Kouji Senzaki

Diversity Revealed by a Novel Family of Cadherins Expressed in Neurons at a Synaptic Complex

In mammals, neurons are highly differentiated and play distinctive functions even in the same brain region. We found a novel cadherin-related neuronal receptor (Cnr) gene family by studying Fyn-binding activity in mouse brain. CNR1 protein is located in the synaptic junction and forms a complex with Fyn. Sequence analysis of eight Cnr products of approximately 20 genes indicates that these comprise a novel cadherin family of the cadherin superfamily. The expression patterns of each member of this novel family were grossly similar to each other but restricted to subpopulations of neurons of the same type. The diversity of the Cnr family genes suggests that there are molecular mechanisms that govern highly differentiated neural networks in the mammalian CNS.

Proteins of the CNR Family Are Multiple Receptors for Reelin

Layering and positioning of neurons require Reelin- and Src family-associated mammalian Disabled (mDab1). Cadherin-related neuronal receptor (CNR) genes are expressed in neurons of the cortical layer, but not in Cajal-Retzius cells expressing Reelin. This leads us to hypothesize that CNRs bound to Fyn of the Src family are receptors for Reelin. Herein we confirm the association and colocalization of CNR proteins with Reelin. This binding is blocked by CR-50 antibody against Reelin, as well as by monoclonal antibodies produced against CNRs. Both disturb the signaling pathway from Reelin to mDab1 and the positioning of cortical neurons in vitro. These results strongly suggest that the CNR family proteins are multiple Reelin receptors. In addition, differential conservation of the Reelin-binding domain among terrestrial vertebrates may be pertinent to the diversity or complexity of brains.

Localization of 5-HT2A Receptor in rat cerebral cortex and olfactory system revealed by immunohistochemistry using two antibodies raised in rabbit and chicken

Serotonin 2A receptor (5-HT2A receptor) is widely distributed in the central nervous system, and has been suggested to be involved in a variety of behavioral conditions and neuropsychiatric disorders. Two polyclonal antibodies were raised against the N-terminus peptide of rat 5-HT2A receptor in chickens (5-HT2A-N) and a glutathione S-transferase fusion protein that contained the C-terminus of the mouse 5-HT2A receptor in rabbits (5-HT2A-C). Affinity-purified 5-HT2A-N and -C antibodies reacted strongly with a single band of 77-78 kDa in postsynaptic density proteins prepared from the rat cortex. The distribution pattern of immunoreactive structures in the rat brain was virtually the same for the two antibodies. The highest levels of immunoreactivity were observed in the olfactory bulb, neocortex, claustrum, piriform cortex, mamillary bodies, pontine nuclei, red nucleus and cranial motor nuclei. In the olfactory bulb, mitral cells were intensely labeled. In the neocortex, many immunoreactive neurons were found in layers II-VI. In layer IV of the neocortex, strong neuropil labeling was observed. In a double-labeling study using chicken 5-HT2A-N and rabbit anti-glial fibrillary acidic protein (GFAP) antibody, a considerable number of GFAP positive cells also showed 5-HT2A immunoreactivity. By using an immunoelectron microscopic technique, 5-HT2A receptor immunoreaction was shown to be localized just beneath the postsynaptic membrane thickening of asymmetric synapses.

The cellular localization of 5-HT2A receptors in the spinal cord and spinal ganglia of the adult rat.

The localization of serotonin2A (5-HT2A) receptors in the adult rat spinal cord and dorsal root ganglia was examined by using a polyclonal antibody that recognizes the C-terminus peptides of the mouse 5-HT2A receptor. Positive cell bodies of 5-HT2A receptor were found in several regions of the spinal cord. Generally, large-to-intermediate sized neuronal cell bodies were intensely immunolabeled. Motoneurons in the ventral horn were the most intensely labeled. Dot-like immunoreactive profiles were located beneath the cell membrane of motoneurons. Neuronal somata in the intermediolateral nucleus of the thoracic spinal cord were moderately labeled. The immunoreactivity in the dorsal horn was weak. A considerable number of glial cell bodies in the white matter were immunostained. The majority of both small and large sized neurons were 5-HT2A immunopositive in the dorsal root ganglion.

Dynamic regulation of the expression of neurotrophin receptors by Runx3

Sensory neurons in the dorsal root ganglion (DRG) specifically project axons to central and peripheral targets according to their sensory modality. However, the molecular mechanisms that govern sensory neuron differentiation and the axonal projections remain unclear. The Runt-related transcription factors, Runx1 and Runx3, are expressed in DRG neuronal subpopulations, suggesting that they might regulate the cell specification and the trajectories of specific axons. Here, we show that parvalbumin-positive DRG neurons fail to differentiate from the onset in Runx3-/- mice. By contrast, TrkC-positive DRG neurons differentiate normally at embryonic day (E) 11.5, but disappear by E13.5 in Runx3-/- mice. Subsequently, TrkC-positive DRG neurons reappear but in smaller numbers than in the wild type. In Runx3-/- mice, central axons of the TrkC-positive DRG neurons project to the dorsal spinal cord but not to the ventral and intermediate spinal cord, whereas the peripheral axons project to skin but not to muscle. These results suggest that Runx3 controls the acquisition of distinct proprioceptive DRG neuron identities, and that TrkC-positive DRG neurons consist of two subpopulations: Runx3-dependent early-appearing proprioceptive neurons that project to the ventral and intermediate spinal cord and muscle; and Runx3-independent late-appearing cutaneous neurons that project to the dorsal spinal cord and skin. Moreover, we show that the number of TrkA-positive DRG neurons is reduced in Runx3-/- mice, as compared with the wild type. These results suggest that Runx3 positively regulates the expression of TrkC and TrkA in DRG neurons.

Serotonin2A receptor-like immunoreactivity in rat cerebellar Purkinje cells

In the present study we examined the distribution pattern of serotonin2A (5-HT2A) receptors in the rat cerebellum. A strong immunoreaction against 5-HT2A receptor protein was observed in Purkinje cells. A dense cluster of immunopositive dendritic profiles of Purkinje cells was located beneath the pia matter of cerebellar cortex. Somal profiles in the cerebellar nuclei had weak to moderate immunoreactions.

Runx1 promotes neuronal differentiation in dorsal root ganglion

Transcription factor Runx1 controls the cell type specification of peptidergic and nonpeptidergic nociceptive dorsal root ganglion (DRG) neurons by repressing TrkA and calcitonin gene-related peptide (CGRP) expression and activating Ret expression during late embryonic and early postnatal periods (Chen et al., 2006b; Kramer et al., 2006; Yoshikawa et al., 2007). Because Runx1 is expressed in DRG from early developmental stages, we examined the roles of Runx1 in the proliferation and the neuronal differentiation of DRG cells. We used transgenic Runx1-deficient (Runx1(-/-)::Tg) mice which are rescued from early embryonic lethality by selective expression of Runx1 in hematopoietic cells under the control of GATA-1 promoter. We found that TrkA-expressing (TrkA(+)) DRG neurons were decreased at embryonic day (E) 12.5 in contrast to the previous study showing that TrkA(+) DRG neurons were increased at E17.5 in Runx1(-/-)::Tg mice (Yoshikawa et al., 2007). The number of DRG neurons which express neuronal markers Hu, NeuN and Islet1 was also reduced in Runx1(-/-)::Tg mice at E12.5, suggesting that the neuronal differentiation was suppressed in these mice. The cell cycle analysis using BrdU/IDU revealed that the number of DRG cells in S-phase and G2/M-phase was increased in Runx1(-/-)::Tg mice at E12.5, while the length of S-phase was not changed between Runx1(+/+)::Tg and Runx1(-/-)::Tg mice, suggesting that Runx1 negatively controls the proliferation of DRG progenitor cell subpopulation in early embryonic period. Hes1 is a negative regulator of neuronal differentiation (Ishibashi et al., 1995; Tomita et al., 1996), and we found that the number of Hes1(+) DRG cells was increased in Runx1(-/-)::Tg mice at E12.5. In summary, the present study suggests a novel function that Runx1 activates the neuronal differentiation of DRG cell subpopulation through the repression of Hes1 expression in early embryonic period.

Subtype specific roles of serotonin receptors in the spine formation of cortical neurons in vitro

Dendritic spines are postsynaptic structures which are formed from filopodia. We examined roles of serotonin (5-HT) receptors in the spine formation. Embryonic rat cortical neurons were cultured for 10 or 14 days and treated by 5-HT receptor agonists for 24 h. At 11 days in vitro, 5-HT(1A) agonist increased filopodia density, whereas 5-HT(2A/2C) agonist increased the density of puncta and spines. At 15 days in vitro, 5-HT(1A) agonist decreased the density of puncta and spines, whereas 5-HT(2A/2C) agonist decreased filopodia density with increase of spines. In conclusion, the present study shows 5-HT receptors have subtype-specific effects on the spine formation.

Roles of serotonin 5-HT3 receptor in the formation of dendrites and axons in the rat cerebral cortex: an in vitro study.

The serotonin type 3 (5-HT(3)) receptor is an only ligand-gated ion channel among 14 serotonin receptors. Here, we examined the roles of the 5-HT(3) receptor in the formation of dendrites and axons, using a dissociation culture of embryonic rat cerebral cortex. Cortical neurons at embryonic day 16 were cultured for 4 days in the presence of a selective 5-HT(3) receptor agonist with or without an antagonist. Neurons were then immunostained by antibodies against microtubule-associated protein 2 (MAP2) and glutamic acid decarboxylase (GAD) 65. All cells expressed MAP2, whereas only limited number of cells expressed GAD65. From the immunoreactivity and the cell shape, we tentatively divided neurons into 3 types; GAD-positive multipolar, GAD-positive bipolar/tripolar and GAD-negative neurons. The total length of axons and dendrites, the number of primary dendrites and the dendritic branching of GAD-negative neurons were decreased by the agonist (10 or 100nM), most of which were reversed by the concomitant treatment of the antagonist. In contrast, no or little effect was observed on the formation of dendrites and axons of GAD-positive multipolar neurons, and the neurite formation of GAD-positive bipolar/tripolar neurons. The present study revealed differential roles of the 5-HT(3) receptor in the formation of dendrites and axons of subtypes of cortical neurons.

Runx3 is required for the specification of TrkC-expressing mechanoreceptive trigeminal ganglion neurons

Sensory neurons project axons to specific peripheral and central targets according to their sensory modality. Runx3 is crucially involved in proprioceptive dorsal root ganglion neuron development. Runx3 is also expressed in trigeminal ganglion (TG) neurons. The role of Runx3 in the TG, however, is largely unknown because the TG does not contain proprioceptive neurons. In Runx3-deficient (Runx3(-/-)) mice, TrkB-expressing TG neurons were increased, whereas TrkC-expressing TG neurons were decreased during TG neuron development. In Runx3(-/-) neonatal mice, TrkC-expressing TG neurons did not project to the Merkel cells in the outer root sheath (ORS) of whisker vibrissae peripherally and the spinal trigeminal nucleus pars interpolaris (Sp5I) centrally. These findings suggest that Runx3 is required for the specification of TrkC-expressing TG neurons, conveying mechanoreceptive signals from the Merkel cells in the ORS of the whisker vibrissae to the Sp5I.