of them remain unresolved. Previous studies utilizing animal models of schizophrenia did not consider the fact that patients with schizophrenia generally cannot discontinue antipsychotic medication due to the high risk of relapse. Here, we used multi-dimensional approaches, including histological analysis of the prelimbic cortex (PL), LC-MS/MS-based in vivo dopamine D2 receptor occupancy analysis for antipsychotics, in vivo calcium imaging, and behavioral analyses of mice using chemogenetics to investigate neural mechanisms and potential therapeutic strategies for working memory deficit in a chronic phencyclidine (PCP) mouse model of schizophrenia. Chronic PCP administration led to alterations in excitatory and inhibitory synapses, specifically in dendritic spines of pyramidal neurons, vesicular glutamate transporter 1 (VGLUT1) positive terminals, and parvalbumin (PV) positive GABAergic interneurons located in layer 2-3 of the PL. Continuous administration of olanzapine, which achieved a sustained therapeutic window of dopamine D2 receptor occupancy (60-80%) in the striatum, did not ameliorate these synaptic abnormalities and working memory deficit in the chronic PCP-treated mice. We demonstrated that chemogenetic activation of PV neurons in the PL, as confirmed by in vivo calcium imaging, ameliorated working memory deficit in this model even under clinically comparable olanzapine treatment which by itself inhibited only PCP-induced psychomotor hyperactivity. Our study suggests that targeting prefrontal PV neurons could be a promising therapeutic intervention for cognitive deficits in schizophrenia in combination with antipsychotic medication.

A novel micro-ECoG recording method for recording multisensory neural activity from the parietal to temporal cortices in mice.

Characterization of inter-regional interactions in brain is essential for understanding the mechanism relevant to normal brain function and neurological disease. The recently developed flexible micro (µ)-electrocorticography (µECoG) device is one prominent method used to examine large-scale cortical activity across multiple regions. The sheet-shaped µECoG electrodes arrays can be placed on a relatively wide area of cortical surface beneath the skull by inserting the device into the space between skull and brain. Although rats and mice are useful tools for neuroscience, current µECoG recording methods in these animals are limited to the parietal region of cerebral cortex. Recording cortical activity from the temporal region of cortex in mice has proven difficult because of surgical barriers created by the skull and surrounding temporalis muscle anatomy. Here, we developed a sheet-shaped 64-channel µECoG device that allows access to the mouse temporal cortex, and we determined the factor determining the appropriate bending stiffness for the µECoG electrode array. We also established a surgical technique to implant the electrode arrays into the epidural space over a wide area of cerebral cortex covering from the barrel field to olfactory (piriform) cortex, which is the deepest region of the cerebral cortex. Using histology and computed tomography (CT) images, we confirmed that the tip of the µECoG device reached to the most ventral part of cerebral cortex without causing noticeable damage to the brain surface. Moreover, the device simultaneously recorded somatosensory and odor stimulus-evoked neural activity from dorsal and ventral parts of cerebral cortex in awake and anesthetized mice. These data indicate that our µECoG device and surgical techniques enable the recording of large-scale cortical activity from the parietal to temporal cortex in mice, including somatosensory and olfactory cortices. This system will provide more opportunities for the investigation of physiological functions from wider areas of the mouse cerebral cortex than those currently available with existing ECoG techniques.

Sevoflurane-induced amnesia is associated with inhibition of hippocampal cell ensemble activity after learning.

General anesthesia could induce amnesia, however the mechanism remains unclear. We hypothesized that suppression of neuronal ensemble activity in the hippocampus by anesthesia during the post-learning period causes retrograde amnesia. To test this hypothesis, two experiments were conducted with sevoflurane anesthesia (2.5%, 30 min): a hippocampus-dependent memory task, the context pre-exposure facilitation effect (CPFE) procedure to measure memory function and in vivo calcium imaging to observe neural activity in hippocampal CA1 during context exploration and sevoflurane/home cage session. Sevoflurane treatment just after context pre-exposure session impaired the CPFE memory, suggesting sevoflurane induced retrograde amnesia. Calcium imaging showed sevoflurane treatment prevented neuronal activity in CA1. Further analysis of neuronal activity with non-negative matrix factorization, which extracts neural ensemble activity based on synchronous activity, showed that sevoflurane treatment reduced the reactivation of neuronal ensembles between during context exploration just before and one day after sevoflurane inhalation. These results suggest that sevoflurane treatment immediately after learning induces amnesia, resulting from suppression of reactivation of neuronal ensembles.

Pcdhß deficiency affects hippocampal CA1 ensemble activity and contextual fear discrimination.

Clustered protocadherins (Pcdhs), a large group of adhesion molecules, are important for axonal projections and dendritic spread, but little is known about how they influence neuronal activity. The Pcdhß cluster is strongly expressed in the hippocampus, and in vivo Ca2+ imaging in Pcdhß-deficient mice revealed altered activity of neuronal ensembles but not of individual cells in this region in freely moving animals. Specifically, Pcdhß deficiency increased the number of large-size neuronal ensembles and the proportion of cells shared between ensembles. Furthermore, Pcdhß-deficient mice exhibited reduced repetitive neuronal population activity during exploration of a novel context and were less able to discriminate contexts in a contextual fear conditioning paradigm. These results suggest that one function of Pcdhßs is to modulate neural ensemble activity in the hippocampus to promote context discrimination.

Selective targeting of mRNA and the following protein synthesis of CaMKIIα at the long-term potentiation-induced site.

Late-phase long-term potentiation (L-LTP) in hippocampus, thought to be the cellular basis of long-term memory, requires new protein synthesis. Neural activity enhances local protein synthesis in dendrites, which in turn mediates long-lasting synaptic plasticity. Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is a locally synthesized protein crucial for this plasticity, as L-LTP is impaired when its local synthesis is eliminated. However, the distribution of Camk2a mRNA during L-LTP induction remains unclear. In this study, we investigated the dendritic targeting of Camk2a mRNA after high-frequency stimulation, which induces L-LTP in synapses of perforant path and granule cells in the dentate gyrus in vivoIn situ hybridization studies revealed that Camk2a mRNA was immediately but transiently targeted to the site receiving high-frequency stimulation. This was associated with an increase in de novo protein synthesis of CaMKIIα. These results suggest that dendritic translation of CaMKIIα is locally mediated where L-LTP is induced. This phenomenon may be one of the essential processes for memory establishment.

Orchestrated ensemble activities constitute a hippocampal memory engram.

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory.

Powerful Homeostatic Control of Oligodendroglial Lineage by PDGFRα in Adult Brain.

Oligodendrocyte progenitor cells (OPCs) are widely distributed cells of ramified morphology in adult brain that express PDGFRα and NG2. They retain mitotic activities in adulthood and contribute to oligodendrogenesis and myelin turnover; however, the regulatory mechanisms of their cell dynamics in adult brain largely remain unknown. Here, we found that global Pdgfra inactivation in adult mice rapidly led to elimination of OPCs due to synchronous maturation toward oligodendrocytes. Surprisingly, OPC densities were robustly reconstituted by the active expansion of Nestin+ immature cells activated in meninges and brain parenchyma, as well as a few OPCs that escaped from Pdgfra inactivation. The multipotent immature cells were induced in the meninges of Pdgfra-inactivated mice, but not of control mice. Our findings revealed powerful homeostatic control of adult OPCs, engaging dual cellular sources of adult OPC formation. These properties of the adult oligodendrocyte lineage and the alternative OPC source may be exploited in regenerative medicine.

Artificial association of memory events by optogenetic stimulation of hippocampal CA3 cell ensembles.

Previous gain-of-function studies using an optogenetic technique showed that manipulation of the hippocampal dentate gyrus or CA1 cell ensembles is important for memory reactivation and to generate synthetic or false memory. However, gain-of-function study manipulating CA3 cell ensembles has not been reported. The CA3 area of the hippocampus comprises a recurrent excitatory circuit, which is thought to be important for the generation of associations among the stored information within one brain region. We investigated whether the coincident firing of cell ensembles in one brain region, hippocampal CA3, associates distinct events. CA3 cell ensembles responding to context exploration and during contextual fear conditioning were labeled with channelrhodopsin-2 (ChR2)-mCherry. The synchronous activation of these ensembles induced freezing behavior in mice in a neutral context, in which a foot shock had never been delivered. The recall of this artificial associative fear memory was context specific. In vivo electrophysiological recordings showed that 20-Hz optical stimulation of ChR2-mCherry-expressing CA3 neurons, which is the same stimulation protocol used in behavioral experiment, induced long-term potentiation at CA3-CA3 synapses. Altogether, these results demonstrate that the synchronous activation of ensembles in one brain region, CA3 of the hippocampus, is sufficient for the association of distinct events. The results of our electrophysiology potentially suggest that this artificial association of memory events might be induced by the strengthening of synaptic efficacy between CA3 ensembles via recurrent circuit.

Adult Neurogenesis Conserves Hippocampal Memory Capacity.

The hippocampus is crucial for declarative memories in humans and encodes episodic and spatial memories in animals. Memory coding strengthens synaptic efficacy via an LTP-like mechanism. Given that animals store memories of everyday experiences, the hippocampal circuit must have a mechanism that prevents saturation of overall synaptic weight for the preservation of learning capacity. LTD works to balance plasticity and prevent saturation. In addition, adult neurogenesis in the hippocampus is proposed to be involved in the down-scaling of synaptic efficacy. Here, we show that adult neurogenesis in male rats plays a crucial role in the maintenance of hippocampal capacity for memory (learning and/or memory formation). Neurogenesis regulated the maintenance of LTP, with decreases and increases in neurogenesis prolonging or shortening LTP persistence, respectively. Artificial saturation of hippocampal LTP impaired memory capacity in contextual fear conditioning, which completely recovered after 14 d, which was the time required for LTP to decay to the basal level. Memory capacity gradually recovered in parallel with neurogenesis-mediated gradual decay of LTP. Ablation of neurogenesis by x-ray irradiation delayed the recovery of memory capacity, whereas enhancement of neurogenesis using a running wheel sped up recovery. Therefore, one benefit of ongoing adult neurogenesis is the maintenance of hippocampal memory capacity through homeostatic renewing of hippocampal memory circuits. Decreased neurogenesis in aged animals may be responsible for the decline in cognitive function with age.SIGNIFICANCE STATEMENT Learning many events each day increases synaptic efficacy via LTP, which can prevent the storage of new memories in the hippocampal circuit. In this study, we demonstrate that hippocampal capacity for the storage of new memories is maintained by ongoing adult neurogenesis through homoeostatic renewing of hippocampal circuits in rats. A decrease or an increase in neurogenesis, respectively, delayed or sped up the recovery of memory capacity, suggesting that hippocampal adult neurogenesis plays a critical role in reducing LTP saturation and keeps the gate open for new memories by clearing out the old memories from the hippocampal memory circuit.

Frequency-specific stimulations induce reconsolidation of long-term potentiation in freely moving rats.

BACKGROUND: When consolidated memories are retrieved, they become labile and a new protein synthesis-dependent reconsolidation process is required to restabilize these memories. So far, most studies conducted on reconsolidation rely on the analyses of animal behavior, leaving the synaptic mechanisms that underlie reconsolidation largely unclear. Here, we examined whether the reconsolidation process occurs in hippocampal long term potentiation (LTP), as a synaptic model that is correlated with long term memories (LTM). RESULTS: We employed LTP system in the dentate gyrus of freely moving rats that lasts for weeks simulating LTM. LTP was induced by high frequency stimulation at 400 Hz (HFS400), and as a reactivation stimulation, we tested a low frequency stimulation at 0.1 Hz (LFS0.1), a theta stimulation at 8 Hz (TS8), or HFS400. Unlike HFS400 reactivation, both LFS0.1 and TS8 induced a reconsolidation-like phenomenon and rendered the LTP labile to protein synthesis inhibition by anisomycin. Without reactivation, LTP remained unaffected by protein synthesis inhibition. In addition, the TS8-induced LTP reconsolidation was NMDAR dependent. CONCLUSION: Our results indicate that, as with behavioral LTM, there are boundary conditions for LTP reconsolidation where only a certain range of frequency stimulations as reactivation can destabilize the consolidated LTP. This LTP reconsolidation system will be useful for future elucidation of the synaptic reconsolidation mechanism.

Targeting of ribosomal protein S6 to dendritic spines by in vivo high frequency stimulation to induce long-term potentiation in the dentate gyrus.

Late phase long-term potentiation (L-LTP) in the hippocampus is believed to be the cellular basis of long-term memory. Protein synthesis is required for persistent forms of synaptic plasticity, including L-LTP. Neural activity is thought to enhance local protein synthesis in dendrites, and one of the mechanisms required to induce or maintain the long-lasting synaptic plasticity is protein translation in the dendrites. One regulator of translational processes is ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. Although polyribosomes containing rpS6 are observed in dendritic spines, it remains unclear whether L-LTP induction triggers selective targeting of the translational machinery to activated synapses in vivo. Therefore, we investigated synaptic targeting of the translational machinery by observing rpS6 immunoreactivity during high frequency stimulation (HFS) for L-LTP induction in vivo. Immunoelectron microscopic analysis revealed a selective but transient increase in rpS6 immunoreactivity occurring as early as 15 min after the onset of HFS in dendritic spine heads at synaptic sites receiving HFS. Concurrently, levels of the rpS6 protein rapidly declined in somata of granule cells, as determined using immunofluorescence microscopy. These results suggest that the translational machinery is rapidly targeted to activated spines and that this targeting mechanism may contribute to the establishment of L-LTP.

Artificial association of pre-stored information to generate a qualitatively new memory.

Memory is thought to be stored in the brain as an ensemble of cells activated during learning. Although optical stimulation of a cell ensemble triggers the retrieval of the corresponding memory, it is unclear how the association of information occurs at the cell ensemble level. Using optogenetic stimulation without any sensory input in mice, we found that an artificial association between stored, non-related contextual, and fear information was generated through the synchronous activation of distinct cell ensembles corresponding to the stored information. This artificial association shared characteristics with physiologically associated memories, such as N-methyl-D-aspartate receptor activity and protein synthesis dependence. These findings suggest that the association of information is achieved through the synchronous activity of distinct cell ensembles. This mechanism may underlie memory updating by incorporating novel information into pre-existing networks to form qualitatively new memories.

Spine formation pattern of adult-born neurons is differentially modulated by the induction timing and location of hippocampal plasticity.

In the adult hippocampus dentate gyrus (DG), newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP) was induced at 12, 16, or 21 days postinfection (dpi), at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn't respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP-induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.

Activity-dependent localization in spines of the F-actin capping protein CapZ screened in a rat model of dementia.

Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.

LTP induction within a narrow critical period of immature stages enhances the survival of newly generated neurons in the adult rat dentate gyrus.

Neurogenesis occurs in the adult hippocampus of various animal species. A substantial fraction of newly generated neurons die before they mature, and the survival rate of new neurons are regulated in an experience-dependent manner. Previous study showed that high-frequency stimulation (HFS) of perforant path fibers to the hippocampal dentate gyrus (DG) induces the long-term potentiation (LTP) in the DG, and enhances the survival of newly generated neurons in the DG. In this study, we addressed whether a time period exists during which the survival of new neurons is maximally sensitive to the HFS. We found that the enhancement of cell survival by HFS was exclusively restricted to the specific narrow period during immature stages of new neurons (7-10 days after birth). Furthermore, the pharmacological blockade of LTP induction suppressed the enhancement of cell survival by the HFS. These results suggest that the LTP induction within a narrow critical period of immature stages enhances the survival of newly generated neurons in rat DG.

Adult neurogenesis modulates the hippocampus-dependent period of associative fear memory.

Acquired memory initially depends on the hippocampus (HPC) for the process of cortical permanent memory formation. The mechanisms through which memory becomes progressively independent from the HPC remain unknown. In the HPC, adult neurogenesis has been described in many mammalian species, even at old ages. Using two mouse models in which hippocampal neurogenesis is physically or genetically suppressed, we show that decreased neurogenesis is accompanied by a prolonged HPC-dependent period of associative fear memory. Inversely, enhanced neurogenesis by voluntary exercise sped up the decay rate of HPC dependency of memory, without loss of memory. Consistently, decreased neurogenesis facilitated the long-lasting maintenance of rat hippocampal long-term potentiation in vivo. These independent lines of evidence strongly suggest that the level of hippocampal neurogenesis play a role in determination of the HPC-dependent period of memory in adult rodents. These observations provide a framework for understanding the mechanisms of the hippocampal-cortical complementary learning systems.

Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP maintenance in vivo.

The dendritic spine is an important site of neuronal plasticity and contains extremely high levels of cytoskeletal actin. However, the dynamics of the actin cytoskeleton during synaptic plasticity and its in vivo function remain unclear. Here we used an in vivo dentate gyrus LTP model to show that LTP induction is associated with actin cytoskeletal reorganization characterized by a long-lasting increase in F-actin content within dendritic spines. This increase in F-actin content is dependent on NMDA receptor activation and involves the inactivation of actin depolymerizing factor/cofilin. Inhibition of actin polymerization with latrunculin A impaired late phase of LTP without affecting the initial amplitude and early maintenance of LTP. These observations suggest that mechanisms regulating the spine actin cytoskeleton contribute to the persistence of LTP.