Generation of TrkA/TrkB Chimeric Receptor Constructs Reveals Molecular Mechanisms Underlying BDNF-Induced Dendritic Outgrowth in Hippocampal Neurons

Neurotrophins (NTs) regulate neuronal survival, differentiation, and synaptic plasticity through tropomyosin receptor kinases (Trks). The molecular mechanisms underlying these functions, however, have remained incompletely understood. In the present study, we first showed that brain-derived neurotrophic factor (BDNF) increased both the number of primary dendrites and dendritic complexity in cultured hippocampal neurons. Since hippocampal neurons predominantly express the BDNF receptor TrkB, but not the nerve growth factor (NGF) receptor Trk, we generated DNA constructs encoding the extracellular domain of TrkA fused with the transmembrane and intracellular domain of TrkB and introduced these constructs into cultured hippocampal neurons. To visualize the dendrites, the TrkA/TrkB fusion proteins were bicistronically expressed with green fluorescence protein (GFP). Interestingly, the GFP-labeled neurons grew dendrites and activated the TrkA/TrkB receptors in response to NGF, but not BDNF. We next generated a series of TrkA/TrkB receptors with mutations at tyrosine residues in the TrkB kinase domain, and sought to identify the signaling pathway required for NT-induced dendrite outgrowth. Sholl analyses demonstrated that TrkB signaling through Shc, but not through PLC-γ, plays a crucial role in NT-elicited dendritic outgrowth in hippocampal neurons.     

Chondroitin sulfate proteoglycans down-regulate spine formation in cortical neurons by targeting tropomyosin-related kinase B (TrkB) protein

Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit axonal sprouting and experience-dependent plasticity. Although protein-tyrosine phosphatase σ (PTPσ) has been proven to be a receptor for CSPGs, its downstream signaling has remained a mystery. Here, we show that CSPGs target and dephosphorylate tropomyosin-related kinase B, the receptor of brain-derived neurotrophic factor (BDNF), via PTPσ in embryonic cortical neurons in vitro. Whereas BDNF promoted dendritic spine formation in embryonic cortical neurons, CSPGs abolished the effects of BDNF and eliminated existing dendritic spines when BDNF was present. The latter effect was dependent on the p75 receptor, presumably because BDNF binding to the p75 receptor elicits elimination of dendritic spines. These results suggest that the inhibitory activity of CSPGs on dendritic spine formation operates through the targeting of neurotrophins at the receptor level.     

A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster

Vertebrate and invertebrate dendrites are information-processing compartments that can be found on both central and peripheral neurons. Elucidating the molecular underpinnings of information processing in the nervous system ultimately requires an understanding of the genetic pathways that regulate dendrite formation and maintenance. Despite the importance of dendrite development, few forward genetic approaches have been used to analyze the latest stages of dendrite development, including the formation of F-actin-rich dendritic filopodia or dendritic spines. We developed a forward genetic screen utilizing transgenic Drosophila second instar larvae expressing an actin, green fluorescent protein (GFP) fusion protein (actin::GFP) in subsets of sensory neurons. Utilizing this fluorescent transgenic reporter, we conducted a forward genetic screen of >4000 mutagenized chromosomes bearing lethal mutations that affected multiple aspects of larval dendrite development. We isolated 13 mutations on the X and second chromosomes composing 11 complementation groups affecting dendrite outgrowth/branching, dendritic filopodia formation, or actin::GFP localization within dendrites in vivo. In a fortuitous observation, we observed that the structure of dendritic arborization (da) neuron dendritic filopodia changes in response to a changing environment.     

Environmental enrichment effects on development of retinal ganglion cell dendritic stratification require retinal BDNF

A well-known developmental event of retinal maturation is the progressive segregation of retinal ganglion cell (RGC) dendrites into a and b sublaminae of the inner plexiform layer (IPL), a morphological rearrangement crucial for the emergence of the ON and OFF pathways. The factors regulating this process are not known, although electrical activity has been demonstrated to play a role. Here we report that Environmental Enrichment (EE) accelerates the developmental segregation of RGC dendrites and prevents the effects exerted on it by dark rearing (DR). Development of RGC stratification was analyzed in a line of transgenic mice expressing plasma-membrane marker green fluorescent protein (GFP) under the control of Thy-1 promoter; we visualized the a and b sublaminae of the IPL by using an antibody selectively directed against a specific marker of cholinergic neurons. EE precociously increases Brain Derived Neurotrophic Factor (BDNF) in the retina, in parallel with the precocious segregation of RGC dendrites; in addition, EE counteracts retinal BDNF reduction in DR retinas and promotes a normal segregation of RGC dendrites. Blocking retinal BDNF by means of antisense oligos blocks EE effects on the maturation of RGC dendritic stratification. Thus, EE affects the development of RGC dendritic segregation and retinal BDNF is required for this effect to take place, suggesting that BDNF could play an important role in the emergence of the ON and OFF pathways.     

A Golgi study of anterior dorsal ventricular ridge in the alligator,Alligator mississippiensis

The anterior dorsal ventricular ridge was examined in the American alligator, Alligator mississippiensis, with cresyl violet and Golgi-Kopsch preparations. Four cytoarchitectonic areas (lateral dorsolateral, medial dorsolateral, intermediolateral, and lateral) can be distinguished by variations in the density of neurons and their tendency to form clusters of neurons with apposed somata. Three distinct types of neurons are distributed throughout these areas. Juxtaependymal neurons lie near the ventricular surface and have dendritic fields paralleling the ependymal layer. Their dendrites bear a moderate density of spines. Spiny neurons all have stellate shaped dendritic fields and dendrites that bear dendritic spines, but they vary greatly in the density of spines and the thickness of their dendrites. A very spiny variety has a high spine density and relatively thick dendrites. A moderately spiny variety has a moderate spine density and thin dendrites. A sparsely spiny variety has a low spine density and thick dendrites. Aspiny neurons have a relatively large number of dendrites that form a gnarled dendritic field and lack spines.     

Spike-timing-dependent BDNF secretion and synaptic plasticity

In acute hippocampal slices, we found that the presence of extracellular brain-derived neurotrophic factor (BDNF) is essential for the induction of spike-timing-dependent long-term potentiation (tLTP). To determine whether BDNF could be secreted from postsynaptic dendrites in a spike-timing-dependent manner, we used a reduced system of dissociated hippocampal neurons in culture. Repetitive pairing of iontophoretically applied glutamate pulses at the dendrite with neuronal spikes could induce persistent alterations of glutamate-induced responses at the same dendritic site in a manner that mimics spike-timing-dependent plasticity (STDP)—the glutamate-induced responses were potentiated and depressed when the glutamate pulses were applied 20 ms before and after neuronal spiking, respectively. By monitoring changes in the green fluorescent protein (GFP) fluorescence at the dendrite of hippocampal neurons expressing GFP-tagged BDNF, we found that pairing of iontophoretic glutamate pulses with neuronal spiking resulted in BDNF secretion from the dendrite at the iontophoretic site only when the glutamate pulses were applied within a time window of approximately 40 ms prior to neuronal spiking, consistent with the timing requirement of synaptic potentiation via STDP. Thus, BDNF is required for tLTP and BDNF secretion could be triggered in a spike-timing-dependent manner from the postsynaptic dendrite.     

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.     

The torus semicircularis in a gekkonid lizard

The cytoarchitecture and neuromorphology of the torus semicircularis in the tokay gecko, Gekko gecko, were examined in Nissl-stained, fiber-stained, and Golgi-impregnated tissues. From a superficial position, the torus semicircularis extends rostrally under the caudal half of the optic tectum. Caudally, the two tori abut upon one another; rostrally, they diverge. The torus semicircularis consists of central, laminar, and superficial nuclei. The central nucleus consists of fusiform, spherical and triangular neurons. Their dendrites are highly branched, with numerous dendritic spines, and are oriented mediolaterally, dorsoventrally, and rostrocaudally. Fusiform and spherical neurons display two dendritic patterns: “single axis,” ramifying in one axis, and “dual axis,” exhibiting higher-order branches perpendicular to the primary dendrites. Triangular neurons exhibit a “radiate” dendritic pattern. In the rostral half of the torus semicircularis, the laminar nucleus caps the central nucleus. The laminar nucleus encircles the central nucleus in the caudal torus semicircularis. The neurons of the laminar nucleus have dendritic arrays oriented parallel to the border of the central nucleus. These dendrites exhibit a paucity of dendritic spines and higher-order branches. Fusiform and spherical neurons exhibit “single axis” and “dual axis” dendritic patterns. Triangular neurons display “radiate” patterns. The caudal superficial nucleus lies dorsal and dorsolateral to the central nucleus. The superficial nucleus is sparsely populated by small fusiform and spherical neurons with moderately branched dendrites and moderate numbers of dendritic spines. These neurons display “single axis” (fusiform neurons) as well as “dual axis” and “radiate” (spherical neurons) dendritic patterns. They are oriented either parallel to or perpendicular to the boundary of the laminar nucleus.     

Brain-derived neurotrophic factor produced by human umbilical tissue-derived cells is required for its effect on hippocampal dendritic differentiation

The potential for nonembryonic cells to promote differentiation of neuronal cells has therapeutic implications for regeneration of neurons damaged by stroke or injury and avoids many ethical and safety concerns. The authors have assessed the capacity of human umbilical tissue-derived cells (hUTC) and human mesenchymal stromal cells (hMSC) to enhance differentiation of rodent hippocampal neurons. Co-culture of hippocampal cells with hUTC or hMSC in transwell inserts for 3 days resulted in increase of several dendritic parameters including the number and length of primary dendrites. The effect of hUTC or hMSC on dendritic maturation was only apparent on neurons grown for 2 weeks in vitro prior to co-culture. Changes in dendritic morphology in the presence of hUTC were also accompanied by increased expression of the presynaptic marker synaptotagmin and the postsynaptic marker postsynaptic density protein 95 kDa (PSD95) suggesting that there may also be an increase in the number of synapses formed in the presence of hUTC. The effect of hUTC and hMSC on hippocampal cells in co-culture was comparable to those induced by treatment with recombinant human brain-derived neurotrophic factor (BDNF) implying that a similar factor may be released from hUTC or hMSC. Analysis of hUTC-conditioned medium by ELISA demonstrated that BDNF was indeed secreted. An antibody that blocks the actions of BDNF partially inhibited the actions of hUTC on dendritic morphology suggesting that BDNF is at least one of the factors secreted from the cells to promote dendritic maturation. These results indicate that hUTC secrete biologically active BDNF, which can affect dendritic morphology.     

Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as brain-derived neurotrophic factor (BDNF). We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GTPase-activating protein neuron-associated developmentally regulated protein (NADRIN), and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.     

The role of LPA1 in formation of synapses among cultured hippocampal neurons

We studied the lysophosphatidic acid receptor-1 (LPA1) gene, which we found to be expressed endogenously in cultured hippocampal neurons, and in vivo in young (1-week-old) rat brain slices. Overexpressed green fluorescent protein (GFP)-tagged, membrane-associated LPA1 accumulated in a punctate manner over the entire dendritic tree and caused an increase in dendritic spine density. About half of the dendritic spines in the LPA1-transfected neurons displayed distinct fluorescent puncta, and this subset of spines was also substantially larger than puncta-free, LPA1-transfected or control GFP spines. This phenotype could also be seen in cells transfected with a ligand-binding, defective mutant and is therefore not dependent on interaction with an ambient ligand. While spontaneous miniature excitatory synaptic currents were of the same amplitudes, they decayed slower in LPA1-transfected neurons compared with GFP controls. We propose that LPA1 may play a role in the formation and modulation of the dendritic spine synapse.     

Cell types within the medial forebrain bundle: a Golgi study of preoptic and hypothalamic neurons in the rat

The morphology of lateral preoptic (POL) and lateral hypothalamic (HLA) neurons was studied in 14- to 200-day-old rats with the chlorate-formaldehyde modification of the Golgi method. Drawings of 91 POL and HLA neurons revealed three distinct neuronal types within the MFB based on somatic size and shape and dendritic morphology. Class I neurons, which accounted for 75-80% of the neurons in the MFB, has fusiform or multipolar somata averaging 21 X 14 micron and 2-5 sparsely branched dendrites with a moderate number of sticklike spines. The extensive dendritic domains of Class I neurons ranged from 700 to 1,500 micron and were usually oriented perpendicular to the longitudinal fibers of the MFB. Both nonoriented and oriented Class I neurons were encountered. Nonoriented Class I neurons had expansive dendritic arbors which reached nearly all regions of the MFB in the coronal plane. Oriented Class I neurons had dendritic domains which were confined to specific regions (e.g., ventral-lateral) of the MFB. Class II neurons, which made up approximately 10% of the MFB neurons, had large multipolar somata averaging 30 X 17 micron and 2-5 stout dendrites which were densely covered with hairlike spines. Class II neurons also exhibited spines on their somata and proximal dendritic trunks and had dendritic domains of 700-1,000 micron. Class III neurons had small somata averaging 15 X 12 micron and restricted dendritic arbors of 500-700 micron in diameter. Class III neurons exhibited both spiny and spine-free dendrites and made up 10% of MFB neurons. Because of the parcellation of chemically coded fiber systems within the MFB, individual POL and HLA neurons may not be homogeneous in the type of afferents they receive from other brain areas.     

Efficient copackaging and cotransport yields postsynaptic colocalization of neuromodulators associated with synaptic plasticity

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy.     

Long-term gene therapy causes transgene-specific changes in the morphology of regenerating retinal ganglion cells

Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5-8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.     

Changes of Dendritic Spine Density and Morphology in the Superficial Layers of the Medial Entorhinal Cortex Induced by Extremely Low-Frequency Magnetic Field Exposure

In the present study, we investigated the effects of chronic exposure (14 and 28 days) to a 0.5 mT 50 Hz extremely low-frequency magnetic field (ELM) on the dendritic spine density and shape in the superficial layers of the medial entorhinal cortex (MEC). We performed Golgi staining to reveal the dendritic spines of the principal neurons in rats. The results showed that ELM exposure induced a decrease in the spine density in the dendrites of stellate neurons and the basal dendrites of pyramidal neurons at both 14 days and 28 days, which was largely due to the loss of the thin and branched spines. The alteration in the density of mushroom and stubby spines post ELM exposure was cell-type specific. For the stellate neurons, ELM exposure slightly increased the density of stubby spines at 28 days, while it did not affect the density of mushroom spines at the same time. In the basal dendrites of pyramidal neurons, we observed a significant decrease in the mushroom spine density only at the later time point post ELM exposure, while the stubby spine density was reduced at 14 days and partially restored at 28 days post ELM exposure. ELM exposure-induced reduction in the spine density in the apical dendrites of pyramidal neurons was only observed at 28 days, reflecting the distinct vulnerability of spines in the apical and basal dendrites. Considering the changes in spine number and shape are involved in synaptic plasticity and the MEC is a part of neural network that is closely related to learning and memory, these findings may be helpful for explaining the ELM exposure-induced impairment in cognitive functions.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.