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![Changes in [Ca2+]in of silent multipolar neurons after bicuculline treatment. Typical changes in [Ca2+]in of silent multipolar neurons were observed using the multisite calcium imaging system at 15 DIV. (A) Effects of continuous application of 20 µm bicuculline. The right-hand figure shows changes in [Ca2+]in of the same neuron after 30 min. (B) Effects of short-term exposure to 20 µm bicuculline. After 2 min exposure to bicuculline, the bath solution was washed out. The right-hand figure shows changes in [Ca2+]in of the same neuron after 30 min treatment with bicuculline. After the removal of bicuculline from the bath, the [Ca2+]in returned to the resting level. (C) Exposure to 30 mm KCl caused only a transient increase in [Ca2+]in. (D) Inhibition by 1 µm TTX of synchronous oscillatory changes in [Ca2+]in induced by 20 µm bicuculline.](profile/Kazuyo-Muramoto/publication/9079495/figure/fig7/AS:267918613151764@1440888183386/Changes-in-Ca2-in-of-silent-multipolar-neurons-after-bicuculline-treatment-Typical.png)
Changes in [Ca2+]in of silent multipolar neurons after bicuculline treatment. Typical changes in [Ca2+]in of silent multipolar neurons were observed using the multisite calcium imaging system at 15 DIV. (A) Effects of continuous application of 20 µm bicuculline. The right-hand figure shows changes in [Ca2+]in of the same neuron after 30 min. (B) Effects of short-term exposure to 20 µm bicuculline. After 2 min exposure to bicuculline, the bath solution was washed out. The right-hand figure shows changes in [Ca2+]in of the same neuron after 30 min treatment with bicuculline. After the removal of bicuculline from the bath, the [Ca2+]in returned to the resting level. (C) Exposure to 30 mm KCl caused only a transient increase in [Ca2+]in. (D) Inhibition by 1 µm TTX of synchronous oscillatory changes in [Ca2+]in induced by 20 µm bicuculline.
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![Figure 2: Spontaneous oscillatory changes in [Ca2+]in of cultured AOB...](profile/Kazuyo-Muramoto/publication/9079495/figure/fig2/AS:267726753103876@1440842440115/Spontaneous-oscillatory-changes-in-Ca2-in-of-cultured-AOB-neurons-Cultured-AOB-neurons_Q320.jpg)
![Figure 3: Changes in [Ca2+]in of silent multipolar neurons after...](profile/Kazuyo-Muramoto/publication/9079495/figure/fig7/AS:267918613151764@1440888183386/Changes-in-Ca2-in-of-silent-multipolar-neurons-after-bicuculline-treatment-Typical_Q320.jpg)
![Figure 4: Changes in [Ca2+]in of a GFP-actin-labelled AOB neuron. (A)...](profile/Kazuyo-Muramoto/publication/9079495/figure/fig8/AS:267923264634928@1440889292927/Changes-in-Ca2-in-of-a-GFP-actin-labelled-AOB-neuron-A-Left-image-of_Q320.jpg)
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...
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... to bicuculline. After exposure to 20 mM bicuculline, 94 AE 3.9% (mean AE SEM; n 400 neurons) of the silent neurons exhibited an increase in [Ca 2 ] in and oscillatory changes in [Ca 2 ] in . The bicuculline-induced [Ca 2 ] in oscillations continued for more than 30 min during the period when cultured AOB neurons were exposed to bicuculline (Fig. 3A). The characteristics of bicuculline-induced [Ca 2 ] in oscillations in silent neurons were similar to those of other non-silent neurons, which appeared spontaneously without bicuculline treatment ( Fig. 2B; cell 1 and cell 2). Moreover, the bicuculline-induced [Ca 2 ] in oscillations in silent neurons synchronized with the ...
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... [Ca 2 ] in oscilla- tions in other non-silent neurons. Although the short-term exposure (2 min) to 20 mM bicuculline induced transient changes in [Ca 2 ] in oscillations of the silent neurons, [Ca 2 ] in returned to the resting level [Ca 2 ] in but failed to induce the oscillatory and long-lasting changes in [Ca 2 ] in of the silent neurons (Fig. 3C). Furthermore, the synchronous and oscil- latory changes in [Ca 2 ] in of the silent neurons induced by bicuculline were completely inhibited by 1 mM TTX, a sodium channel blocker (Fig. 3D). We analysed neurons in 2±3 culture dishes in one experi- ment and obtained similar results in four independent culture series. To identify the ...
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... [Ca 2 ] in returned to the resting level [Ca 2 ] in but failed to induce the oscillatory and long-lasting changes in [Ca 2 ] in of the silent neurons (Fig. 3C). Furthermore, the synchronous and oscil- latory changes in [Ca 2 ] in of the silent neurons induced by bicuculline were completely inhibited by 1 mM TTX, a sodium channel blocker (Fig. 3D). We analysed neurons in 2±3 culture dishes in one experi- ment and obtained similar results in four independent culture series. To identify the cell type of these silent neurons, cultured AOB neurons were immunostained with the anti-GAD antibody immediately after the analysis of [Ca 2 ] in and were observed under a confocal laser ...
Citations
... Several studies have convincingly demonstrated that elevating synaptic or network activity leads to enhanced spine outgrowth (Engert and Bonhoeffer, 1999;Maletic-Savatic et al., 1999;Jourdain et al., 2003;Kato-Negishi et al., 2003;Hamilton et al., 2012). The signaling pathways linking enhanced neural activity to new spine growth are currently being elucidated; many converge upon Rho GTPases as critical regulators of spinogenesis (Tolias et al., 2011;Penzes and Cahill, 2012;Saneyoshi and Hayashi, 2012;Lai and Ip, 2013;Um et al., 2014;Kim et al., 2015;Nishiyama and Yasuda, 2015;Lee et al., 2016). ...
The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.
... The resulting abnormal elevation of intracellular Ca 2+ levels can lead to the activation of apoptotic pathways and neuritic degeneration (Kawahara et al. 2011 ). Other possible consequences of altered neuronal Ca 2+ homeostasis include membrane disruption and changes in the dendritic spine number, morphology and synaptic plasticity, eventually leading to synaptic impairment (Kato-Negishi et al. 2003 ). Ultimately, cytosolic and mitochondrial Ca 2+ overload can occur, contributing to the oxidative damage and apoptosis (Querfurth and LaFerla 2010 ). ...
Aggregation of amyloid-beta (Aβ) peptide is the major event underlying neuronal damage in Alzheimer's disease (AD). Specific lipids and their homeostasis play important roles in this and other neurodegenerative disorders. The complex interplay between the lipids and the generation, clearance or deposition of Aβ has been intensively investigated and is reviewed in this chapter. Membrane lipids can have an important influence on the biogenesis of Aβ from its precursor protein. In particular, increased cholesterol in the plasma membrane augments Aβ generation and shows a strong positive correlation with AD progression. Furthermore, apolipoprotein E, which transports cholesterol in the cerebrospinal fluid and is known to interact with Aβ or compete with it for the lipoprotein receptor binding, significantly influences Aβ clearance in an isoform-specific manner and is the major genetic risk factor for AD. Aβ is an amphiphilic peptide that interacts with various lipids, proteins and their assemblies, which can lead to variation in Aβ aggregation in vitro and in vivo. Upon interaction with the lipid raft components, such as cholesterol, gangliosides and phospholipids, Aβ can aggregate on the cell membrane and thereby disrupt it, perhaps by forming channel-like pores. This leads to perturbed cellular calcium homeostasis, suggesting that Aβ-lipid interactions at the cell membrane probably trigger the neurotoxic cascade in AD. Here, we overview the roles of specific lipids, lipid assemblies and apolipoprotein E in Aβ processing, clearance and aggregation, and discuss the contribution of these factors to the neurotoxicity in AD.
... Next, we investigated the activity-dependence of the expression of V2Rs by pharmacologically blocking the neuronal activities. The most of AOB neurons show spontaneous activities under the culture conditions, as previously reported [16,8]. Similarly, cultured VSNs occasionally show spontaneous activities [data not shown]. ...
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.
... The disruption of calcium homeostasis could trigger the membrane disruption, the formation of reactive oxygen species (ROS), and induce other adverse effects which are often observed after exposure to AβP. It is widely known that the increase in [Ca 2+ ] i induced changes in the number of spines, their morphology, and the number of synapses [35]. Considering that AβP and APP coexist in the synapses [36], calcium imbalances in the synaptic compartment could directly influence neuronal activities and cause synaptic impairment (synaptotoxicity). ...
... Their activity was blocked by Zn 2+ , which is abundantly present in the brain [52]. Other neurotoxic peptide fragments of AβP, including AβP (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and AβP , were reported to form calcium-permeable pores on artificial lipid bilayers as well as AβP [53,54]. The characteristics of amyloid channels formed by AβP pores which consist of tetrameric and hexameric β-sheet subunits from the observations in NMR [57]. ...
Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP) play crucial roles in the pathogenesis of Alzheimer's disease (AD). Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels ("amyloid channels"), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.
... The olfactory bulb is being widely used for studies on adult neurogenesis (see Effect of GABA on Neurite Extension During Adult Neurogenesis in this review), but few papers also reported GABAergic effects on dendritic growth in immature cultures. Opposite GABAergic effects to those described in systems outlined in previous sections were reported in primary cultures of embryonic accessory olfactory bulb, where blockade (by bicuculline) rather than activation of GABA A receptors induces the formation of fi lopodia in non GABAergic, presumed mitral cells (Kato-Negishi et al., 2003). However, these same cells were quiescent in control conditions and they developed glutamatemediated sustained Ca 2+ oscillations in the presence of bicuculline , suggesting that GABA is actually hyperpolarizing in these cultures. ...
... Moreover, once GABA has switched to its mature inhibitory role, it can no longer induce neurite growth (Marty et al., 1996; Gascon et al., 2006). In the few cases where GABA inhibited neurite growth, either direct (Kato-Negishi et al., 2003) or indirect (Liu et al., 1997 ) evidence demonstrates that the effect was due to hyperpolarizing GABAergic signaling. One study reported that GABAergic inhibition promoted dendritic growth in the Xenopus optic tectum (Shen et al., 2009). ...
During development, Gamma-aminobutyric acidergic (GABAergic) neurons mature at early stages, long before excitatory neurons. Conversely, GABA reuptake transporters become operative later than glutamate transporters. GABA is therefore not removed efficiently from the extracellular domain and it can exert significant paracrine effects. Hence, GABA-mediated activity is a prominent source of overall neural activity in developing CNS networks, while neurons extend dendrites and axons, and establish synaptic connections. One of the unique features of GABAergic functional plasticity is that in early development, activation of GABA(A) receptors results in depolarizing (mainly excitatory) responses and Ca(2+) influx. Although there is strong evidence from several areas of the CNS that GABA plays a significant role in neurite growth not only during development but also during adult neurogenesis, surprisingly little effort has been made into putting all these observations into a common framework in an attempt to understand the general rules that regulate these basic and evolutionary well-conserved processes. In this review, we discuss the current knowledge in this important field. In order to decipher common, universal features and highlight differences between systems throughout development, we compare findings about dendritic proliferation and remodeling in different areas of the nervous system and species, and we also review recent evidence for a role in axonal elongation. In addition to early developmental aspects, we also consider the GABAergic role in dendritic growth during adult neurogenesis, extending our discussion to the roles played by GABA during dendritic proliferation in early developing networks versus adult, well established networks.
... The morphologies of neurons observed in our mouse AOB culture system closely resembles the cells observed by the Ichikawa laboratory in cultures of rat AOB (Kato-Negishi et al., 2003). In primary culture of the rat AOB, two primary groups of neurons (MAP2-positive cells) were observed, medium-sized multipolar neurons with thick dendrites and smaller unipolar and bipolar neurons with thinner dendrites (Kato-Negishi et al., 2003). ...
... The morphologies of neurons observed in our mouse AOB culture system closely resembles the cells observed by the Ichikawa laboratory in cultures of rat AOB (Kato-Negishi et al., 2003). In primary culture of the rat AOB, two primary groups of neurons (MAP2-positive cells) were observed, medium-sized multipolar neurons with thick dendrites and smaller unipolar and bipolar neurons with thinner dendrites (Kato-Negishi et al., 2003). Of the neurons in our cultures, nearly all resembled either the multipolar neurons or the uni/bipolar morphology observed in rat AOB culture. ...
Glutamate and norepinephrine (NE) are believed to mediate the long-lasting synaptic plasticity in the accessory olfactory bulb (AOB) that underlies pheromone recognition memory. The mechanisms by which these neurotransmitters bring about the synaptic changes are not clearly understood. In order to study signals that mediate synaptic plasticity in the AOB, we used AOB neurons in primary culture as a model system. Because induction of pheromone memory requires coincident glutamatergic and noradrenergic input to the AOB, and requires new protein synthesis, we reasoned that glutamate and NE must induce gene expression in the AOB. We used a combination of agonists that stimulate alpha1 and alpha2 adrenergic receptors in combination with N-methyl-d-aspartic acid and tested expression of the immediate-early gene (IEG) c-Fos. We found that the glutamatergic and noradrenergic stimulation caused significant induction of c-Fos mRNA and protein. Induction of c-Fos was significantly reduced in the presence of inhibitors of protein kinase C, mitogen-activated protein kinase (MAPK) and phospholipase C. These results suggest that glutamate and NE induce gene expression in the AOB through a signaling pathway mediated by protein kinase C and MAPK.
... Primary cultures of dissociated AOB cells were prepared from Wistar rats at embryonic day (E)19 following a previously reported procedure (Muramoto et al., , 2004Kato-Negishi et al., 2003). In brief, the pregnant rats were killed by decapitation after inhaling excess diethyl ether, and then their uteri were dissected out. ...
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.
... Furthermore, there is no report that, to the best of our knowledge, has ever comprehensively clarified the distinct subsets of downstream genes in response to different routes of Ca 2þ entry, although progress has been gradually achieved in the mechanisms of ion channels for excitation-transcription coupling. Earlier reports demonstrated that the induction of bursts of action potential firing in neuronal populations by g-aminobutyric acid (GABA) A receptor antagonists, represented a model for investigating the mechanisms of synaptic plasticity and activity-dependent neuroprotection (Hardingham et al., 2001(Hardingham et al., , 2002Lu et al., 2001;Ehlers, 2003;Fujino et al., 2003;Kato-Negishi et al., 2003;Arnold et al., 2005;Guan et al., 2005;Lee et al., 2005;Papadia et al., 2005). Using such model, therefore, we took a genome-wide microarray analysis in cultured rat cortical neurons, to gain a global view of the activity-dependent changes in gene expression, as well as their temporal dynamics underlying modulation of synaptic efficacy. ...
... The potassium channel blocker 4AP (5 mM) further enhanced the Bic-induced bursts, which could be abolished by the addition of the sodium channel blocker TTX (1 mM), verifying the physiological basis for the observed bursts caused by disinhibition of neurons. These observations were in agreement with multiple references (Hardingham et al., 2001(Hardingham et al., , 2002Ehlers, 2003;Kato-Negishi et al., 2003;Arnold et al., 2005;Lee et al., 2005;Papadia et al., 2005). The role of bursting has been implicated in various phenomena, including induction of synaptic plasticity (Pike et al., 1999), selective communication between neurons (Izhikevich et al., 2003), dysfunctional states such as epileptic seizures (McCormick and Contreras, 2001), and even for sensory information transmission (Krahe and Gabbiani, 2004). ...
... As demonstrated, the model employed in this study was implicated in synaptic plasticity (Hardingham et al., 2001;Arnold et al., 2005). Kato-Negishi et al. (2003) also showed that Bic induced synapse formation on primary cultured accessory olfactory bulb neurons, which was associated with long-term synaptic plasticity (Yuste and Bonhoeffer, 2001). The studies of Fujino et al. (2003) and Guan et al. (2005), using another two kinds of GABA A receptor antagonists, picrotoxin and lindane, respectively, to induce activity-dependent gene transcription, further supported the involvement of synaptic plasticity in the model. ...
Neuronal activity-dependent gene transcription is a key feature of long-lasting synaptic strengthening associated with learning and memory, as well as activity-dependent neuroprotection. To comprehensively determine the molecular alterations, we carried out genome-wide microarray analysis in cultured rat cortical neurons treated with specific pharmacological agents, a model with alterations in neuronal activity, which were monitored by multi-site electrophysiological recordings. Of the approximately 27,000 genes, the expression of 248 genes was strongly changed in response to enhanced activity. These genes encompass a large number of members of distinct families, including synaptic vesicle proteins, ion channels, signal transduction molecules, synaptic growth regulators, and others. Two subsets of these genes were further confirmed to be specifically induced by Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors and L-type voltage-gated Ca2+ channels (VGCCs). In addition, those genes dynamically regulated by the enhanced activity were also elucidated, as well as those candidate genes associated with synaptic plasticity and neuroprotection. Our findings therefore would help define the molecular mechanisms that occur in response to neuronal activity and identify specific clusters of genes that contribute to activity-dependent and Ca2+-inducible modulation of brain development and function. J. Cell. Physiol. 212: 126–136, 2007. © 2007 Wiley-Liss, Inc.
... Primary cultures of dissociated AOB cells were prepared following a previously reported procedure (Muramoto et al., , 2004Kato-Negishi et al., 2003;Moriya-Ito et al., 2005) with some modifications. Briefly, AOBs were dissected out from Wistar rats at embryonic day 20 (E20) and pooled. ...
... Spontaneous calcium oscillations occurred synchronously among cultured AOB neurons without VNPs as previously reported (Muramoto et al., 2001;Kato-Negishi et al., 2003). At 14 -28 DIV, cultured AOB neurons were observed for intracellular Ca 2ϩ changes using the above-described system. ...
... Such Ca 2ϩ increases were periodically observed in many cultured AOB neurons and not linked with electrical stimulation (Fig. 6E, H). These spontaneous Ca 2ϩ oscillations could be observed from about 7 DICC (data not shown) and appeared even in AOB-alone cultures without VNPs around 14 DIV ( Fig. 7; Muramoto et al., 2001;Kato-Negishi et al., 2003). The application of 25 M AP5 completely blocked the spontaneous Ca 2ϩ oscillations but only partially affected evoked Ca 2ϩ increases in cultured AOB neurons (Fig. 6B, F). ...
To investigate the interaction between vomeronasal receptor neurons and accessory olfactory bulb neurons during pheromonal signal processing and specific synapse formation, partially dissociated rat vomeronasal receptor neurons were co-cultured with accessory olfactory bulb neurons. Between 7 and 14 days in co-culture, a few bundles of fibers from a spherical structure, termed the vomeronasal pocket, of cultured vomeronasal receptor neurons extended to the accessory olfactory bulb neurons. An optical recording of the intracellular Ca(2+) concentration was used to monitor the synaptic activation of cultured accessory olfactory bulb neurons. Electrical stimulation of the vomeronasal pocket between 7 and 14 days in co-culture had no effects on most of the cultured neurons tested, although it occasionally evoked weak responses in a small number of neurons. In contrast, vomeronasal pocket stimulation after 21 days in co-culture evoked clear calcium transients in a substantial number of cultured accessory olfactory bulb neurons. These responses of accessory olfactory bulb neurons were reversibly suppressed by the application of 6-cyano-7-nitroquinoxaline-2,3-dione; the calcium transients disappeared in most of the neurons and were diminished in the others. The application of d-2-amino-5-phosphonopentanoic acid partially affected the calcium transients, but blocked spontaneous calcium increases, which were observed repeatedly in accessory olfactory bulb-alone cultures. The application of both 6-cyano-7-nitroquinoxaline-2,3-dione and d-2-amino-5-phosphonopentanoic acid completely blocked the evoked calcium transients. These results suggest that functional glutamatergic synapses between vomeronasal receptor neurons and accessory olfactory bulb neurons were formed at around 21 days in co-culture.
... In the past decade, we developed primary culture systems for VN organ and AOB for studying the functional roles of VN and AOB neurons in pheromonal signal perception and processing ( Osada et al., 1999; Muramoto et al., 2003 Muramoto et al., , 2004 Negishi-Kato et al., 2003; Moriya-Ito et al., 2005). We identified two types of neuron in the AOB culture system. ...
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.
