Regulation of neurotrophin-induced axonal responses via Rho GTPases

Nerve growth factor (NGF) and related neurotrophins induce differential axon growth patterns from embryonic sensory neurons (Lentz et al. [1999] J. Neurosci. 19:1038-1048; Ulupinar et al. [2000a] J. Comp. Neurol 425:622-630). In wholemount explant cultures of embryonic rat trigeminal ganglion and brainstem or in dissociated cell cultures of the trigeminal ganglion, exogenous supply of NGF leads to axonal elongation, whereas neurotrophin-3 (NT-3) treatment leads to short branching and arborization (Ulupinar et al. [2000a] J. Comp. Neurol. 425:622-630). Axonal responses to neurotrophins might be mediated via the Rho GTPases. To investigate this possibility, we prepared wholemount trigeminal pathway cultures from E15 rats. We infected the ganglia with recombinant vaccinia viruses that express GFP-tagged dominant negative Rac, Rho, or constitutively active Rac or treated the cultures with lysophosphatitic acid (LPA) to activate Rho. We then examined axonal responses to NGF by use of the lipophilic tracer DiI. Rac activity induced longer axonal growth from the central trigeminal tract, whereas the dominant negative construct of Rac eliminated NGF-induced axon outgrowth. Rho activity also significantly reduced, and the Rho dominant negative construct increased, axon growth from the trigeminal tract. Similar alterations in axonal responses to NT-3 and brain-derived neurotrophic factor were also noted. Our results demonstrate that Rho GTPases play a major role in neurotrophin-induced axonal differentiation of embryonic trigeminal axons.     

Local neurotrophin effects on central trigeminal axon growth patterns

In dissociated cell and wholemount explant cultures of the embryonic trigeminal pathway NGF promotes exuberant elongation of trigeminal ganglion (TG) axons, whereas NT-3 leads to precocious arborization [J. Comp. Neurol. 425 (2000) 202]. In the present study, we investigated the axonal effects of local applications of NGF and NT-3. We placed small sepharose beads loaded with either NGF or NT-3 along the lateral edge of the central trigeminal tract in TG-brainstem intact wholemount explant cultures prepared from embryonic day 15 rats. Labeling of the TG with carbocyanine dye, DiI, revealed that NGF induces local defasciculation and diversion of trigeminal axons. Numerous axons leave the tract, grow towards the bead and engulf it, while some axons grow away from the neurotrophin source. NT-3, on the other hand, induced localized interstitial branching and formation of neuritic tangles in the vicinity of the neurotrophin source. Double immunocytochemistry showed that axons responding to NGF were predominantly TrkA-positive, whereas both TrkA and TrkC-positive axons responded to NT-3. Our results indicate that localized neurotrophin sources along the routes of embryonic sensory axons in the central nervous system, far away from their parent cell bodies, can alter restricted axonal pathways and induce elongation, arborization responses.     

Cells Expressing mRNA for Neurotrophins and their Receptors During Embryonic Rat Development

In situ hybridization analysis of cells expressing messenger RNAs (mRNAs) for the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their high-affinity receptors (trk, trkB and trkC) in the rat embryo revealed a complex but specific expression pattern for each of these mRNAs. For all mRNAs a developmentally regulated expression was seen in many different tissues. BDNF and NT-3 mRNAs were expressed in the sensory epithelia of the cochlea and vestibule macula of the sacculus and utricle, and both trkB and trkC mRNA were expressed in the spiral and vestibule ganglia innervating these sensory structures. NGF and NT-3 mRNA were found in the iris, innervated by the sympathetic neurons of the superior cervical ganglion and sensory neurons from the trigeminal ganglion, which expressed both trk and trkC mRNAs. Both NGF and NT-3 mRNAs were also expressed in other target fields of the trigeminal ganglion, the epithelium of the whisker follicles (NT-3 mRNA) and in the epithelium of the nose, tongue and jaw. NT-3 mRNA was found in the cerebellar external granule layer and trkC mRNA in the Purkinje layer of the cerebellar primordia. These sites of synthesis are consistent with a target-derived neurotrophic interaction for NGF, BDNF and NT-3. However, in some cases mRNAs for both the neurotrophins and their high-affinity receptors were detected in the same tissue, including the dorsal root, geniculate, superior, jugular, petrose and nodose ganglia, as well as in the hippocampus, frontal cortical plate and pineal recess, implying a local mode of action. Combined, these data suggest a broad function for the neurotrophins and their receptors in supporting neural innervation during embryonic development. The results also identify several novel neuronal systems that are likely to depend on the neurotrophins in vivo.     

Embryonic sensory development: Local expression of neurotrophin-3 and target expression of nerve growth factor

Development and maintenance of peripheral sensory and sympathetic neurons are regulated by target-derived neurotrophins, including nerve growth factor (NGF). To determine whether trophins are potentially critical prior to and during target innervation, for neuronal survival or axon guidance, in situ hybridization was performed in the rat embryo. We examined the expression of genes encoding NGF, neurotrophin-3 (NT-3), and their putative high-affinity receptors, trk A and trk C, respectively. Trks A and C were detected in dorsal root sensory ganglia (DRG) on embryonic day 12.5 (E12.5), implying early responsiveness to NGF and NT-3. NGF mRNA was expressed in the central spinal cord target and by the peripheral somite, at this early time, which thereby may function as a transient “guidepost” target for sensory fibers. Somitic expression was transient and was undetectable by E17.5. NT-3 was expressed in the DRG itself from E13.5 to 17.5, suggesting local transient actions on sensory neurons. NT-3 was also expressed in the ventral spinal cord at low levels on E13.5. We examined the trigeminal ganglion to determine whether cranial sensory neurons are similarly regulated. Trk A was detected in the trigeminal ganglion, while NGF was expressed in the central myelencephalon target, paralleling observations in the DRG and spinal cord. However, NT-3 and trk C were undetectable, in contrast to DRG, suggesting that the environment or different neural crest lineages govern expression of different trophins and trks. Apparently, multiple trophins regulate sensory neuron development through local as well as transient target mechanisms prior to innervation of definitive targets.     

The cytokine/neurotrophin axis in peripheral axon outgrowth

Inflammation is part of the physiological wound healing response following mechanical lesioning of the peripheral nervous system. However, cytokine effects on axonal regeneration are still poorly understood. Because cytokines influence the expression of neurotrophins and their receptors, which play a major role in axonal outgrowth after lesioning, we investigated the hypothesis that cytokines influence specifically neurotrophin-dependent axon elongation. Therefore, we have characterized neurotrophin-dependent neurite outgrowth of murine dorsal root ganglia (DRG) in vitro and investigated the influence of pro- and anti-inflammatory cytokines on these outgrowth patterns. Embryonic day 13 (E13) DRG were cultured in Matrigel for 2 days and axonal morphology, density and elongation were determined using an image analysis system. Nerve growth factor (NGF), neurotrophin-3 (NT-3) and -4 (NT-4) were applied alone (50 ng/mL), in double or in triple combinations. NT-3, NT-4 and NT-3 + NT-4 combined induced a moderate increase in axonal outgrowth (P < 0.001) compared with controls, while NGF and all combinations including NGF induced an even more pronounced increase in axonal outgrowth (P < 0.001). After characterizing these outgrowth patterns, interleukin (IL)-1beta, IL-4, IL-6, interferon-gamma (IFNgamma) and tumour necrosis factor-alpha (TNFalpha) (50 or 500 ng/mL) were added to the different neurotrophin combinations. Low doses of TNFalpha and IL-6 influenced neurite extension induced by endogenous neurotrophins. IL-4 increased NT-4-induced outgrowth. IL-6 stimulated NT-3 + NT-4-induced outgrowth. IFNgamma stimulated neurite extension in the presence of NT-3 + NT-4 and NT-3 + NGF. TNFalpha inhibited NT-3-, NT-3 + NGF-, NT-4 + NGF- and NT-3 + NT-4 + NGF-induced outgrowth. These data suggest that inflammation following nerve injury modulates re-innervation via a cytokine/neurotrophin axis.     

BDNF and NT-4 differentially modulate neurite outgrowth in developing retinal ganglion cells.

We show here that neurite outgrowth of ganglion cells (RGCs) was selectively enhanced following treatment with BDNF or NT-4 in short-term cultures of dissociated cells derived from the neuroretina of postnatal rats. NT-4 was more effective than BDNF. The effect of NT-3 was variable, whereas NGF and CNTF had no effects upon neurite elongation. The neuritogenic responses of RGCs to both BDNF and NT-4 were prevented by competition with soluble TrkB receptor, and abolished by K252a, a selective inhibitor of the tyrosine kinase activity of Trks. These results indicate that the differentiating effects of BDNF and NT-4 are mediated by TrkB receptors, naturally expressed by RGCs. Developing RGCs treated with these TrkB ligands displayed distinct, albeit partially overlapping, patterns of neurite morphology. BDNF supported predominantly polarized outgrowth, whereas NT-4 induced the appearance of intensely branched symmetrical arbors. The lack of RGCs showing combined morphologies (e.g., highly arborized unipolar cells) suggests distinct mechanisms underlying either elongation or branching, and implicates distinct responses of RGC subsets. We conclude that neurite growth in vitro is extensively promoted by neurotrophins in developing RGCs. Moreover, highly homologous neurotrophins such as BDNF and NT-4, presumably activating via TrkB receptors, selectively control the differentiation of distinct ganglion cell neuritic morphologies.     

Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and trigeminal axons

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in vitro sensitivity of trigeminal axons to neurotrophins and target-derived diffusible factors and now report on these properties for geniculate axons. GDNF, BDNF, and NT-4, but not NT-3 or NGF, stimulate geniculate axon outgrowth during the ages investigated, embryonic days 12-14. Sensitivity to effective neurotrophins is developmentally regulated and different from that of the trigeminal ganglion. In vitro coculture studies revealed that geniculate axons were repelled by branchial arch explants that were previously shown to be repellent to trigeminal axons (Rochlin and Farbman [1998] J Neurosci 18:6840-6852). In addition, some branchial arch explants and untransfected COS7 cells repelled geniculate but not trigeminal axons. Sema3A, a ligand for neuropilin-1, is effective in repelling geniculate and trigeminal axons, and antineuropilin-1, but not antineuropilin-2, completely blocks the repulsion by arch explants that repel axon outgrowth from both ganglia. Sema3A mRNA is concentrated in branchial arch epithelium at the appropriate time to mediate the repulsion. In Sema3A knockout mice, geniculate and trigeminal afferents explore medial regions of the immature tongue and surrounding territories not explored in heterozygotes, supporting our previous hypothesis that Sema3A-based repulsion mediates the early restriction of sensory afferents away from midline structures.     

Peripheral target-specific influences on embryonic neurite growth vigor and patterns

We examined axon–target interactions in cocultures of embryonic rat trigeminal, dorsal root, nodose, superior cervical ganglia or retina with a variety of native or foreign peripheral targets such as the whisker pad, forepaw, and heart explants. Axon growth into these peripheral target tissues was analyzed by the use of lipophilic tracer DiI. Embryonic day 15 dorsal root and trigeminal axons grew into isochronic normal and foreign cutaneous targets. Both axon populations avoided the same age heart tissue, but grew profusely into younger (embryonic day 13) or older (postnatal) heart explants. In contrast, embryonic day 15 superior cervical or nodose ganglion axons grew heavily into the same age heart and forepaw explants and to a lesser extent into the whisker pad explants. Embryonic day 15 retinal axons grew into all three peripheral targets used in this study. Primary sensory and sympathetic axons, but not retinal axons, formed target-specific patterns in the whisker pad and forepaw explants. DiI-labeling and immunostaining of primary sensory neurons in coculture revealed that these neurons retain their bipolar characteristics, and express class-specific markers such as parvalbumin, calcitonin gene-related peptide and TrkA receptors. In the whisker pad explants, axons positive for all three markers were seen to form patterns around the follicles. Our results indicate that developing peripheral targets can attract and support axon growth from a variety of sources. Whereas neurotrophins play a major role in attracting and supporting survival of subpopulations of sensory neurons, other substrate-bound or locally released molecules must regulate sensory neurite growth into specific peripheral and central targets. J. Comp. Neurol. 399:427–439, 1998. © 1998 Wiley-Liss, Inc.     

Epidermal growth factor receptor inhibitors promote CNS axon growth through off-target effects on glia

Administration of epidermal growth factor receptor (EGFR) inhibitors (e.g. AG1478/PD168393) promotes central nervous system (CNS) axon regeneration in vivo by an unknown mechanism. Here, we show that EGFR activation is not required for AG1478-/PD168393-induced neurite outgrowth in cultures of dorsal root ganglion neurons (DRGN) with added inhibitory CNS myelin extract (CME), but is mediated by the paracrine and autocrine actions of the glia-/neuron-derived neurotrophins (NT) NGF, BDNF and NT-3 through Trk signalling in DRGN potentiated by elevated cAMP levels. The DRGN neurite growth seen in CME-inhibited cultures treated with AG1478 is eradicated by blocking Trk signalling but undiminished after siRNA knockdown of > 90% EGFR. Moreover, addition of the combined triplet of NT restores neurite outgrowth in CME-inhibited cultures, when cAMP levels are raised. Accordingly, we suggest that chemical EGFR inhibitors act independently of EGFR, inducing glia and neurons to secrete NT and raising cAMP levels in DRG cultures, leading to Trk-dependent disinhibited DRGN neurite outgrowth.     

Excess nerve growth factor in the periphery does not obscure development of whisker-related patterns in the rodent brain

We have addressed the issue of whether or not peripherally expressed nerve growth factor (NGF) influences the formation of whisker-specific patterns in the brain by regulating the survival of sensory neurons. Transgenic mice that overexpress an NGF cDNA in the skin were examined. In these animals, excess NGF expression is controlled by promoter and enhancer sequences of a keratin gene, thus restricting the higher levels of NGF expression to basal keratinocytes of the epidermis. Twice the number of trigeminal sensory neurons survive in transgenic mice as in normal animals, and a corresponding hyperinnervation of the whisker pad is noted, both around the vibrissa follicles and along the intervibrissal epidermis. However, the increased survival of sensory neurons and the enhanced peripheral projections do not interfere with the development of whisker-specific patterns in the trigeminal brainstem, in the ventrobasal thalamic complex or in the face-representation region of the primary somatosensory (SI) cortex. These results demonstrate that vibrissa-related central patterns are able to form in the virtual absence of trigeminal ganglion cell death and suggest that mechanisms other than a selective elimination of sensory neurons control the development of whisker-specific neural patterns in the brain. © 1996 Wiley-Liss, Inc.     

Axonal transport of neurotrophins by visceral afferent and efferent neurons of the vagus nerve of the rat

The receptor-mediated axonal transport of [¹²⁵I]-labeled neurotrophins by afferent and efferent neurons of the vagus nerve was determined to predict the responsiveness of these neurons to neurotrophins in vivo. [¹²⁵I]-labeled neurotrophins were administered to the proximal stump of the transected cervical vagus nerve of adult rats. Vagal afferent neurons retrogradely transported [¹²⁵I]neurotrophin-3 (NT-3), [¹²⁵I]nerve growth factor (NGF), and [¹²⁵I]neurotrophin-4 (NT-4) to perikarya in the ipsilateral nodose ganglion, and transganglionically transported [¹²⁵I]NT-3, [¹²⁵I]NGF, and [¹²⁵I]NT-4 to the central terminal field, the nucleus tractus solitarius (NTS). Vagal afferent neurons showed minimal accumulation of [¹²⁵I]brain-derived neurotrophic factor (BDNF). In contrast, efferent (parasympathetic and motor) neurons located in the dorsal motor nucleus of the vagus and nucleus ambiguus retrogradely transported [¹²⁵I]BDNF, [¹²⁵I]NT-3, and [¹²⁵I]NT-4, but not [¹²⁵I]NGF. The receptor specificity of neurotrophin transport was examined by applying [¹²⁵I]-labeled neurotrophins with an excess of unlabeled neurotrophins. The retrograde transport of [¹²⁵I]NT-3 to the nodose ganglion was reduced by NT-3 and by NGF, and the transport of [¹²⁵I]NGF was reduced only by NGF, whereas the transport of [¹²⁵I]NT-4 was significantly reduced by each of the neurotrophins. The competition profiles for the transport of NT-3 and NGF are consistent with the presence of TrkA and TrkC and the absence of TrkB in the nodose ganglion, whereas the profile for NT-4 suggests a p75 receptor-mediated transport mechanism. The transport profiles of neurotrophins by efferent vagal neurons in the dorsal motor nucleus of the vagus and nucleus ambiguus are consistent with the presence of TrkB and TrkC, but not TrkA, in these nuclei. These observations describe the unique receptor-mediated axonal transport of neurotrophins in adult vagal afferent and efferent neurons and thus serve as a template to discern the role of specific neurotrophins in the functions of these visceral sensory and motor neurons in vivo. J. Comp. Neurol. 393:102–117, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US government work and, as such, is in the public domain in the United States of America.

     

Neurotrophin-3, but not nerve growth factor, promotes survival of human intestinal mast cells

Neurotrophins are potent regulators of neuronal cell survival and function. Nerve growth factor (NGF) was shown to reduce apoptosis in cord blood-derived mast cells. Here, we examined the effect of the neurotrophins NGF and neurotrophin (NT)-3 on survival and mediator release of human intestinal mast cells. Mast cells isolated from normal intestinal tissue were cultured in the presence of NGF, NT-3, or stem cell factor (SCF) alone or in the presence of SCF together with each neurotrophin. NGF or NT-3 alone did not promote mast cell survival. In contrast, mast cell recovery was increased twofold when mast cells were cultured with NT-3 in addition to SCF for 14 days compared with control. Mast cell recovery was further increased following a combined addition of NT-3, SCF and IL-4. NT-3 mediated mast cell growth was dependent on the primary receptor for NT-3 TrkC. NGF in combination with SCF or with SCF and IL-4 showed no effect on mast cell survival. Histamine release and histamine content per mast cell remained unchanged, whereas leukotriene C4 release decreased if mast cells were cultured with NGF or NT-3 in addition to SCF. In summary, NT-3 affects mature human mast cells by promoting mast cell survival, whereas NGF does not.     

Neurotrophins alter the numbers of neurotransmitter-ir mature vagal/glossopharyngeal visceral afferent neurons in vitro

Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10–100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.     

Distinct usages of phospholipase C gamma and Shc in intracellular signaling stimulated by neurotrophins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrophin family, bind to and activate TrkA, TrkB and TrkC, respectively, members of the Trk receptor tyrosine kinase family, to exert various effects including promotion of differentiation and survival, and regulation of synaptic plasticity in neuronal cells. Many reports have suggested that different neurotrophins show distinct biological functions, although molecular mechanisms by which neurotrophins exert their different functions remain unclear. In the present study, we found distinct usages of phospholipase Cgamma (PLCgamma) and Shc in intracellular signaling stimulated by neurotrophins. BDNF stimulated much stronger interactions of PLCgamma with Trk than NGF and NT-3 in PC12 cells stably expressing TrkB and cultured cerebral cortical neurons, respectively, although BDNF, NGF and NT-3 induced similar levels of tyrosine phosphorylation of Trk. Furthermore, the cultured cortical neurons showed large PLCgamma-dependent increases in intracellular Ca(2+) levels in response to BDNF compared with NT-3. In Shc signaling, NGF, but not BDNF, displayed interactions between Trk and Shc in a phenylarsine oxide (PAO; an inhibitor of tyrosine phosphatase)-dependent manner in TrkB-expressing PC12 cells. These results indicated that neurotrophins stimulate distinct kinds of interactions between Trk and PLCgamma and between Trk and Shc. These differences may lead to the distinct biological functions of neurotrophins.     

Neurotrophins alter the numbers of neurotransmitter-ir mature vagal/glossopharyngeal visceral afferent neurons in vitro

Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10-100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.     

Expression of neurotrophins and the low-affinity NGF receptor in septal and hippocampal reaggregate cultures: local physiologic effects of NGF synthesized in the septal region.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of a family of trophic factors designated the neurotrophins, each of which can bind to the low-affinity NGF receptor (LNGFR). To investigate the mechanisms that regulate the expression of the neurotrophins and the LNGFR in the developing brain, we grew cells from the embryonic mouse septum and hippocampus in reaggregating cell culture and compared neurotrophin and LNGFR expression in developing reaggregates with that seen in the developing septum and hippocampus in situ. NGF, BDNF, NT-3 and LNGFR were each expressed in septal and hippocampal reaggregates as well as the native septum and hippocampus. Additionally, the temporal expression profiles observed in reaggregates were generally similar to those seen in the respective brain regions in situ. In order to determine whether NGF can modulate neurotrophin or LNGFR expression, reaggregates were cultured in the continual presence of either exogenous NGF or anti-NGF antibodies. NGF-treated septal cultures expressed twice the level of LNGFR mRNA as was seen in untreated septal cultures; on the other hand, septal cultures grown in the presence of anti-NGF antibodies, to neutralize endogenously synthesized NGF, displayed a 3-fold decrease in LNGFR mRNA expression compared to untreated cultures. No effects of NGF or anti-NGF were observed on LNGFR expression in hippocampal reaggregates, or on neurotrophin mRNA expression in either reaggregate type. These results suggest that regulatory mechanisms intrinsic to the septal and hippocampal regions control neurotrophin and LNGFR expression. NGF is likely to be one of these regulatory cues since it acts locally in septal reaggregates to control the developmental expression of LNGFR mRNA. The possible roles of locally synthesized NGF and other neurotrophins in the development of septal neurons are discussed.     

Nerve growth factor and neurotrophin-3 modulate the rabies infection of adult sensory neurons in primary cultures

With the aim of determining if the proportion of rabies virus (RV)-infected adult neurons from dorsal root ganglion are affected by in vitro treatment with different neurotrophins, experiments using Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) or Neurotrophin-3 (NT-3) as supplements for cells in culture were performed. Cultures treated with three different concentrations of each of the neurotrophins mentioned were infected with Challenge Virus Standard RV strain. An indirect immunoperoxidase technique was performed for the detection and counting of infected cells. NGF (2 ngml(-1) and 10 ngml(-1)) and NT-3 (1 ngml(-1) and 5 ngml(-1)) induced a significant reduction of infected neurons. None of the cultures treated with BDNF showed changes in the percentage of infected neurons. Likewise, the proportion of infected non-neuronal cells (Schwann cells and fibroblasts) was not altered by the treatment with neurotrophins. In addition, morphometric analysis of total and virus-immunoreactive neurons in culture were carried out, the neurotrophin treatment induced variations in the profile of neurons preferentially infected, since cell diameters in the infected cell population are different in the presence of NGF and NT-3. Data presented here could indicate a putative participation of neurotrophin receptor or biochemical modifications induced by neurotrophin treatment that affect the infection. The primary culture of dorsal root ganglion cells from adult mice is a very useful model for studying the basic phenomena of the RV-neuron interaction.     

Neuronal signals regulate neurotrophin expression in oligodendrocytes of the basal forebrain

Previous studies suggest that oligodendrocytes express trophic molecules, including neurotrophins. These molecules have been shown to influence nearby neurons. To determine whether neuronal signals may, in turn, affect oligodendrocyte-derived trophins, we examined regulation of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNA expression in cultured oligodendrocytes of the basal forebrain. Neuronal signals had distinct effects on individual neurotrophins. KCl elicited increases in BDNF mRNA, but did not affect expression of NGF or NT-3. The cholinergic agonist, carbachol, increased expression of NGF, but did not affect expression of BDNF or NT-3. Glutamate elicited a decrease in BDNF, but did not affect expression of NGF or NT-3. This glutamate effect is not due to toxicity, since the number of total cells was unchanged, while the number of mature myelin basic protein positive (MBP+) cells increased. Our observations suggest that individual neuronal signals distinctly influence the trophic function of oligodendrocytes.     

Expression of neurotrophins and Trk receptors in the avian retina

Using the RNase protection assay, we have found that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are expressed in the avian retina during development. The expression peaks around embryonic days 12–15, with decreasing levels at later stages of development. Abundant levels of NGF and BDNF but low levels of NT-3 mRNA were found in the adult retina. We also found that light/darkness regulated the levels of NGF and BDNF mRNAs but not the levels of NT-3 mRNA in the 5-day-old chicken retina. It was demonstrated that NGF and BDNF mRNA levels were up-regulated by light exposure. The cellular localization of mRNA expression for the neurotrophins and neurotrophin receptors TrkA, TrkB, and TrkC in the retina was studied using in situ hybridization. The patterns of NGF and trkA mRNA expression were very similar and were localized to the external part of the inner nuclear layer on the border with the outer plexiform layer and corresponded to the localization of horizontal cells. NT-3 labeling was also found over the external part of the inner nuclear layer, whereas trkC mRNA was found over all layers in the retina. BDNF labeling was found over all layers in the retina, whereas TrkB labeling was intense over cells in the ganglion cell layer, which is in agreement with the response of ganglion cells to BDNF stimulation. Functional neurotrophin receptors were suggested by the response of retinal explants to neurotrophin stimulation. These data indicate that the neurotrophins play local roles in the retina that involve interactions between specific neuronal populations, which were identified by the localization of the Trk receptor expression. The data also suggest that NGF and BDNF expression is regulated by normal neuron usage in the retina. © 1996 Wiley-Liss, Inc.