Projections of hippocampal CA2 pyramidal neurons: Distinct innervation patterns of CA2 compared to CA3 in rodents.

The hippocampus is a center for spatial and episodic memory formation in rodents. Understanding the composition of subregions and circuitry maps of the hippocampus is essential for elucidating the mechanism of memory formation and recall. For decades, the trisynaptic circuit (entorhinal cortex layer II-dentate gyrus - CA3-CA1) has been considered the neural network substrate responsible for learning and memory. Recently, CA2 has emerged as an important area in the hippocampal circuitry, with distinct functions from those of CA3. In this article, we review the historical definition of the hippocampal area CA2 and the differential projection patterns between CA2 and CA3 pyramidal neurons. We provide a concise and comprehensive map of CA2 outputs by comparing (1) ipsi versus contra projections, (2) septal versus temporal projections, and (3) lamellar structures of CA2 and CA3 pyramidal neurons.

Dark Rearing in the Visual Critical Period Causes Structural Changes in Myelinated Axons in the Adult Mouse Visual Pathway.

An appropriate sensory experience during the early developmental period is important for brain maturation. Dark rearing during the visual critical period delays the maturation of neuronal circuits in the visual cortex. Although the formation and structural plasticity of the myelin sheaths on retinal ganglion cell axons modulate the visual function, the effects of dark rearing during the visual critical period on the structure of the retinal ganglion cell axons and their myelin sheaths are still unclear. To address this question, mice were reared in a dark box during the visual critical period and then normally reared to adulthood. We found that myelin sheaths on the retinal ganglion cell axons of dark-reared mice were thicker than those of normally reared mice in both the optic chiasm and optic nerve. Furthermore, whole-mount immunostaining with fluorescent axonal labeling and tissue clearing revealed that the myelin internodal length in dark-reared mice was shorter than that in normally reared mice in both the optic chiasm and optic nerve. These findings demonstrate that dark rearing during the visual critical period affects the morphology of myelin sheaths, shortens and thickens myelin sheaths in the visual pathway, despite the mice being reared in normal light/dark conditions after the dark rearing.

Perivascular macrophages produce type I collagen around cerebral small vessels under prolonged hypertension in rats.

Hypertension leads to structural remodeling of cerebral blood vessels, which has been implicated in the pathophysiology of cerebrovascular diseases. The remodeling and progression of arteriolosclerosis under hypertension involve fibrosis along with the production of type I collagen around cerebral arterioles. However, the source and regulatory mechanisms of this collagen production remain elusive. In this study, we examined if perivascular macrophages (PVMs) are involved in collagen production around cerebral small vessels in hypertensive SHRSP/Izm rats. Immunoreactivity for type I collagen around cerebral small vessels in 12-week-old hypertensive rats tended to higher than those in 4-week-old hypertensive and 12-week-old control rats. In ultrastructural analyses using transmission electron microscopy, the substantial deposition of collagen fibers could be observed in the intercellular spaces around PVMs near the arterioles of rats with prolonged hypertension. In situ hybridization analyses revealed that cells positive for mRNA of Col1a1, which comprises type I collagen, were observed near cerebral small vessels. The Col1a1-positive cells around cerebral small vessels were colocalized with immunoreactivity for CD206, a marker for PVMs, but not with those for glial fibrillary acidic protein or desmin, markers for other perivascular cells such as astrocytes and vascular smooth muscle cells. These results demonstrated that enhanced production of type I collagen is observed around cerebral small vessels in rats with prolonged hypertension and Col1a1 is expressed by PVMs, and support the concept that PVMs are involved in collagen production and vascular fibrosis under hypertensive conditions.

Rearing-environment-dependent hippocampal local field potential differences in wild-type and inositol trisphosphate receptor type 2 knockout mice.

KEY POINTS: Mice reared in an enriched environment are demonstrated to have larger hippocampal gamma oscillations than those reared in isolation, thereby confirming previous observations in rats. To test whether astrocytic Ca2+ surges are involved in this experience-dependent LFP pattern modulation, we used inositol trisphosphate receptor type 2 (IP3 R2)-knockout (KO) mice, in which IP3 /Ca2+ signalling in astrocytes is largely diminished. We found that this experience-dependent gamma power alteration persists in the KO mice. Interestingly, hippocampal ripple events, the synchronized events critical for memory consolidation, are reduced in magnitude and frequency by both isolated rearing and IP3 R2 deficiency. ABSTRACT: Rearing in an enriched environment (ENR) is known to enhance cognitive and memory abilities in rodents, whereas social isolation (ISO) induces depression-like behaviour. The hippocampus has been documented to undergo morphological and functional changes depending on these rearing environments. For example, rearing condition during juvenility alters CA1 stratum radiatum gamma oscillation power in rats. In the present study, hippocampal CA1 local field potentials (LFP) were recorded from bilateral CA1 in urethane-anaesthetized mice that were reared in either an ENR or ISO condition. Similar to previous findings in rats, gamma oscillation power during theta states was higher in the ENR group. Ripple events that occur during non-theta periods in the CA1 stratum pyramidale also had longer intervals in ISO mice. Because astrocytic Ca2+ elevations play a key role in synaptic plasticity, we next tested whether these changes in LFP are also expressed in inositol trisphosphate receptor type 2 (IP3 R2)-knockout (KO) mice, in which astrocytic Ca2+ elevations are largely diminished. We found that the gamma power was also higher in IP3 R2-KO-ENR mice compared to IP3 R2-KO-ISO mice, suggesting that the rearing-environment-dependent gamma power alteration does not necessarily require the astrocytic IP3 /Ca2+ pathway. By contrast, ripple events showed genotype-dependent changes, as well as rearing condition-dependent changes: ISO housing and IP3 R2 deficiency both lead to longer inter-ripple intervals. Moreover, we found that ripple magnitude in the right CA1 tended to be smaller in IP3 R2-KO. Because IP3 R2-KO mice have been reported to have depression phenotypes, our results suggest that ripple events and the mood of animals may be broadly correlated.

The defects of the hippocampal ripples and theta rhythm in depression, and the effects of physical exercise on their amelioration.

Adverse environmental stress causes depressive symptoms with the impairments of memory formation, cognition, and motivation, however, their underlying neural bases have not been well understood, especially based on the observation of living animals. In the present study, therefore, the mice model of restraint-induced stress was examined electrophysiologically to investigate the alterations of hippocampal sharp wave ripples (SWRs) and theta rhythms. In addition, the therapeutic effects of physical exercise on the amelioration of those hippocampal impairments were examined in combination with a series of behavioral tests. The data demonstrated that chronic restraint stress caused the reductions of occurrence and amplitude of hippocampal SWRs and the decreases of occurrence, duration, and power of theta rhythms, while physical exercise significantly reverted them to the levels of healthy control. Furthermore, hippocampal adult neurogenesis and microglial activation, previously reported to be involved in the etiology of depression, were histologically examined in the mice. The results showed that the impairment of neurogenesis and alleviation of microglial activation were induced in the depressed mice. On the other hand, physical exercise considerably ameliorated those pathological conditions in the affected brain. Consistently, the data of behavioral tests in mice suggested that physical exercise ameliorated the symptomatic defects of motivation, memory formation, and cognition in the depressed mice. The impairments of hippocampal SWRs and theta rhythms in the affected hippocampus are linked with the symptomatic impairments of cognition and motivation, and the defect of memory formation, respectively, in depression. Taken together, this study demonstrated the implications of impairment of the hippocampal SWRs and theta rhythms in the etiology of depression and their usefulness as diagnostic markers of depression.

Regulation of synaptic currents by mGluR2 at reciprocal synapses in the mouse accessory olfactory bulb.

The throughput of information from the accessory olfactory bulb (AOB) to downstream structures is controlled by reciprocal dendrodendritic inhibition of mitral cells by granule cells. Given the high expression levels of mGluR2, a metabotropic glutamate receptor, in the AOB and the fact that the activation of mGluR2 permits the formation of a specific olfactory memory, we reasoned that mGluR2 might play an important role in regulating dendrodendritic inhibition. To test this hypothesis, we examined the effects of pharmacological and genetic manipulations of mGluR2 on synaptic responses measured from mitral or granule cells in slice preparations from 23- to 36-day-old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from -70 to 0 mV or a threshold current stimulus adjusted to elicit action potential(s) was applied to a mitral cell using either a nystatin-perforated or conventional whole-cell configuration. We found that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG-IV [(2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine], suppressed, whereas the mGluR2/mGluR3 antagonist LY341495 [(αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid] enhanced dendrodendritic inhibition. Genetic ablation of mGluR2 markedly impaired the effects of DCG-IV and LY341495 on dendrodendritic inhibition. DCG-IV reduced both the frequency and the amplitude of spontaneous miniature excitatory postsynaptic currents recorded from granule cells. Additionally, DCG-IV inhibited high-voltage-activated calcium currents in both mitral and granule cells. These results suggest that mGluR2 reduces dendrodendritic inhibition by inhibiting synaptic transmission between mitral cells and granule cells in the AOB.

Correlative light and electron microscopic observation of calcium phosphate particles in a mouse kidney formed under a high-phosphate diet.

Calcium phosphate forms particles under excessive urinary excretion of phosphate in the kidney. While the formation of calcium phosphate particles (CaPs) has been implicated in the damage to renal tubular cells and renal dysfunction, clarifying the ultrastructural information and the elemental composition of the small CaPs in the wide areas of kidney tissue has been technically difficult. This study introduces correlative and sequential light as well as electron microscopic CaP observation in the kidney tissue by combining fluorescent staining for CaPs and energy-dispersive X-ray spectroscopy (EDS) in scanning electron microscopy (SEM) on resin sections prepared using high-pressure freezing and freeze substitution. CaPs formed in mouse kidneys under long-term feeding of a high-phosphate diet were clearly visualized on resin sections by fluorescence-conjugated alendronate derivatives and toluidine blue metachromasia. These CaPs were verified by correlative observation with EDS. Furthermore, small CaPs formed in the kidney under short-term feeding were detected using fluorescent probes. The elemental composition of the particles, including calcium and magnesium, was identified following EDS analyses. These results suggest that the correlative microscopy approach is helpful for observing in situ distribution and elemental composition of CaPs in the kidney and contributing to studies regarding CaP formation-associated pathophysiology.

Quantification of postsynaptic density proteins: glutamate receptor subunits and scaffolding proteins.

The postsynaptic density (PSD) protein complex has long been a major target of proteomics in neuroscience. As the number of glutamate receptors on a synapse is one of the main determinants of synaptic efficacy, determining the absolute numbers of receptors in the PSD is necessary for estimating the amplitude of the excitatory postsynaptic current (EPSC) in individual synapses. Moreover, as the receptor molecules are embedded in a macromolecular complex within the PSD, stoichiometry between the receptors and other PSD proteins could help explain the functional and regional specialization of the synapses and their possible roles in synaptic plasticity. Here, I review various studies concerned with the quantification of PSD proteins.

Right-hemispheric dominance of spatial memory in split-brain mice.

Left-right asymmetry of human brain function has been known for a century, although much of molecular and cellular basis of brain laterality remains to be elusive. Recent studies suggest that hippocampal CA3-CA1 excitatory synapses are asymmetrically arranged, however, the functional implication of the asymmetrical circuitry has not been studied at the behavioral level. In order to address the left-right asymmetry of hippocampal function in behaving mice, we analyzed the performance of "split-brain" mice in the Barnes maze. The "split-brain" mice received ventral hippocampal commissure and corpus callosum transection in addition to deprivation of visual input from one eye. In such mice, the hippocampus in the side of visual deprivation receives sensory-driven input. Better spatial task performance was achieved by the mice which were forced to use the right hippocampus than those which were forced to use the left hippocampus. In two-choice spatial maze, forced usage of left hippocampus resulted in a comparable performance to the right counterpart, suggesting that both hippocampal hemispheres are capable of conducting spatial learning. Therefore, the results obtained from the Barnes maze suggest that the usage of the right hippocampus improves the accuracy of spatial memory. Performance of non-spatial yet hippocampus-dependent tasks (e.g. fear conditioning) was not influenced by the laterality of the hippocampus.

Hippocampal CA3 and CA2 have distinct bilateral innervation patterns to CA1 in rodents.

Ipsilateral and contralateral hippocampal CA3-CA1 and CA2-CA1 projections were investigated in adult male Long-Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2. The contralateral and ipsilateral anterior-posterior extents of CA3 innervation to CA1 were similar. Fifteen to twenty per cent of the hippocampus proper cells that give rise to CA1 stratum oriens innervation were CA2 pyramidal cells, whereas CA2 cells were a mere 3% for CA1 stratum radiatum innervation. The preferred projection of CA2 pyramidal cells to the CA1 stratum oriens was also manifested in transgenic mice that express GFP under the control of the CACNG5 promoter, in which CA2 cells express high amounts of GFP. The ratios of ipsilateral to contralateral projections were compared. For the CA3-CA1 connection, we found that dorsal CA1 stratum radiatum received more ipsilateral projections whereas CA1 stratum oriens received more contralateral innervation. Interestingly, ipsilateral connections dominated for both CA2-CA1 stratum oriens and CA2-CA1 stratum radiatum. These results demonstrate that the primary intrahippocampal target of CA2 pyramidal cells is the ipsilateral CA1 stratum oriens, in contrast to CA3 cells which project more diversely to bilateral CA1 regions. Such innervation patterns may suggest differential dendritic information processing in apical and basal dendrites of CA1 pyramidal cells.

Heterogeneity and regulation of oligodendrocyte morphology.

Oligodendrocytes form multiple myelin sheaths in the central nervous system (CNS), which increase nerve conduction velocity and are necessary for basic and higher brain functions such as sensory function, motor control, and learning. Structures of the myelin sheath such as myelin internodal length and myelin thickness regulate nerve conduction. Various parts of the central nervous system exhibit different myelin structures and oligodendrocyte morphologies. Recent studies supported that oligodendrocytes are a heterogenous population of cells and myelin sheaths formed by some oligodendrocytes can be biased to particular groups of axons, and myelin structures are dynamically modulated in certain classes of neurons by specific experiences. Structures of oligodendrocyte/myelin are also affected in pathological conditions such as demyelinating and neuropsychiatric disorders. This review summarizes our understanding of heterogeneity and regulation of oligodendrocyte morphology concerning central nervous system regions, neuronal classes, experiences, diseases, and how oligodendrocytes are optimized to execute central nervous system functions.

miRNA profiling of bilateral rat hippocampal CA3 by deep sequencing.

MicroRNAs (miRNAs) have been demonstrated to be potent post-trascriptional modulators of protein expression. miRNA expression was profiled in the left and right dorsal hippocampal CA3 of mature rats by high-throughput deep sequencing. Among the sequenced and cross-mapped small RNAs, 88% belonged to the miRNAs annotated in the miRBase 15 database. Nearly half of the small RNAs belonged to the let-7 family miRNA. Seven percent of the sequenced small RNAs were not annotated in miRBase 15. Bioinformatic analysis of the unannotated small RNA sequences suggested seventeen novel miRNA candidates with relatively high expression levels (>100 tags per million). The left:right expression ratios were similar for all highly expressed miRNAs with less than 10% differences. These results provide a basic idea of the relative expression strengths of known and unknown miRNAs in the dorsal hippocampal CA3.

Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors.

Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits.

Macroscopic detection of demyelinated lesions in mouse PNS with neutral red dye.

Lysophosphatidylcholine (LPC)-induced demyelination is a versatile animal model that is frequently used to identify and examine molecular pathways of demyelination and remyelination in the central (CNS) and peripheral nervous system (PNS). However, identification of focally demyelinated lesion had been difficult and usually required tissue fixation, sectioning and histological analysis. Recently, a method for labeling and identification of demyelinated lesions in the CNS by intraperitoneal injection of neutral red (NR) dye was developed. However, it remained unknown whether NR can be used to label demyelinated lesions in PNS. In this study, we generated LPC-induced demyelination in sciatic nerve of mice, and demonstrated that the demyelinated lesions at the site of LPC injection were readily detectable at 7 days postlesion (dpl) by macroscopic observation of NR labeling. Moreover, NR staining gradually decreased from 7 to 21 dpl over the course of remyelination. Electron microscopy analysis of NR-labeled sciatic nerves at 7 dpl confirmed demyelination and myelin debris in lesions. Furthermore, fluorescence microscopy showed NR co-labeling with activated macrophages and Schwann cells in the PNS lesions. Together, NR labeling is a straightforward method that allows the macroscopic detection of demyelinated lesions in sciatic nerves after LPC injection.

Adrenergic inhibition facilitates normalization of extracellular potassium after cortical spreading depolarization.

Cortical spreading depolarization (CSD) is a propagating wave of tissue depolarization characterized by a large increase of extracellular potassium concentration and prolonged subsequent electrical silencing of neurons. Waves of CSD arise spontaneously in various acute neurological settings, including migraine aura and ischemic stroke. Recently, we have reported that pan-inhibition of adrenergic receptors (AdRs) facilitates the normalization of extracellular potassium after acute photothrombotic stroke in mice. Here, we have extended that mechanistic study to ask whether AdR antagonists also modify the dynamics of KCl-induced CSD and post-CSD recovery in vivo. Spontaneous neural activity and KCl-induced CSD were visualized by cortex-wide transcranial Ca2+ imaging in G-CaMP7 transgenic mice. AdR antagonism decreased the recurrence of CSD waves and accelerated the post-CSD recovery of neural activity. Two-photon imaging revealed that astrocytes exhibited aberrant Ca2+ signaling after passage of the CSD wave. This astrocytic Ca2+ activity was diminished by the AdR antagonists. Furthermore, AdR pan-antagonism facilitated the normalization of the extracellular potassium level after CSD, which paralleled the recovery of neural activity. These observations add support to the proposal that neuroprotective effects of AdR pan-antagonism arise from accelerated normalization of extracellular K+ levels in the setting of acute brain injury.

Neural-activity-dependent release of S100B from astrocytes enhances kainate-induced gamma oscillations in vivo.

S100B is the principal calcium-binding protein of astrocytes and known to be secreted to extracellular space. Although secreted S100B has been reported to promote neurite extension and cell survival via its receptor [receptor for advanced glycation end products (RAGE)], effects of extracellular S100B on neural activity have been mostly unexplored. Here, we demonstrate that secreted S100B enhances kainate-induced gamma oscillations. Local infusion of S100B in S100B(-/-) mice enhanced hippocampal kainate-induced gamma oscillations in vivo. In a complementary set of experiments, local application of anti-S100B antibody in wild-type mice attenuated the gamma oscillations. Both results indicate that the presence of extracellular S100B enhances the kainate-induced gamma oscillations. In acutely isolated hippocampal slices, kainate application increased S100B secretion in a neural-activity-dependent manner. Further pharmacological experiments revealed that S100B secretion was critically dependent on presynaptic release of neurotransmitter and activation of metabotropic glutamate receptor 3. Moreover, the kainate-induced gamma oscillations were attenuated by the genetic deletion or antibody blockade of RAGE in vivo. These results suggest RAGE activation by S100B enhances the gamma oscillations. Together, we propose a novel pathway of neuron-glia communications--astrocytic release of S100B modulates neural network activity through RAGE activation.

Potential implication of SGK1-dependent activity change in BV-2 microglial cells.

It has recently been established that microglial activation is involved in the pathophysiology of various neurological and psychiatric disorders such as amyotrophic lateral sclerosis and schizophrenia. The pathological molecular machineries underlying microglial activation and its accelerating molecules have been precisely described in the diseased central nervous system (CNS). However, to date, the details of physiological mechanism, which represses microglial activation, are still to be elucidated. Our latest report demonstrated that serum- and glucocorticoid-inducible kinases (SGK1 and SGK3) were expressed in multiple microglial cell lines, and their inhibitor enhanced the toxic effect of lipopolysaccharide on microglial production of inflammatory substances such as TNFα and iNOS. In the present report, we prepared SGK1-lacked microglial cell line (BV-2) and demonstrated that deficiency of SGK1 in microglia induced its toxic conversion, in which it took amoeboid morphology characteristic of reactive microglia, increased CD68 expression, quickened its proliferation, and showed higher susceptibility to ATP and subsequent cell death. Our data indicate that SGK1 plays pivotal roles in inhibiting its pathological activation, and suggest its potential function as a therapeutic target for the treatment of various disorders related to the inflammation in the CNS.

Conversion of a (sp3)C-F bond of alkyl fluorides to (sp3)C-X (X = Cl, C, H, O, S, Se, Te, N) bonds using organoaluminium reagents.

A simple method for the conversion of (sp(3))C-F bonds of alkyl fluorides to (sp(3))C-X (X = Cl, C, H, O, S, Se, Te, N) bonds has been achieved by the use of a hexane solution of organoaluminum reagents having Al-X bonds.

Network mechanisms of hippocampal laterality, place coding, and goal-directed navigation.

The hippocampus and associated structures are responsible for episodic memory in humans. In rodents, the most prominent behavioral correlate of hippocampal neural activity is place coding, which is thought to underlie spatial navigation. While episodic memory is considered to be unique to humans in a restricted context, it has been proposed that the same neural circuitry and algorithms that enable spatial coding and navigation also support episodic memory. Here we review the recent progress in neural circuit mechanisms of hippocampal activity by introducing several topics: (1) cooperation and specialization of the bilateral hippocampi, (2) the role of synaptic plasticity in gamma phase-locking of spikes and place cell formation, (3) impaired goal-related activity and oscillations in a mouse model of mental disorders, and (4) a prefrontal-thalamo-hippocampal circuit for goal-directed spatial navigation.