Abstract: Dendritic and axonal processes are input and output sites, respectively, of neuronal information, and detailed visualization of these processes may be indispensable for elucidating the neuronal circuits and revealing the principles of neuronal functions. To establish a method for completely visualizing dendritic processes, we first developed green fluorescent protein (GFP)-based proteins and, by using lentivirus with a neuron-specific promoter, examined whether or not the protein fully visualized the dendritic processes of infected neurons. When GFP with a palmitoylation (palGFP) or myristoylation/palmitoylation site (myrGFP) was expressed in rat brain with lentiviruses, myrGFP labeled dendritic membrane better than palGFP. Subsequently, dendrite-targeting efficiencies of three basolateral membrane-sorting and three putative dendrite-targeting domains, which were attached to myrGFP C-terminus, were examined in striatonigral GABAergic and corticothalamic glutamatergic neurons, and in cultured cortical neurons. Of the six domains, C-terminal cytoplasmic domain of low density lipoprotein receptor (LDLRct) was most efficient in targeting the protein to dendrites, showing 8.5-15-fold higher efficiency in striatonigral neurons compared with myrGFP. Finally, dendritic membrane-targeting potency of myrGFP-LDLRct was confirmed in transgenic mice using Thy1 or Gad1 expression cassette. Thus, myrGFP-LDLRct is an excellent synthetic protein for dendritic visualization, and may be a useful tool for the morphological analysis of neuronal circuits.

Abstract: By altering their morphology, astrocytes, including those involved in the maintenance and plasticity of neurons and in clearance of transmitter, play important roles in synaptic transmission; however, the mechanism that regulates the morphological plasticity of astrocytes remains unclear. Recently, we reported that T1, a subtype of TrkB (a family of BDNF-specific receptors), altered astrocytic morphology through the control of Rho GTPases in primary astrocyte cultures. In this study, we extended this observation to investigate acute neocortical slices from adult rats. T1 siRNA-expression vectors were electroporated into astrocytes in neocortical layer I of living rats. In both normal slices and control vector-electroporated slices, BDNF induced the elongation of the astrocytic processes and increased the branching of processes in slices after 1 h incubation. In contrast, in T1 siRNA-electroporated slices, no such significant morphological changes were observed in the astrocytes. In addition, the number of synaptophysin+ sites in contact with GFAP+ processes increased in a BDNF-T1-dependent manner without the increase in the total synaptophysin+ sites. Therefore, the present study provides evidence of the regulation of layer I astrocytic morphology by the BDNF-T1 signal in adult rat neocortical slices.

Abstract: In the field of basic and clinical neurosciences, it is important to develop a method for easy delivery and persistent expression of transgene in central neurons. We firstly generated lentiviral vectors with five kinds of neuron-specific promoters, such as synapsin I (SYN), calcium/calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet-derived growth factor beta chain promoters and then novel hybrid promoters by fusing cytomegalovirus enhancer (E) to those neuron-specific promoters. Neuron-specific expression of green fluorescent protein (GFP) with those promoters was examined in vivo by injecting the lentiviral vectors into the rat neostriatum, thalamus and neocortex. Among all the promoters, SYN promoter displayed the highest specificity for neuronal expression in all the regions examined (more than 96%). Although GFP production by the hybrid promoters was about 2-4 times larger than the non-enhanced promoters, the neuronal specificity was significantly decreased in most cases. However, the neuronal specificity of E/SYN hybrid promoter exhibited the least decrease only in the thalamus. Furthermore, the transcriptional activity and neuronal specificity of E/SYN promoter were sustained for up to 8 weeks. Thus, lentivirus with E/SYN promoter is the best vector for strong persistent expression in neurons.

Abstract: Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton.

Abstract: A truncated TrkB receptor, T1, which is one of the receptors for brain-derived neurotrophic factor, has been shown to regulate the morphology of neurons and glial cells in primary cultures and/or slices overexpressing T1 in the recent past. However, in vivo localization of T1 at protein level remains unclear. In the present study, we examined the localization of T1 in the primary motor and prefrontal cortices of adult monkeys by using immunohistochemistry. In the primary motor cortex, T1 immunoreactivity was observed mainly in the pyramidal neurons of layers II-VI, especially Betz cells of layer V. The apical and basal dendrites and cell bodies of Betz cells were strongly stained. In addition, we found that the interneurons were also T1-immunopositive and that there were no T1-positive astrocytes. In the prefrontal cortex, we observed strong immunoreactivity of T1 in astrocytes as well as pyramidal neurons of layer V. The pyramidal neurons and interneurons in layers II/III were faintly immunoreactive for T1. Thus, these findings, together with the fact that T1 is involved in morphological control of neurons and glial cells, suggest that the prefrontal cortex might possess a different degree of morphological plasticity than the primary motor cortex in the adult monkey.

Abstract: Through tropomyosin-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Abstract: In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.

Abstract: BDNF and its specific receptor TrkB are concerned with synaptic plasticity as well as maintenance of the nervous system. TrkB has three subtypes: full-length TrkB (TK+), which has a tyrosine kinase containing intracellular domain, and two truncated TrkBs (TK-; T1 and T2), which lack tyrosine kinases. To understand the molecular interaction among these subtypes, we investigated the expression and distribution of BDNF, TK+, and T1 in the adult monkey cerebellum by single and double immunohistochemistry and Western blot analysis. We observed by single immunohistochemistry that BDNF, TK+, and T1 are distributed in almost all the somata and dendrites of Purkinje and granule cells. In the double-stained sections, three kinds of regions were observed: TK+ >T1; TK+ =T1; TK+ <T1. Moreover, three types of TrkB dimers (TK+/TK+ homodimer, TK+/TK- heterodimer, and TK-/TK- homodimer) were induced by stimulating with exogenous BDNF. These observations suggest that the functions of BDNF may be modified by interaction among subtypes of TrkB in each region of the Purkinje cells.

Abstract: To date, two subtypes of TrkB, a BDNF receptor, have been described. One is full-length TrkB (TK+), which has a tyrosine kinase-containing intracellular domain. The other is truncated TrkB (TK-), which has a short intracellular domain lacking the tyrosine kinase. In this study, we investigated the dimerization of TrkB subtypes in the developing monkey prefrontal cortex by means of cross-linking. At embryonic day 120, the TK+/TK+ and the 100 kDa/100 kDa homodimers were observed with BDNF stimulation. At the newborn stage, the TK+/TK+ and the TK-/TK- homodimers were observed with BDNF stimulation. At the adult stage, the TK-/TK- homodimer and the TK+/TK- heterodimer were formed by BDNF stimulation. The levels of all dimers increased in proportion to the concentration of BDNF. Moreover, the dimers were clearly formed within 5 min of treatment with BDNF. BDNF and NT-4/5 induced the dimers, whereas NT-3 formed slight dimers but NGF did not. Furthermore, anti-BDNF antibody inhibited the TrkB dimerization. Moreover, the intercellular binding proteins of TrkB were not cross-linked by BS3. Therefore, these results suggest that the change in dimerization among TrkB subtypes occurs during development of the monkey prefrontal cortex.

Abstract: We investigated the changes of brain-derived neurotrophic factor (BDNF)-immunoreactive structures in the hippocampal formation of aged macaques (Macaca fuscata fuscata). At adult stages (10 and 12 years), BDNF immunoreactivity occurred in the neurons of the dentate gyrus, the pyramidal neurons in the CA1, CA2, CA3 subfields and the subiculum, and the neurons in the CA4 subfield and the entorhinal cortex. The apical dendrites were also BDNF immunopositive. In aged monkeys (26, 30 and 32 years), the intensity of the BDNF-immunoreactivity declined significantly in cell bodies and dendrites of the neurons in the hippocampal formation except the CA2 pyramidal neurons. These findings indicate that BDNF is one of the vulnerable signal molecules during the aging process of the primate hippocampal formation.

Abstract: Cholesterol- and glycolipid-enriched microdomains within the plasma membrane of animal cells, including neurons, have been purified and used as a low-density membrane domain after extraction with Triton X-100 (raft), or after subcellular fractionation without detergent (LDM). In this study, we compared the protein compositions in the raft and the LDM. Membrane receptors, acylated- and glycosylphosphatidylinositol (GPI)- anchored proteins were enriched in the LDM. Further treatment of the LDM with Triton X-100 excluded the membrane receptors, TrkBs and insulin receptor beta. In the presence of calcium ions, the endogenous tyrosine kinase activities in the LDM and the raft were enhanced, suggesting an important role of calcium ions in the signal transduction via the LDM and the raft.

Abstract: Distribution and morphological changes of cells containing the signal transducing neurotrophin receptor, full-length Trk B (fl-Trk B), were investigated in the hippocampal formation of the macaque monkey between embryonic day 140 and the adult stage. Western blot analysis showed that one main protein band, which migrated at 141 kDa, was detected in both the embryonic and adult hippocampal formation. In the pyramidal cells in CA1 and CA3 subfields, the subiculum, and the entorhinal cortex, fl-Trk B-immunoreactive dendrites were observable in the embryonic stage. In contrast, in the granule cells of the dentate gyrus, few dendrites were immunoreactive during embryonic and early developmental stages. This difference may be due to the later growth of the granule cells of the dentate gyrus. The existence of fl-Trk B immunoreactivity in the cell body and dendrites in the embryonic hippocampal neurons, suggests that BDNF and/or NT4/5 act on the hippocampal cells by autocrine/paracrine mechanisms. In the entorhinal cortex, fl-Trk B immunoreactivity became localized in the stellate cells in layer II and the pyramidal cells in layers III, V and VI in adulthood. This indicates that BDNF and/or NT4/5 are important for the maintenance of the projection neurons in the entorhinal cortex at the adult stage. The strongest fl-Trk B immunoreactivity in the hippocampal neurons occurred at postnatal month 4, corresponding to the period of greatest synapse production in the monkey hippocampus, suggesting that BDNF and/or NT4/5 with fl-Trk B may play a role in synapse formation in the monkey hippocampus.

Abstract: We examined the expression of full-length TrkB (TrkBTK+) and truncated TrkB (TrkBTK-) in the central nervous system (CNS) of the macaque monkey (Macaca fascicularis) using a western blot analysis. At the adult stage, the levels of TrkBTK+ in cerebral cortices were higher than those in other structures of CNS and the expressions of TrkBTK+ in the association cortices (except area PE) were relatively lower than those in the primary cortices. In contrast, TrkBTK- in the hippocampus and the cerebellum was significantly higher than in other structures. In various developing cerebral cortices, TrkBTK+ was detected at the same levels from embryonic day 120 (E120) to the adult period. In contrast, the expression of TrkBTK- increased remarkably after the newborn stage (NB), reached the maximum level at postnatal day 60 (P60) and maintained the same level into adulthood. The peaks of TrkBTK- in the association cortices were more delayed than in the primary cortices. The expression of TrkBTK- occurred at a time that correlates with the elimination of axons and the down-regulation of neuropeptides. The present study suggests that TrkBTK- plays an important role in the axonal remodelling and that it may act as a negative effector of TrkBTK+ in the primate CNS, reducing responsiveness to BDNF and/or NT-4/5.