Olfactory signals from the main olfactory bulb converge with taste information from the chorda tympani nerve in the agranular insular cortex of rats.

Gustation and olfaction are integrated into flavor, which contribute to detection and identification of foods. We focused on the insular cortex (IC), as a possible center of flavor integration, because the IC has been reported to receive olfactory in addition to gustatory inputs. In the present report, we tested the hypothesis that these two chemosensory signals are integrated in the IC. We examined the spatiotemporal dynamics of cortical responses induced by stimulating the chorda tympani nerve (CT) and the main olfactory bulb (mOB) in male Sprague-Dawley rats by in vivo optical imaging with a voltage-sensitive dye (VSD). CT stimulation elicited responses in the rostral part of the dysgranular IC (DI), while responses to mOB stimulation were observed in the agranular IC (AI) as well as in the piriform cortex (PC). To characterize the temporal specificity of these responses, we performed combined mOB and CT stimulation with three different timings: simultaneous stimulation and the stimulation of the mOB 150 ms before or after CT stimulation. Simultaneous stimulation increased the signal amplitude in AI additively. These results indicate that the AI and DI contribute to the convergence of gustatory and olfactory information. Of them the DI predominantly processes the taste information, whereas the AI is more sensitive to the olfactory signal.

Potentiating a non-neuronal cardiac cholinergic system reinforces the functional integrity of the blood brain barrier associated with systemic anti-inflammatory responses.

We previously reported that the heart-specific choline acetyltransferase (ChAT) gene overexpressing mice (ChAT tg) show specific phenotypes including ischemic tolerance and the CNS stress tolerance. In the current study, we focused on molecular mechanisms responsible for systemic and localized anti-inflammatory phenotypes of ChAT tg. ChAT tg were resistant to systemic inflammation induced by lipopolysaccharides due to an attenuated cytokine response. In addition, ChAT tg, originally equipped with less reactive Kupffer cells, were refractory to brain cold injury, with decreased blood brain barrier (BBB) permeability and reduced inflammation. This is because ChAT tg brain endothelial cells expressed more claudin-5, and their astrocytes were less reactive, causing decreased hypertrophy. Moreover, reconstruction of the BBB integrity in vitro confirmed the consolidation of ChAT tg. ChAT tg were also resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neuronal toxicity due to lower mortality rate and neuronal loss of substantia nigra. Additionally, ChAT tg subjected to MPTP showed attenuated BBB disruption, as evident from reduced sodium fluorescein levels in the brain parenchyma. The activated central cholinergic pathway of ChAT tg lead to anti-convulsive effects like vagus nerve stimulation. However, DSP-4, a noradrenergic neuron-selective neurotoxin against the CNS including the locus ceruleus, abrogated the beneficial phenotype and vagotomy attenuated expression of claudin-5, suggesting the link between the cholinergic pathway and BBB function. Altogether, these findings indicate that ChAT tg possess an anti-inflammatory response potential, associated with upregulated claudin-5, leading to the consolidation of BBB integrity. These characteristics protect ChAT tg against systemic and localized inflammatory pathological disorders, which targets the CNS.

Intracortical signal processing of periodontal ligament sensations in rat.

The somatosensory information from the orofacial region, including the periodontal ligament (PDL), is processed in a manner that differs from that used for other body somatosensory information in the related cortices. It was reported that electrical stimulation to rat PDL elicited activation of the insular oral region (IOR) and the primary (S1) and secondary (S2) somatosensory cortices. However, the physiological relationship between S1 and S2/IOR is not well understood. To address this issue, we performed in vivo optical imaging using a voltage-sensitive dye. Our results demonstrated that the electrical stimulation to the PDL of the mandibular incisor evoked the simultaneous activation of S1 and the S2/IOR. The stimulation to the initial response area of the S1 evoked responses in the S2/IOR, and vice versa. An injection of tetrodotoxin (TTX) to the cortical region between S1 and S2/IOR attenuated such elicited responses only in the non-stimulated cortical partner site. The cortico-cortical interaction between S1 and S2/IOR was suppressed by the application of TTX, indicating that these two cortical regions bi-directionally communicate the signal processing of PDL sensations. An injection of FluoroGold™ (FG) to the initial response area in S1 or the S2/IOR showed that FG-positive cells were scattered in the non-injected cortical counterpart. This morphological result demonstrated the presence of a bi-directional intracortical connection between the initial response areas in S1 and the S2/IOR. These findings suggest the presence of a mutual connection between S1 and the S2/IOR as an intracortical signal processing network for orofacial nociception.

Non-neuronal cardiac cholinergic system influences CNS via the vagus nerve to acquire a stress-refractory propensity.

We previously developed cardiac ventricle-specific choline acetyltransferase (ChAT) gene-overexpressing transgenic mice (ChAT tgm), i.e. an in vivo model of the cardiac non-neuronal acetylcholine (NNA) system or non-neuronal cardiac cholinergic system (NNCCS). By using this murine model, we determined that this system was responsible for characteristics of resistance to ischaemia, or hypoxia, via the modulation of cellular energy metabolism and angiogenesis. In line with our previous study, neuronal ChAT-immunoreactivity in the ChAT tgm brains was not altered from that in the wild-type (WT) mice brains; in contrast, the ChAT tgm hearts were the organs with the highest expression of the ChAT transgene. ChAT tgm showed specific traits in a central nervous system (CNS) phenotype, including decreased response to restraint stress, less depressive-like and anxiety-like behaviours and anti-convulsive effects, all of which may benefit the heart. These phenotypes, induced by the activation of cardiac NNCCS, were dependent on the vagus nerve, because vagus nerve stimulation (VS) in WT mice also evoked phenotypes similar to those of ChAT tgm, which display higher vagus nerve discharge frequency; in contrast, lateral vagotomy attenuated these traits in ChAT tgm to levels observed in WT mice. Furthermore, ChAT tgm induced several biomarkers of VS responsible for anti-convulsive and anti-depressive-like effects. These results suggest that the augmentation of the NNCCS transduces an effective and beneficial signal to the afferent pathway, which mimics VS. Therefore, the present study supports our hypothesis that activation of the NNCCS modifies CNS to a more stress-resistant state through vagus nerve activity.

Accessory olfactory bulb neurons are required for maintenance but not induction of V2R vomeronasal receptor gene expression in vitro.

Many mammals detect pheromones by a sensory organ, the vomeronasal organ (VNO). In a previous study using immunoblot and immunocytochemical analyses, we reported that cocultures of VNOs with accessory olfactory bulb (AOB) neurons resulted in the maturation of vomeronasal sensory neurons (VSNs) and a greater expression of V2R family vomeronasal receptors than cultures with VNO alone. To further characterize the V2R expression, we here investigated the time course of the expression of V2R mRNA in the presence or absence of AOB neurons using RT-PCR analysis. The expression of V2R mRNA was already detectable not only in the VNO cocultured with AOB neurons for 3 days in coculture but also in the VNO cultured alone for the same number of days. However, the expression of V2R mRNA in the VNO cultured alone was remarkably decreased during the additional culture period, although that in the cocultured VNO showed sustained expression. Moreover, the application of 2 µM TTX to the cocultured VNO resulted in a marked decrease in the V2R mRNA expression to a level equal to that in the VNO cultured alone for 14 days in coculture. Our previous working hypothesis was that the expression of V2Rs in VSNs was induced by interacting with AOB neurons. However, the present results suggest that the receptor expression in VSNs is independent of the interaction with AOB neurons in the early developmental stage, but is maintained by the active interaction with AOB neurons.

Ameliorative effects of Eriobotrya japonica seed extract on cellular aging in cultured rat fibroblasts.

To investigate the effects of Eriobotrya japonica seed extract (ESE) on cellular aging, intracellular calcium homeostasis in young and senescent cells was analyzed using a rat fibroblast culture as an in vitro model system and a calcium imaging technique. The application of bradykinin (BK) transiently elicited intracellular calcium ion (Ca(2+)) increased in most of the young fibroblasts, whereas these responses were scarcely observed or were significantly attenuated in senescent cells. However, the long-term treatment of senescent cells with ESE (for 7 days) dose-dependently increased the amplitude of BK-induced responses and the percentage of BK-responding cells. In particular, most senescent cells could respond to BK with long-term treatment with ESE (1.0% or 2.0%), an effect that reinstated the percentage of BK-responding cells to the same level as that in young cells. The effects of ESE on amplitude or percentage of responding cells were not observed in young cells. Moreover, the time to half decay, which was significantly longer in senescent cells than that in young cells, was shortened in senescent cells with long-term treatment with ESE. These results suggest that treatment with an adequate concentration of ESE renders BK-induced Ca(2+) dynamics in senescent cells similar to those in young cells. Therefore, ESE can retard and/or protect against cellular aging and may be useful for elucidating the antiaging processes.

The effect of vapor of propylene glycol on rats.

Propylene glycol (PG) is commonly used as a solvent for odorous chemicals employed in studies of the olfactory system because PG has been considered to be odorless for humans and other animals. However, if laboratory rats can detect the vapor of PG and if exposure to this influences behaviors, such effects might confound data obtained from experiments exposing conscious rats to odorants dissolved in PG. Therefore, we examined this issue using differences in the acoustic startle reflex (ASR) as an index. We also conducted a habituation/dishabituation test to assess the ability of rats to detect the vapor of PG. In addition, we observed Ca(2+) responses of vomeronasal neurons (VNs) in rats exposed to PG using the confocal Ca(2+)-imaging approach. Pure PG vapor significantly enhanced the ASR at a dose of 1 x 10(-4) M, which was much lower than the dose for efficiently detecting. In Ca(2+) imaging, VNs were activated by PG at a dose of 1 x 10(-4) M or lower. These results suggest that PG vapor acts as an aversive stimulus to rats at very low doses, even lower than those required for its detection, indicating that we should consider such effect of PG when it is employed as a solvent for odorants in studies using conscious rats. In addition, our study suggests that some non-pheromonal volatile odorants might affect animal behaviors via the vomeronasal system.

A HIF-1alpha-related gene involved in cell protection from hypoxia by suppression of mitochondrial function.

Recently, we reported that acetylcholine-induced hypoxia-inducible factor-1alpha protects cardiomyocytes from hypoxia; however, the downstream factors reducing hypoxic stress are unknown. We identified apoptosis inhibitor (AI) gene as being differentially expressed between von Hippel Lindau (VHL) protein-positive cells with high levels of GRP78 expression and VHL-negative cells with lower GRP levels, using cDNA subtraction. AI decreased GRP78 level, suppressed mitochondrial function, reduced oxygen consumption and, ultimately, suppressed hypoxia-induced apoptosis. By contrast, knockdown of the AI gene increased mitochondrial function. Hypoxic cardiomyocytes and ischemic myocardium showed increased AI mRNA expression. These findings suggest that AI is involved in suppressing mitochondrial function, thereby leading to cellular stress eradication and consequently to protection during hypoxia.

Target regulation of V2R expression and functional maturation in vomeronasal sensory neurons in vitro.

Vomeronasal receptors from the V1R and V2R gene families mediate the detection of chemical stimuli such as pheromones via the vomeronasal organ (VNO). The differential expression of vomeronasal receptors might contribute in part to a variety of pheromonal effects, which are different sexually, developmentally and even individually. However, little is known about the mechanisms controlling vomeronasal receptor expression. Cultured vomeronasal sensory neurons (VSNs) bear phenotypic resemblance to the intact VNO but they remain immature. Because indices of VSN maturation are increased by coculture with the target cells for VSNs, accessory olfactory bulb (AOB) neurons, AOB neurons may regulate vomeronasal receptor expression and functional maturation in VSNs. To test this hypothesis, we examined the expression of V2R-type vomeronasal receptors (VR1 and VR4) and chemosensory responsiveness in VNOs cocultured with AOB neurons. Immunoblot and immunocytochemical analysis revealed that the coculture of VNOs with AOB neurons resulted in a greater expression of VR1 and VR4 after 10 days than VNOs cultured alone. Moreover, calcium imaging analysis showed that cocultured VNOs responded to urine components applied iontophoretically into their cavities with a time course similar to the V2R expression, in contrast to singly cultured VNOs that displayed no response. These results demonstrate that AOB neurons induce the expression of vomeronasal receptors in VSNs, allowing them to function.

Modification of synapse formation of accessory olfactory bulb neurons by coculture with vomeronasal neurons.

Previously, a coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal (VN) neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of VN neurons on the development of AOB neurons was examined in a coculture system. Spine density was quantitatively measured for various culture periods of 21, 28, 36, and 42 days in vitro. The densities of dendritic spines were lower in the coculture than in single culture for all periods in vitro. Synapse formation on spines was analyzed immunocytochemically using an anti-synaptophysin antibody. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. These changes were not observed in the coculture in which there was no physical contact between AOB neurons and VN neurons. These observations suggest that synapse formation on the spines of AOB neurons is modified by physical contact with VN neurons.

Efferent vagal nerve stimulation protects heart against ischemia-induced arrhythmias by preserving connexin43 protein.

BACKGROUND: Myocardial ischemia (MI) leads to derangements in cellular electrical stability and the generation of lethal arrhythmias. Vagal nerve stimulation has been postulated to contribute to the antifibrillatory effect. Here, we suggest a novel mechanism for the antiarrhythmogenic properties of vagal stimulation during acute MI. METHODS AND RESULTS: Under anesthesia, Wistar rats underwent 30 minutes of left coronary artery (LCA) ligation with vagal stimulation (MI-VS group, n=11) and with sham stimulation (MI-SS group, n=12). Eight of the 12 rats in the MI-SS group had ventricular tachyarrhythmia (VT) during 30-minute LCA ligation; on the other hand, VT occurred in only 1 of the 11 rats in the MI-VS group (67% versus 9%, respectively). Atropine administration abolished the antiarrhythmogenic effect of vagal stimulation. Immunoblotting revealed that the MI-SS group showed a marked reduction in the amount of phosphorylated connexin43 (Cx43), whereas the MI-VS group showed only a slight reduction compared with the sham operation and sham stimulation group (37+/-20% versus 79+/-18%). Immunohistochemistry confirmed that the MI-induced loss of Cx43 from intercellular junctions was prevented by vagal stimulation. In addition, studies with rat primary-cultured cardiomyocytes demonstrated that acetylcholine effectively prevented the hypoxia-induced loss of phosphorylated Cx43 and ameliorated the loss of cell-to-cell communication as determined by Lucifer Yellow dye transfer assay, which supports the in vivo results. CONCLUSIONS: Vagal nerve stimulation exerts antiarrhythmogenic effects accompanied by prevention of the loss of phosphorylated Cx43 during acute MI and thus plays a critical role in improving ischemia-induced electrical instability.

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons.

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.

Differences in development and cellular composition between neuronal cultures of rat accessory and main olfactory bulbs.

We previously established a primary culture system of the accessory olfactory bulb (AOB) to investigate the functional roles of individual types of neuron in pheromonal signal processing. However, the detailed characteristics of cultured AOB neurons were not yet apparent. In the present study, we address the cytological aspects of cultured AOB neurons using immunocytochemical staining methods. Cultured AOB neurons were compared with cultured main olfactory bulb (MOB) neurons in neuronal composition, maturational time course, and cell size. The number of total neurons, measured by microtubule-associated protein 2 (MAP2) immunostaining, progressively decreased, and glutamic acid decarboxylase positive (GAD+) interneurons were scarcely changed in their number in both AOB and MOB cultures over the culture periods. In contrast, the number of tyrosine hydroxylase positive (TH+) neurons in AOB cultures showed a slight, but significant, increase over time in culture, while those in MOB cultures remarkably decreased. The numbers of total neurons and GAD+ neurons were significantly greater in AOB cultures than in MOB cultures at all investigated time points. However, the numbers of TH+ neurons were lower at 7 days in vitro (DIV) and greater at 21 DIV in AOB cultures than in MOB cultures. The somatic sizes of all types of neurons at 14 DIV were significantly larger in AOB cultures than in MOB cultures. Furthermore, the frequency distributions of somatic sizes of total, GAD+, and TH+ neurons were significantly different between AOB and MOB cultures. These subtle differences in vitro may reflect in vivo differences between the AOB and MOB.

Developmental changes of GABAergic synapses formed between primary cultured cortical neurons.

The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.

[Assay system for synapse formation between primary cultured neurons].

Synapse plasticity, in particular, formation of new synapses, plays crucial roles in learning and memory. We have developed a convenient assay system for measuring the number of newly formed synapses between cultured rat cerebrocortical neurons using the multisite fluorometry system of intracellular calcium. We found that cultured neurons exhibited spontaneous oscillatory changes in intracellular calcium levels and that the frequency of the oscillation was strongly correlated with synaptic density. Combined with immunohistochemical studies, this assay system enables us to study the molecular mechanism of synapse formation, in particular, the involvement of ecto-protein kinase. Other applications of the assay system are discussed here.

Bicuculline induces synapse formation on primary cultured accessory olfactory bulb neurons.

To investigate the roles of the GABAergic inhibitory system of accessory olfactory bulb (AOB) in pheromonal memory formation, we have developed a primary culture system of AOB neurons, which had numerous excitatory and inhibitory synapses. Using this culture system of AOB neurons, we examined the correlation in rats between neuronal excitation and synaptic morphology by bicuculline-induced disinhibition of cultured AOB neurons. The exposure to bicuculline induced long-lasting oscillatory changes in the intracellular calcium level ([Ca2+]in) of cultured non-GABAergic multipolar neurons, which were identified as mitral/tufted cells (MT cells). These MT cells exhibited the appearance of dendritic filopodia structures after a 10-min treatment with bicuculline. By labelling presynaptic terminals with FM4-64, the appearance of new presynaptic terminals was clearly observed on newly formed filopodia after 120 min treatment with bicuculline. These results suggest that bicuculline-induced [Ca2+]in oscillation of MT cells induces the growth of filopodia and subsequently the formation of new presynaptic terminals. Furthermore, tetrodotoxin or the deprivation of extracellular calcium blocked bicuculline-induced synapse formation. The present results indicate that the long-lasting [Ca2+]in oscillation caused by bicuculline-induced disinhibition of cultured MT cells is significantly implicated in the mechanism underlying synapse formation on cultured AOB neurons. Our established culture system of AOB neurons will aid in clarifying the mechanism of synapse formation between AOB neurons and the molecular mechanism of pheromonal memory formation.

Localization of microtubule-associated protein (MAP) 1B in the postsynaptic densities of the rat cerebral cortex.

1. Although microtubule-associated protein (MAP) 1B and its phosphorylation have been suggested to be important for synapse formation among cortical neurons, the localization of MAP1B in synapses has not yet been confirmed. In this report, we examine the localization of MAP1B in synaptic regions. 2. The localization of MAP1B was observed by immunohistochemical and electron microscopic techniques using specific antibodies against MAP1B. 3. MAP1B immunoreactivities were widely distributed in the cerebral cortex and were observed in the postsynaptic area but not in presynaptic terminals. 4. These synapses were classified as the asymmetrical type. 5. Only some synapses exhibited MAP1B immunoreactivities. MAP1B-immunopositive synapses accounted for about half of the total synapses. 6. Such a localization suggests MAP1B's important roles in synaptic functions.

Thyroid hormone enhances the formation of synapses between cultured neurons of rat cerebral cortex.

1. Thyroid hormones play important roles in the development of the brain. Increasing evidence suggests that the deprivation of thyroid hormones in the early developmental stage causes structural and functional deficits in the CNS, but the precise mechanism underlying this remains elusive. In this study, we investigated the effects of thyroid hormones on synapse formation between cultured rat cortical neurons, using a system to estimate functional synapse formation in vitro. 2. Exposure to 10(-9) M thyroid hormones, 3,5,3'-triiodothyronine or thyroxine, caused an increase in the frequency of spontaneous synchronous oscillatory changes in intracellular calcium concentration, which correlated with the number of synapses formed. 3. The detection of synaptic vesicle-associated protein synapsin I by immunocytochemical and immunoblot analysis also confirmed that exposure to thyroxine facilitated synapse formation. 4. The presence of amiodarone, an inhibitor of 5'-deiodinase, or amitrole, a herbicide, inhibited the synapse formation in the presence of thyroxine. 5. In conclusion, we established a useful in vitro assay system for screening of miscellaneous chemicals that might interfere with synapse formation in the developing CNS by disrupting the thyroid system.