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.

Requirement for hippocampal CA3 NMDA receptors in artificial association of memory events stored in CA3 cell ensembles.

The N-methyl-D-aspartate receptors (NRs) in hippocampal CA3 are crucial for the synaptic transmission and plasticity within the CA3 recurrent circuit, which supports the hippocampal functions, such as pattern completion, and reverberatory association of sensory inputs. Previous study showed that synchronous activation of distinct cell populations in CA3, which correspond to distinct events, associated independent events, suggesting that the recurrent circuit expressing NRs in CA3 mediates the artificial association of memory events stored in CA3 ensembles. However, it is still unclear whether CA3 NRs are crucial for the artificial association of memory events stored in the CA3 ensembles. Here we report that the triple transgenic mice (cfos-tTA/KA1-Cre/NR1 flox/flox), which specifically lack NRs in the CA3 cell ensembles, showed impairment in artificial association between two events, which in control mice triggered artificial association. This result indicates that NRs in the hippocampal CA3 are required for the artificial association of memory events stored in the CA3 cell ensembles.

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.

MicroLED neural probe for effective in vivo optogenetic stimulation.

The MicroLED probe enables optogenetic control of neural activity in spatially separated brain regions. Understanding its heat generation characteristics is important. In this study, we investigated the temperature rise (ΔT) characteristics in the brain tissue using a MicroLED probe. The ΔT strongly depended on the surrounding environment of the probe, including the differences between the air and the brain, and the area touching the brain tissue. Through animal experiments, we suggest an in situ temperature monitoring method using temperature dependence on electrical characteristics of the MicroLED. Finally, optical stimulation by MicroLEDs proved effective in controlling optogenetic neural activity in animal models.

A cortical cell ensemble in the posterior parietal cortex controls past experience-dependent memory updating.

When processing current sensory inputs, animals refer to related past experiences. Current information is then incorporated into the related neural network to update previously stored memories. However, the neuronal mechanism underlying the impact of memories of prior experiences on current learning is not well understood. Here, we found that a cellular ensemble in the posterior parietal cortex (PPC) that is activated during past experience mediates an interaction between past and current information to update memory through a PPC-anterior cingulate cortex circuit in mice. Moreover, optogenetic silencing of the PPC ensemble immediately after retrieval dissociated the interaction without affecting individual memories stored in the hippocampus and amygdala. Thus, a specific subpopulation of PPC cells represents past information and instructs downstream brain regions to update previous memories.

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.

Overlapping memory trace indispensable for linking, but not recalling, individual memories.

Memories are not stored in isolation from other memories but are integrated into associative networks. However, the mechanisms underlying memory association remain elusive. Using two amygdala-dependent behavioral paradigms-conditioned taste aversion (CTA) and auditory-cued fear conditioning (AFC)-in mice, we found that presenting the conditioned stimulus used for the CTA task triggered the conditioned response of the AFC task after natural coreactivation of the memories. This was accompanied through an increase in the overlapping neuronal ensemble in the basolateral amygdala. Silencing of the overlapping ensemble suppressed CTA retrieval-induced freezing. However, retrieval of the original CTA or AFC memory was not affected. A small population of coshared neurons thus mediates the link between memories. They are not necessary for recalling individual memories.

Cellular tagging as a neural network mechanism for behavioural tagging.

Behavioural tagging is the transformation of a short-term memory, induced by a weak experience, into a long-term memory (LTM) due to the temporal association with a novel experience. The mechanism by which neuronal ensembles, each carrying a memory engram of one of the experiences, interact to achieve behavioural tagging is unknown. Here we show that retrieval of a LTM formed by behavioural tagging of a weak experience depends on the degree of overlap with the neuronal ensemble corresponding to a novel experience. The numbers of neurons activated by weak training in a novel object recognition (NOR) task and by a novel context exploration (NCE) task, denoted as overlapping neurons, increases in the hippocampal CA1 when behavioural tagging is successfully achieved. Optical silencing of an NCE-related ensemble suppresses NOR-LTM retrieval. Thus, a population of cells recruited by NOR is tagged and then preferentially incorporated into the memory trace for NCE to achieve behavioural tagging.

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.

Neuronal stimulation induces autophagy in hippocampal neurons that is involved in AMPA receptor degradation after chemical long-term depression.

Many studies have reported the roles played by regulated proteolysis in synaptic plasticity and memory, but the role of autophagy in neurons remains unclear. In mammalian cells, autophagy functions in the clearance of long-lived proteins and organelles and in adaptation to starvation. In neurons, although autophagy-related proteins (ATGs) are highly expressed, autophagic activity markers, autophagosome (AP) number, and light chain protein 3-II (LC3-II) are low compared with other cell types. In contrast, conditional knock-out of ATG5 or ATG7 in mouse brain causes neurodegeneration and behavioral deficits. Therefore, this study aimed to test whether autophagy is especially regulated in neurons to adapt to brain functions. In cultured rat hippocampal neurons, we found that KCl depolarization transiently increased LC3-II and AP number, which was partially inhibited with APV, an NMDA receptor (NMDAR) inhibitor. Brief low-dose NMDA, a model of chemical long-term depression (chem-LTD), increased LC3-II with a time course coincident with Akt and mammalian target of rapamycin (mTOR) dephosphorylation and degradation of GluR1, an AMPA receptor (AMPAR) subunit. Downstream of NMDAR, the protein phosphatase 1 inhibitor okadaic acid, PTEN inhibitor bpV(HOpic), autophagy inhibitor wortmannin, and short hairpin RNA-mediated knockdown of ATG7 blocked chem-LTD-induced autophagy and partially recovered GluR1 levels. After chem-LTD, GFP-LC3 puncta increased in spines and in dendrites when AP-lysosome fusion was blocked. These results indicate that neuronal stimulation induces NMDAR-dependent autophagy through PI3K-Akt-mTOR pathway inhibition, which may function in AMPAR degradation, thus suggesting autophagy as a contributor to NMDAR-dependent synaptic plasticity and brain functions.

N-acetyltransferase ARD1-NAT1 regulates neuronal dendritic development.

ARD1 and NAT1 constitute an N-acetyltransferase complex where ARD1 holds the enzymatic activity of the complex. The ARD1-NAT1 complex mediates N-terminal acetylation of nascent polypeptides that emerge from ribosomes after translation. ARD1 may also acetylate the internal lysine residues of proteins. Although ARD1 and NAT1 have been found in the brain, the physiological role and substrates of the ARD1-NAT1 complex in neurons remain unclear. Here we investigated role of N-acetyltransferase activity in the process of neuronal development. Expression of ARD1 and NAT1 increased during dendritic development, and both proteins colocalized with microtubules in dendrites. The ARD1-NAT1 complex displayed acetyltransferase activity against a purified microtubule fraction in vitro. Inhibition of the complex limited the dendritic extension of cultured neurons. These findings suggest that the ARD1-NAT1 complex has acetyltransferase activity against microtubules in dendrites. Regulation by acetyltransferase activity is a novel mechanism that is required for dendritic arborization during neuronal development.