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.

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.

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.

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

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

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

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

Prioritized experience replays on a hippocampal predictive map for learning.

Hippocampal cells are central to spatial and predictive representations, and experience replays by place cells are crucial for learning and memory. Nonetheless, how hippocampal replay patterns dynamically change during the learning process remains to be elucidated. Here, we designed a spatial task in which rats learned a new behavioral trajectory for reward. We found that as rats updated their behavioral strategies for a novel salient location, hippocampal cell ensembles increased theta-sequences and sharp wave ripple-associated synchronous spikes that preferentially replayed salient locations and reward-related contexts in reverse order. The directionality and contents of the replays progressively varied with learning, including an optimized path that had never been exploited by the animals, suggesting prioritized replays of significant experiences on a predictive map. Online feedback blockade of sharp wave ripples during a learning process inhibited stabilizing optimized behavior. These results implicate learning-associated experience replays that act to learn and reinforce specific behavioral strategies.

Astrocytic cAMP modulates memory via synaptic plasticity.

Astrocytes play a key role in brain homeostasis and functions such as memory. Specifically, astrocytes express multiple receptors that transduce signals via the second messenger cAMP. However, the involvement of astrocytic cAMP in animal behavior and the underlying glial-neuronal interactions remains largely unknown. Here, we show that an increase in astrocytic cAMP is sufficient to induce synaptic plasticity and modulate memory. We developed a method to increase astrocytic cAMP levels in vivo using photoactivated adenylyl cyclase and found that increased cAMP in hippocampal astrocytes at different time points facilitated memory formation but interrupted memory retention via NMDA receptor-dependent plasticity. Furthermore, we found that the cAMP-induced modulation of memory was mediated by the astrocyte-neuron lactate shuttle. Thus, our study unveils a role of astrocytic cAMP in brain function by providing a tool to modulate astrocytic cAMP in vivo.

Analyzing schizophrenia-related phenotypes in mice caused by autoantibodies against NRXN1α in schizophrenia.

The molecular pathological mechanisms underlying schizophrenia remain unclear; however, genomic analysis has identified genes encoding important risk molecules. One such molecule is neurexin 1α (NRXN1α), a presynaptic cell adhesion molecule. In addition, novel autoantibodies that target the nervous system have been found in patients with encephalitis and neurological disorders. Some of these autoantibodies inhibit synaptic antigen molecules. Studies have examined the association between schizophrenia and autoimmunity; however, the pathological data remain unclear. Here, we identified a novel autoantibody against NRXN1α in patients with schizophrenia (n = 2.1%) in a Japanese cohort (n = 387). None of the healthy control participants (n = 362) were positive for anti-NRXN1α autoantibodies. Anti-NRXN1α autoantibodies isolated from patients with schizophrenia inhibited the molecular interaction between NRXN1α and Neuroligin 1 (NLGN1) and between NRXN1α and Neuroligin 2 (NLGN2). Additionally, these autoantibodies reduced the frequency of the miniature excitatory postsynaptic current in the frontal cortex of mice. Administration of anti-NRXN1α autoantibodies from patients with schizophrenia into the cerebrospinal fluid of mice reduced the number of spines/synapses in the frontal cortex and induced schizophrenia-related behaviors such as reduced cognition, impaired pre-pulse inhibition, and reduced social novelty preference. These changes were improved through the removal of anti-NRXN1α autoantibodies from the IgG fraction of patients with schizophrenia. These findings demonstrate that anti-NRXN1α autoantibodies transferred from patients with schizophrenia cause schizophrenia-related pathology in mice. Removal of anti-NRXN1α autoantibodies may be a therapeutic target for a subgroup of patients who are positive for these autoantibodies.

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.

Evidence that endpoint feedback facilitates intermanual transfer of visuomotor force learning by a cognitive strategy.

Humans continuously adapt their movement to a novel environment by recalibrating their sensorimotor system. Recent evidence, however, shows that explicit planning to compensate for external changes, i.e., a cognitive strategy, can also aid performance. If such a strategy is planned in external space, it should improve performance in an effector-independent manner. We tested this hypothesis by examining whether promoting a cognitive strategy during a visual-force adaptation task performed in one hand can facilitate learning for the opposite hand. Participants rapidly adjusted the height of visual bar on screen to a target level by isometrically exerting force on a handle using their right hand. Visuomotor gain increased during the task and participants learned the increased gain. Visual feedback was continuously provided for one group, whereas for another group only the endpoint of the force trajectory was presented. The latter has been reported to promote cognitive strategy use. We found that endpoint feedback produced stronger intermanual transfer of learning and slower response times than continuous feedback. In a separate experiment, we found evidence that aftereffects are reduced when only endpoint feedback is provided, a finding that has been consistently observed when cognitive strategies are used. The results suggest that intermanual transfer can be facilitated by a cognitive strategy. This indicates that the behavioral observation of intermanual transfer can be achieved either by forming an effector-independent motor representation or by sharing an effector-independent cognitive strategy between the hands.NEW & NOTEWORTHY The causes and consequences of cognitive strategy use are poorly understood. We tested whether a visuomotor task learned in a manner that may promote cognitive strategy use causes greater generalization across effectors. Visual feedback was manipulated to promote cognitive strategy use. Learning consistent with cognitive strategy use for one hand transferred to the unlearned hand. Our result suggests that intermanual transfer can result from a common cognitive strategy used to control both hands.

Delayed reinforcement hinders subsequent extinction.

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

Contextual Fear Memory Retrieval Is Vulnerable to Hippocampal Noise.

Memory retrieval depends on reactivation of memory engram cells. Inadvertent activation of these cells is expected to cause memory-retrieval failure, but little is known about how noisy activity of memory-irrelevant neurons impacts mnemonic processes. Here, we report that optogenetic nonselective activation of only tens of hippocampal CA1 cells (∼0.01% of the total cells in the CA1 pyramidal cell layer) impairs contextual fear memory recall. Memory recall failure was associated with altered neuronal reactivation in the basolateral amygdala. These results indicate that hippocampal memory retrieval requires strictly regulated activation of a specific neuron ensemble and is easily disrupted by the introduction of noisy CA1 activity, suggesting that reactivating memory engram cells as well as silencing memory-irrelevant neurons are both crucial for memory retrieval.

The subiculum sensitizes retrosplenial cortex layer 2/3 pyramidal neurons.

KEY POINTS: Neurons in the retrosplenial cortex (RSC), a cerebral region that connects synaptically with various brain regions, are known to increase neuronal activity in accordance with hippocampal sharp wave-ripples. Pyramidal cells in granular RSC (gRSC) layer 2/3, but not layer 5, exhibit slowly ramping depolarization and considerably delayed spikes in response to a step-pulse current injection. The latencies of delayed spikes in RSC layer 2/3 pyramidal neurons were shortened by a preceding current injection. This effect was mimicked by activation of axonal afferents from the subiculum, but not of neocortical afferents. The subiculum is likely to facilitate information processing and flow in the RSC. ABSTRACT: The retrosplenial cortex (RSC), a cerebral region involved in diverse cognitive functions, is an anatomical hub that forms monosynaptic connections with various brain areas. Here, we report a unique form of short-term intrinsic plasticity in mouse granular RSC layer 2/3 pyramidal cells. These cells exhibited delayed spikes in response to somatic current injection, but the spike latencies were shortened by a preceding brief depolarization (priming). This priming-induced sensitization is distinct from desensitization, which is commonly observed in other cortical neurons. The facilitatory priming effect lasted for more than 3 s, providing a time window for increased sensitivity to RSC inputs. Based on in vitro and in vivo patch-clamp recordings following optogenetic stimulation of axonal fibres, we found that preactivation of subicular afferents replicated the facilitatory priming effect. The results suggest that subicular inputs to RSC layer 2/3 neurons may modulate subsequent information integration in the RSC layer 2/3 circuits.

Neuronal brain-derived neurotrophic factor manipulates microglial dynamics.

Brain-derived neurotrophic factor (BDNF), a main member of the neurotrophin family that is active in the brain, supports neuronal survival and growth. Microglial BDNF affects both the structural and functional properties of neurons. In contrast, whether and how neuronal BDNF affects microglial dynamics remain largely undetermined. Here, we examined the effects of BDNF on the properties of microglia in the CA3 region of the hippocampus. We chose this site because the axonal boutons of hippocampal mossy fibers, which are mostly formed in the CA3 region, contain the highest levels of BDNF in the rodent brain. We transfected mouse dentate granule cells with an adeno-associated virus that encodes both a BDNF short hairpin RNA (shRNA) and red fluorescent protein to examine the effects of mossy fiber-derived BDNF on microglia. Based on immunohistochemistry, BDNF knockdown with an shRNA resulted in an increase in microglial density in the mossy fiber pathway and increased engulfment of mossy fiber axons by microglia. In addition, we performed time-lapse imaging of microglial processes in hippocampal slice cultures to examine the effects of BDNF on microglial motility. Time-lapse imaging revealed increases in the motility of microglial processes and the engulfment of mossy fiber synapses by microglia when BDNF signaling was pharmacologically blocked. Thus, neuronal BDNF prevents microglia from engulfing mossy fiber synapses in the hippocampus.

Highly active neurons emerging in vitro.

Mean firing rates vary across neurons in a neuronal network. Although most neurons infrequently emit spikes, a small fraction of neurons exhibit extremely high frequencies of spikes; this fraction of neurons plays a pivotal role in information processing, however, little is known about how these outliers emerge and whether they are maintained over time. In primary cultures of mouse hippocampal neurons, we traced highly active neurons every 24 h for 7 wk by optically observing the fluorescent protein dVenus; the expression of dVenus was controlled by the promoter of Arc, an immediate early gene that is induced by neuronal activity. Under default-mode conditions, 0.3%-0.4% of neurons were spontaneously Arc-dVenus positive, exhibiting high firing rates. These neurons were spatially clustered, exhibited intermittently repeated dVenus expression, and often continued to express Arc-dVenus for approximately 2 wk. Thus, highly active neurons constitute a few select functional subpopulations in the neuronal network.NEW & NOTEWORTHY The overdispersion of neuronal activity levels can often be attributed to very few neurons exhibiting extremely high firing rates, but due to technical difficulty, no studies have examined how these outliers are selected during development and whether they are maintained over time. We optically monitored highly active neurons for as long as 7 wk in vitro and found that they constituted a unique population that was different from other "mediocre" neurons with normal firing rates.

Hippocampal beta oscillations predict mouse object-location associative memory performance.

Memorizing the locations of environmental cues is crucial for survival and depends on the hippocampus. We recorded local field potentials (LFPs) from the hippocampus of freely moving mice during an object location task. The power of beta-band (23-30 Hz) oscillations increased immediately before approaching objects in a memory-encoding phase. The exploration-induced beta oscillations gradually decreased during the memory-encoding session. Mice that exhibited stronger beta oscillation power exhibited better performance in the subsequent memory-retrieval test. These results suggest that beta oscillations in the hippocampal CA1 region are involved in the memory encoding of object-location associations.

The effects of microglia- and astrocyte-derived factors on neurogenesis in health and disease.

Hippocampal neurogenesis continues throughout life and has been suggested to play an essential role in maintaining spatial cognitive function under physiological conditions. An increasing amount of evidence has indicated that adult neurogenesis is tightly controlled by environmental conditions in the neurogenic niche, which consists of multiple types of cells including microglia and astrocytes. Microglia maintain the environment of neurogenic niche through their phagocytic capacity and interaction with neurons via fractalkine-CX3CR1 signaling. In addition, microglia release growth factors such as brain-derived neurotrophic factor (BDNF) and cytokines such as tumor necrosis factor (TNF)-α to support the development of adult born neurons. Astrocytes also manipulate neurogenesis by releasing various soluble factors including adenosine triphosphate and lactate. Whereas, under pathological conditions such as Alzheimer's disease, depression, and epilepsy, microglia and astrocytes play a leading role in inflammation and are involved in attenuating the normal process of neurogenesis. The modulation of glial functions on neurogenesis in these brain diseases are attracting attention as a new therapeutic target. This review describes how these glial cells play a role in adult hippocampal neurogenesis in both health and disease, especially focusing glia-derived factors.

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

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