The induction of neurotrophin and trk receptor mRNA expression during early avian embryogenesis

Nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, designated neurotrophins, are a family of neurotrophic factors, having important functions in the survival of embryonic and adult neuronal subpopulations. Through the trk family of receptors, these neurotrophins utilize phosphotyrosine-mediated signal transduction. We have used RT-PCR to detect the expression of mRNA for the above neurotrophins and their respective receptors, namely trkA, trkB and trkC in embryonic stages 1–8 of chicken development. While trkA and trkC mRNAs were expressed from stage 1 onwards, NGF and NT-3 mRNAs were expressed only at stages 3 and 5, respectively. In contrast, BDNF mRNA was expressed at stage 1, being the only neurotrophin expressed prior to expression of its respective receptor trkB. However, the latter was not expressed until stage 8. These results indicate an earlier expression of some but not all trk proto-oncogenes, suggesting that the two different receptor mRNAs expressed i.e. trkA and trkC in conjunction with BDNF, at stage 1, may act in aspects of very early embryonic development, such as gastrulation. Thereafter, mRNAs for trkB, NGF and NT-3 are expressed reflecting their later action in early embryonic development.     

Developmentally Regulated Expression of mRNA for Neurotrophin High-Affinity (trk) Receptors within Chick Trigeminal Sensory Neurons

To investigate the distribution of neurons within the developing trigeminal sensory system which express mRNA for each of the three known high-affinity neurotrophin receptors (trk, trkB and trkC), we have performed in situ hybridization histochemistry on serial sections through the trigeminal ganglion and trigeminal mesencephalic nucleus at various ages of development using specific antisense oligonucleotide probes. We show that trkC mRNA is first expressed in the chicken embryo at stage 13, in presumptive neurons prior to the formation of the ganglion, that trkB mRNA labelling is initially observed within peripheral neurons slightly later, at stage 19, and that trk mRNA expression is not detectable until around embryonic day 3.5 (stage 21/22). The neurons which exhibit mRNA labelling for each of the high-affinity receptors occupy discrete regions within the ganglion, indicating that the ganglion comprises distinct neuronal subpopulations, each of which has a different capacity to respond to the different neurotrophins. Neurons which express trk mRNA are confined to the proximal region of the ganglion, whereas those which express trkB mRNA and trkC mRNA are located in two distinct regions within the distal aspect and also within the trigeminal mesencephalic nucleus. From the estimation of the number of neurons which exhibit labelling between embryonic days 9 and 18, we determined that the expression of mRNA for the high-affinity receptors changes during embryonic development of the ganglion. This is consistent with the observed differences in the response to neurotrophins in vitro.     

Function and evolution in the NGF family and its receptors.

The gene family of neurotrophins includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Recently, neurotrophin-5 (NT-5), a possible mammalian homologue to NT-4 described in the frog Xenopus, has been cloned in man and rat. The neurotrophins stimulate survival and differentiation of a range of target neurons by binding to cell surface receptors. The structure of NGF has recently been clarified from crystallographic data. The similarities between the different neurotrophins are substantial with the variable regions, giving specificity to each of the family members, being localized to some exposed loop regions. Low-affinity binding (Kd of 10(-9) M) of all tested neurotrophins is mediated via a 75 K glycoprotein (LNGFR) that has been cloned and characterized. A 140 K tyrosine protein kinase encoded by the proto-oncogene trk has been found to bind NGF with high affinity (Kd of 10(-11) M) and to evoke the cellular neurotrophic responses. In addition, a protein encoded by the trk-related gene trkB has been shown to bind BDNF. Recently, a third member of the trk family, trkC, has been cloned and demonstrated to function as a high-affinity receptor for NT-3. The expression of trk and LNGFR mRNA are co-localized in the rat brain to the medial septal nucleus and the nucleus of Broca's diagonal band containing the NGF-responsive magnocellular cholinergic neurons projecting to hippocampus and cerebral cortex. In sharp contrast, the pattern of expression of trkB is widely spread in many areas of the cortex as well as lateral septum. The trkB protein might serve general functions in large areas of the cortex. Site-directed mutagenesis and expression of recombinant chimaeric neurotrophin proteins have made it possible to localize a likely region for the interaction between NGF and the LNGFR. This region could be altered, resulting in the total loss of LNGFR binding by the mutant NGF protein without affecting the binding to the trk receptor which was sufficient for the full biological activity. Cladistic analysis of likely phylogenies within the neurotrophins shows BDNF and NT-4 to be most closely related whereas NGF may be the sister group to NT-3, BDNF, and NT-4. Neurotrophins offer obvious clinical possibilities for treatment of neurodegenerative diseases.     

Distribution and colocalization of NGF and GDNF family ligand receptor mRNAs in dorsal root and nodose ganglion neurons of adult rats

To understand the dependence of primary sensory neurons on neurotrophic factors, we examined the distribution and colocalization of mRNAs for receptors of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) family ligands in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons of adult rats by in situ hybridization (ISH) histochemistry using serial sections. About 35, 10, and 20% of the lumbar DRG neurons expressed trkA, trkB and trkC mRNAs, respectively. Messenger RNA signals for c-ret, a common signaling receptor of GDNF family ligands, were seen in about 60% of DRG neurons, and some of these neurons expressed trkA, trkB, or trkC mRNAs. Most (97%) of the DRG neurons observed were positive to at least one of these four mRNAs. About 50, 20, and 20% of DRG neurons expressed GDNF family receptor alpha1 (GFR alpha1), GFR alpha2, and GFR alpha3 mRNAs, respectively, and most of these neurons were positive to c-ret mRNA. Interestingly, GFR alpha2 and GFR alpha3 mRNA signals were frequently seen in the same neurons, which lack GFR alpha1 mRNA signals. On the other hand, 98% of NG neurons expressed trkB mRNA and 30-40% of NG neurons co-expressed c-ret and GFR alpha1 mRNAs. However, mRNA signals for other receptors (TrkA, TrkC, GFR alpha2, GFR alpha3) were seen in only a few NG neurons. These findings suggest that all the DRG neurons in adult rats depend on at least one of the NGF and GDNF family ligands, and that some DRG neurons depend on two ligands or more. In contrast, NG neurons were suggested to be divided into two major groups; one group depends on brain-derived neurotrophic factor (BDNF)/neurotrophin-4/5 (NT-4/5), and the other depends on both BDNF/NT-4/5 and GDNF.     

Each sensory nerve arising from the geniculate ganglion expresses a unique fingerprint of neurotrophin and neurotrophin receptor genes

Neurons in the geniculate ganglion, like those in other sensory ganglia, are dependent on neurotrophins for survival. Most geniculate ganglion neurons innervate taste buds in two regions of the tongue and two regions of the palate; the rest are cutaneous nerves to the skin of the ear. We investigated the expression of four neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and NT-4, and five neurotrophin receptors, trkA, trkB, trkC, p75, and truncated trkB (Trn-B) in single sensory neurons of the adult rat geniculate ganglion associated with the five innervation fields. For fungiform papillae, a glass pipette containing biotinylated dextran was placed over the target papilla and the tracer was iontophoresed into the target papilla. For the other target fields, Fluoro-Gold was microinjected. After 3 days, geniculate ganglia were harvested, sectioned, and treated histochemically (for biotinylated dextran) or immunohistochemically (for Fluoro-Gold) to reveal the neurons containing the tracer. Single labeled neurons were harvested from the slides and subjected to RNA amplification and RT-PCR to reveal the neurotrophin or neurotrophin receptor genes that were expressed. Neurons projecting from the geniculate ganglion to each of the five target fields had a unique expression profile of neurotrophin and neurotrophic receptor genes. Several individual neurons expressed more than one neurotrophin receptor or more than one neurotrophin gene. Although BDNF is significantly expressed in taste buds, its primary high affinity receptor, trkB, was not prominently expressed in the neurons. The results are consistent with the interpretation that at least some, perhaps most, of the trophic influence on the sensory neurons is derived from the neuronal somata, and the trophic effect is paracrine or autocrine, rather than target derived. The BDNF in the taste bud may also act in a paracrine or autocrine manner on the trkB expressed in taste buds, as shown by others.     

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.     

(±)3,4-methylenedioxymethamphetamine ("ecstasy") treatment modulates expression of neurotrophins and their receptors in multiple regions of adult rat brain

(±)3,4-Methylenedioxymethamphetamine (MDMA), a widely used drug of abuse, rapidly reduces serotonin levels in the brain when ingested or administered in sufficient quantities, resulting in deficits in complex route-based learning, spatial learning, and reference memory. Neurotrophins are important for survival and preservation of neurons in the adult brain, including serotonergic neurons. In this study, we examined the effects of MDMA on the expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their respective high-affinity receptors, tropomyosin receptor kinase (trk)B and trkC, in multiple regions of the rat brain. A serotonergic-depleting dose of MDMA (10 mg/kg × 4 at 2-hour intervals on a single day) was administered to adult Sprague-Dawley rats, and brains were examined 1, 7, or 24 hours after the last dose. Messenger RNA levels of BDNF, NT-3, trkB, and trkC were analyzed by using in situ hybridization with cRNA probes. The prefrontal cortex was particularly vulnerable to MDMA-induced alterations in that BDNF, NT-3, trkB, and trkC mRNAs were all upregulated at multiple time points. MDMA-treated animals had increased BDNF expression in the frontal, parietal, piriform, and entorhinal cortices, increased NT-3 expression in the anterior cingulate cortex, and elevated trkC in the entorhinal cortex. In the nigrostriatal system, BDNF expression was upregulated in the substantia nigra pars compacta, and trkB was elevated in the striatum in MDMA-treated animals. Both neurotrophins and trkB were differentially regulated in several regions of the hippocampal formation. These findings suggest a possible role for neurotrophin signaling in the learning and memory deficits seen following MDMA treatment.     

Developmental expression of neurotrophins and their receptors in postnatal rat ventral midbrain

Neurotrophins are a group of structurally related polypeptides that support the survival, differentiation, and maintenance of neuronal populations that express the appropriate high-affinity neurotrophin receptors. Two members of the neurotrophin family, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been shown to increase the survival of dopaminergic neurons from the ventral midbrain in vitro. Evidence suggests that ventral midbrain neurons might be able to derive support from these trophic factors in vivo through paracrine or autocrine interactions. Both BDNF and NT-3 mRNAs and their receptor mRNAs, trkB and trkC mRNAs, respectively, have been localized to the ventral mesencephalon. However, the relative expression levels of the neurotrophins and their receptor mRNAs throughout ontogeny and in adulthood have not been elucidated. In the present study, the postnatal developmental expression of BDNF, NT-3, trkB, and trkC mRNAs was analyzed via in situ hybridization to gain insight into the possible roles of these factors in vivo. We found that there was a developmental decline in the expression of BDNF and NT-3 mRNAs in the ventral mesencephalon. In contrast, no alterations in the expression of midbrain trkB or trkC mRNAs could be discerned. The present results suggest a role for BDNF and NT-3 in the earlier postnatal developmental events of responsive populations. The continued, albeit lower, expression of the neurotrophins in the ventral mesencephalon in adulthood also suggests a role for these factors in mature neuronal systems.     

Expression of trkB and trkC receptors and their ligands brain-derived neurotrophic factor and neurotrophin-3 in the murine amygdala

The neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their cognate receptors, trkB and trkC, have a variety of physiological brain functions, ranging from cell survival to mechanisms involved in learning and memory and long-term potentiation (LTP). LTP can be induced in the cortex and hippocampus, as well as within the amygdala. However, the role of neurotrophins in amygdalar LTP is largely unknown. Expression patterns of BDNF and NT-3 and their cognate receptors in the adult mouse amygdala have not been analyzed in detail. We have therefore examined the expression of trkB, trkC, BDNF, and NT-3 mRNA and protein in different amygdalar nuclei as well as in the hippocampal areas CA1-CA3 and the dentate gyrus. The distribution pattern of trkB, trkC, BDNF, and NT-3 mRNA in the murine hippocampus is comparable to that seen in rats. Within most amygdalar nuclei, a moderate BDNF mRNA expression was found; however, BDNF mRNA was virtually absent from the central nucleus. No expression of NT-3 mRNA was found within the amygdala, but trkC mRNA-expressing cells were widely distributed within this brain region. trkB mRNA was strongly expressed in the amygdala. Because trkB is expressed in a full-length and a truncated form (the latter form is also expressed by nonneuronal cells), we also investigated the distribution of full-length trkB mRNA-expressing cells and could demonstrate that this version of trkB receptors is also widely expressed in the amygdala. These results can serve as a basis for studies elucidating the physiological roles of these receptors in the amygdala.     

NGF, NT-3 and Trk C mRNAs, but not TrkA mRNA, are upregulated in the paraventricular structures in experimental hydrocephalus

OBJECTS: This study was designed to detect possible alterations in the expression of neurotrophins and trks in kaolin-induced hydrocephalus by in situ hybridization. METHODS AND RESULTS: Sixteen rats were treated by injection of 25 mg kaolin suspended in 0.1 ml of physiological saline into the cisterna magna. Four rats were injected with saline and served as controls. The kaolin-treated rats were divided into two groups studied 1 and 4 weeks after treatment. Rats were anesthetized and killed, and their brains were rapidly dissected and frozen. DNA oligonucleotide probes for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and trkA, trkB, and C were labeled with [(35)S]dATP using terminal deoxyribonucleotidyl transferase for in situ hybridization. Hydrocephalic brains were also classified according to the degree of ventricular enlargement. The results observed were as follows. (1) The medial septal and striatal NGF mRNA levels increased with severity in animals. (2) Hippocampal trkB and BDNF mRNA levels increased with time in animals with moderate ventricular enlargement. (3) Expression of hippocampal trkB, trkC, and NT-3 mRNA increased in animals with moderate ventricular enlargement, while it apparently decreased in the large ventricular enlargement group reaching normal ranges. (4) In the corpus callosum there was an apparent increase in NGF, NT-3 and trkC mRNA, but not in trkA, in hydrocephalic animals. NT-3 EIA confirmed the presence of NT-3 protein increases in corpus callosum. It is therefore possible that simultaneous NGF, NT-3, and trkC receptor upregulation occurred in glial elements of the white matter. CONCLUSIONS: These results demonstrate that neurotrophins and their receptors are overexpressed in many damaged structures of the severely hydrocephalic brain. There were discrepancies in the distribution of NGF and trkA mRNA, and we hypothesize that NGF mRNA in the damaged white matter structure might be due to the reduced availability of other receptors, such as the low-affinity NGF receptors.     

Anatomical evidence supporting the potential for modulation by multiple neurotrophins in the majority of adult lumbar sensory neurons.

Neurotrophins exert effects on sensory neurons through receptor tyrosine kinases (trks) and a common neurotrophin receptor (p75). Quantitative in situ hybridization studies were performed on serial sections to identify neurons expressing single or multiple neurotrophin trk receptor mRNA(s) in adult lumbar dorsal root ganglion (DRG) in order to examine the possibility of multi-neurotrophin modulation of phenotype via different trk receptors or various trk isoforms. Expression of mRNA encoding trkA, trkB, trkC, or p75 is restricted to select subpopulations representing approximately 41%, 33%, 43%, and 79% of DRG neurons, respectively. Colocalization studies reveal that approximately 10% of DRG neurons coexpress trkA and trkB mRNA; 19% coexpress trkA and trkC mRNA; and 18% coexpress trkB and trkC mRNA. Trilocalization of all three trk mRNAs is rare, with approximately 3-4% of neurons in this category. Overall incidence of expression of more than one full length trk mRNA occurs in approximately 40% of DRG neurons, whereas expression of individual trk mRNA is found in approximately 34%. Full length trk receptor mRNA is rarely detected without p75, implicating the latter in neuronal response to neurotrophins. Examination of two full-length isoforms of trkA reveal that they are coexpressed with relative levels of expression positively correlated. TrkC mRNAs corresponding to 14- or 39-amino acid insert isoforms colocalize with the non-insert trkC isoform, but the converse is not necessarily true. The data suggest that substantial subpopulations of adult sensory neurons may be modulated through interactions with multiple neurotrophins, the consequences of which are largely unknown.     

Alterations in neurotrophin and neurotrophin receptor gene expression patterns in the rat central nervous system following perinatal Borna disease virus infection

Infection of newborn rats with Borna disease virus (BDV) leads to persistence in the absence of overt signs of inflammation. BDV persistence, however, causes cerebellar hypoplasia and hippocampal dentate gyrus neuronal cell loss, which are accompanied by diverse neurobehavioral abnormalities. Neurotrophins and their receptors play important roles in the differentiation and survival of hippocampal and cerebellar neurons. We have examined whether BDV can cause alterations in the neurotrophin network, thus promoting neuronal damage. We have used RNase protection assay to measure mRNA levels of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their trkC and trkB receptors, as well as the growth factors insulin-like growth factor I (IGF-1) and basic fibroblast growth factor (bFGF), in the cerebellum and hippocampus of BDV-infected and control rats at different time points p.i. Reduced mRNA expression levels of NT-3, BDNF and NGF were found after day 14 p.i. in the hippocampus, but not in the cerebellum, of newborn infected rats. Three weeks after infection, trkC mRNA expression levels were reduced in both hippocampus and cerebellum of infected rats, whereas decreased trkB mRNA levels were only observed in the cerebellum. Reduced trkC mRNA expression was confined to the dentate gyrus of the hippocampus, as assessed by in situ hybridization. TUNEL assay revealed massive apoptotic cell death in the dentate gyrus of infected rats at days 27 and 33 p.i. Increased numbers of apoptotic cells were also detected in the cerebellar granular layer of infected rats after 8 days p.i. Moreover, a dramatic loss of cerebellar Purkinje cells was seen after day 27 p.i. Our results support the hypothesis, that BDV-induced alterations in neurotrophin systems might contribute to selective neuronal cell death.     

Brain-derived Neurotrophic Factor is a Survival Factor for Cultured Rat Cerebellar Granule Neurons and Protects them Against Glutamate-induced Neurotoxicity

We have studied the effects of different neurotrophins on the survival and proliferation of rat cerebellar granule cells in culture. These neurons express trkB and trkC, the putative neuronal receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) respectively. Binding studies using iodinated BDNF and NT-3 demonstrated that both BDNF and NT-3 bind to the cerebellar granule neurons with a similar affinity of ˜ 2x10^-9 M. The number of receptors per granule cell was surprisingly high, ∼30x10^-4 and 2x 10⁵ for BDNF and NT-3, respectively. Both NT-3 and BDNF elevated c-fos mRNA in the granule neurons, but only BDNF up-regulated the mRNA encoding the low-affinity neurotrophin receptor (p75). In contrast to NT-3, BDNF acted as a survival factor for the granule neurons. BDNF also induced sprouting of the granule neurons and significantly protected them against neurotoxicity induced by high (1 mM) glutamate concentrations. Cultured granule neurons also expressed low levels of BDNF mRNA which were increased by kainic acid, a glutamate receptor agonist. Thus, BDNF, but not NT-3, is a survival factor for cultured cerebellar granule neurons and activation of glutamate receptor(s) up-regulates BDNF expression in these cells.     

Overexpression of Nerve Growth Factor in Skin Increases Sensory Neuron Size and Modulates Trk Receptor Expression

Sensory neuron development and differentiation is dependent on a family of growth factors known as neurotrophins. Neurotrophins modulate neuron development via trk tyrosine kinase receptor proteins trkA, trkB and trkC. To determine how elevated levels of a target-derived neurotrophin might affect neuronal differentiation, we analysed trk expression in the trigeminal ganglion of transgenic mice that overexpressed nerve growth factor (NGF) in the skin. increased levels of NGF caused a five-fold increase in neurons expressing trkA mRNA and a two-fold increase in neurons expressing trkC. In control mice, cell size distributions of neuronal subpopulations expressing each trk mRNA showed the three subpopulations distributed over a narrow, overlapping range. In contrast, cell size distribution in NGF-transgenic mice was significantly divergent due in large part to hypertrophy of trkA neurons and, to a lesser extent, trkC neurons. In addition, we examined neurons that bound the isolectin B4 from Bandeiraea simplicifolia (BS-IB4) because most of these neurons do not express any trk receptor in the adult. There was a significant increase in the size of BS-IB4–positive neurons in transgenic mice; however, there was no increase in their number. These studies indicate that an increased level of target-derived NGF affects the development of sensory neurons that in the adult express trkA or trkC, as well as neurons that do not express trk receptors.     

Colocalization of NGF binding sites, trk mRNA, and low-affinity NGF receptor mRNA in primary sensory neurons: responses to injury and infusion of NGF.

The distributions of mRNAs for the protooncogene trk and the low-affinity NGF receptor (LNGFR) were studied by hybridization with oligonucleotide probes on sections of adult rat primary sensory and sympathetic ganglia. For comparison with high-affinity binding sites, adjacent sections were processed for NGF receptor radioautography. Among neurons in lumbar dorsal root ganglia and trigeminal ganglia, trk mRNA and NGF-binding sites were closely colocalized; this finding together with previous direct evidence in other cell types is taken to indicate that trk protein is an essential component of the high-affinity NGF receptor in adult sensory neurons. In lumbar dorsal root ganglia and trigeminal ganglia, abundant LNGFR mRNA was found in all neurons with strong 125I-NGF labeling and on additional neurons lacking high-affinity NGF-binding sites. The presence of abundant LNGFR in neurons with high-affinity receptors could be the cause and/or consequence of their ability to respond to NGF. Neurons with abundant LNGFR mRNA but few high-affinity NGF-binding sites may have receptors for other members of the neurotrophin family. In nodose ganglia, neurons with high concentrations of LNGFR mRNA greatly outnumbered the small percentage with abundant trk mRNA. Following intrathecal infusion of NGF to otherwise normal dorsal root ganglia, the concentrations of LNGFR mRNA but not those of trk mRNA and NGF-binding sites were increased in NGF-responsive neurons. The usual single normal pattern of frequency histograms of LNGFR labeling indices became bimodal in response to NGF. Concentrations of NGF-binding sites, LNGFR mRNA, and trk mRNA were all decreased by peripheral nerve transection and restored by exogenous NGF, the restoration being complete for LNGFR mRNA and partial for trk mRNA and NGF-binding sites. The data indicate that NGF can regulate both LNGFR and trk mRNAs but do not clarify the possible contribution of the LNGFR protein to high-affinity binding sites.     

Regionally specific effects of BDNF on oligodendrocytes

To define the effects of neurotrophins on oligodendrocytes, we monitored NGF, BDNF and NT-3 actions on basal forebrain (BF) and cortical populations. NGF, BDNF and NT-3 applied to BF oligodendrocytes elicited increases in expression of myelin basic protein (MBP) and enhanced the numbers of MBP+ cells, without affecting total cell numbers. In the cortex, however, while NGF and NT-3 influenced MBP expression, BDNF was without effect. To explore this apparent regional difference in BDNF action, we compared expression of the neurotrophin receptors trkA, trkB and trkC. While BF cells expressed all three trks, cortical cells did not express the full-length BDNF receptor, trkB. Interestingly, in no case was any receptor expressed by all oligodendrocytes, indicating that oligodendrocytes may be heterogeneous within a brain region. The data suggest that BF oligodendrocytes are influenced by BDNF to express MBP and are distinct in this ability from cortical cells.     

BDN F Down-regulates Neurotrophin Responsiveness, TrkB Protein and TrkB mRNA Levels in Cultured Rat Hippocampal Neurons

Regulation of Trk receptors by their ligands, the neurotrophins, was investigated in dissociated cultures of embryonic day 18 rat hippocampal neurons. Cultures were exposed to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NT-4/5 for 24 h upon plating followed by factor washout. As determined by immunohistochemical staining and phosphotyrosine blotting, the functional responses to acute stimulation with BDNF, NT-3 and NT-4/5, including c-Fos induction and phosphorylation of Trk and extracellular signal-regulated kinase (ERK) proteins, were significantly decreased after 6 days in culture by prior exposure to BDNF. As determined by Western and Northern blot analysis respectively, there was a parallel down-regulation of TrkB protein as well as of trkB and trkC mRNA levels in BDNF-pretreated cultures. Exposure to NT-3 or NT-4/5 at the same concentrations as BDNF did not down-regulate any of the measured cellular responses or TrkB protein and/or trkB and trkC mRNA levels. Regulation of hippocampal neuronal TrkB protein does not appear to be just a developmental phenomenon, as infusion of BDNF into the hippocampus of adult rats for 6 days produced an 80% decrease in levels of full-length TrkB protein. We thus show that exposure of hippocampal neurons to BDNF, both in culture and in the adult brain, results in down-regulation of TrkB. At least in vitro , this leads to long-term functional desensitization to BDNF. NT-3 and NT-4/5. as well as down-regulation of trkB and trkC mRNA.