The Neurotrophins BDNF, NT-3 and NT-4/5, But Not NGF, Up-regulate the Cholinergic Phenotype of Developing Motor Neurons

Although developing motor neurons express low-affinity nerve growth factor (NGF) receptors, there is no known biological effect of NGF on developing or adult motor neurons. In this study, we found that, unlike NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) stimulated cholinergic phenotype by increasing choline acetyltransferase (CAT) activity in cultures enriched with embryonic rat motor neurons. Ciliary neurotrophic factor (CNTF) also stimulated CAT activity. The effects of BDNF and NT-4/5 on CAT activity appeared to be synergistic with that of CNTF. Cotreatment with BDNF and NT-3 resulted in an additive effect, suggesting that signal transduction was mediated through different high-affinity receptors tyrosine kinases B and C (Trk B and Trk C). However, cotreatment with BDNF and NT-4/5 did not result in an increase in CAT activity greater than that of either BDNF or NT-4/5 alone, suggesting that their effects were mediated via the same receptor Trk B. Supporting our findings that spinal cholinergic neurons are responsive to trophic actions of members of the neurotrophin family, motor neuron-enriched cultures were found to express mRNA for Trk B and Trk C, which have been identified as high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively.     

Neurotrophin-induced trk receptor phosphorylation and cholinergic neuron response in primary cultures of embryonic rat brain neurons.

Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.     

Highly selective effects of nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3 on intact and injured basal forebrain magnocellular neurons

Cholinergic neurons of the basal nucleus complex (BNC) respond to nerve growth factor (NCF), the first member of a polypeptide gene family that also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), NGF, BDNF, and NT-3 are enriched in hippocampus. In addition, NGF and, more recently, BDNF have been shown to stimulate the cholinergic differentiation and enhance the survival of BNC cells in vitro. The present investigation was designed to test, in a comparative fashion, the in vivo effects of human recombinant NGF, BDNF, and NT-3 with confirmed activities in vitro on cholinergic and γ-aminobutyric acid (GABA)-ergic BNC neurons. The specific questions asked were whether and, to what extent, biologically active recombinant neurotrophins stimulate the transmitter phenotypes of intact cholinergic and GABAergic neurons of the BNC, and whether, and to what extent, recombinant neurotrophins protect the transmitter phenotypes of axotomized cholinergic and GABAergic neurons of the BNC following complete transections of the fimbria-fornix (measured by ChAT mRNA hybridization). Our results confirm the profound stimulatory and p^75NGFR expression in both intact and axotomized cholinergic neurons and to exert minor effects on some cholinergic markers (e.g., ChAT immunoreactivity). NT-3 had no influence on GABAergic neurons. Taken together, these results indicate that, despite their significant sequence homologies and their shared abundance in target fields of BNC neurons, NGF, BDNF, and NT-3 show striking differences in their efficacies as cholinergic trophic factors. GABAergic neurons of the BNC are resistant to neurotrophins. The result of the present investigation establish that NGF excels among neurotrophins as a trophic factor for intact and injured basal forebrain cholinergic neurons. © 1994 Wiley-Liss, Inc.     

Enhanced survival and morphological features of basal forebrain cholinergic neurons in vitro: role of neurotrophins and other potential cortically derived cholinergic trophic factors

The present study examined survival- and growth-enhancing effects of cortical cells on basal forebrain cholinergic neurons (BFCNs) in culture and the degree to which endogenous nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) contribute to those trophic effects. When fetal (17 days of gestation) basal forebrain (BF) cells were grown for 5 days in coculture with cortical neurons, staining for acetylcholinesterase (AChE) showed a threefold increase in the number of BFCNs relative to BF cultures without cortex. Most of these labeled cells also displayed enhanced somatic, dendritic, and axonal growth. Coculturing cortical neurons with BF cells taken from postnatal animals produced similar results but with a somewhat greater degree of morphologic enhancement. Function-neutralizing antibodies to NGF, BDNF, and NT-3 were employed to determine whether they would block the trophic effects of cortical neurons on postnatal BFCNs. Although no significant changes in numbers or morphological features of AChE(+) neurons were observed with treatment with individual antibodies, cocultures treated with a combination of all three antibodies displayed fewer morphologically enhanced AChE(+) cells and more nonenhanced cells; the total number of AChE(+) neurons was not significantly changed. Treatment of pure BF cultures with exogenous NGF, BDNF, and NT-3 increased the number of AChE(+) neurons but did not reproduce the morphologic enhancement of cortical cells on BFCNs. These results suggest that neurotrophins by themselves can increase survival of postnatal BFCNs in culture and may work in concert with other unknown cortically derived factors to enhance BFCN morphologic differentiation. The unidentified cortical factors may also have strong survival-enhancing effects on BFCNs that are independent of the known neurotrophins.     

NGF, BDNF and NT-5, but not NT-3 protect against MPP toxicity and oxidative stress in neonatal animals

A growing body of evidence suggests that neurotrophic factors can protect neurons against neuronal death. In the present study we examined whether systemic administration of members of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 5 (NT-5) and basic fibroblast growth factor (bFGF) could protect against 1-methyl-4-phenylpyridinium (MPP⁺) induced striatal damage in neonatal rats. Systemic administration of NGF, BDNF and NT-5 produced significant neuroprotective effects, whereas NT-3 was ineffective. Systemic administration of bFGF had significant neuroprotective effects as assessed by T₂-weighted magnetic resonance imaging and measurements of n-acetylaspartate and lactate using chemical shift magnetic resonance imaging. Systemic administration of NGF, BDNF and bFGF, but not NT-3 attenuated MPP⁺ induced increases in hydroxyl radical generation as assessed by the conversion of salicylate to 2,3- or 2,5-dihydroxybenzoic acid (DHBA). These results show that systemic administration of several neurotrophins and bFGF can attenuate neuronal damage induced by chemical hypoxia in vivo by a mechanism which may involve attenuation of oxidative stress.     

Synergistic effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on cultured basal forebrain cholinergic neurons from postnatal 2-week-old rats.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, and ciliary neurotrophic factor (CNTF), a member of the neurocytokine family, are known to have synergistic effects on motoneurons, but such synergistic effect has not been studied in detail especially in the brain. In the present study, we examined the synergistic effects of BDNF and CNTF on the survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. Although BDNF is well-known to promote the survival of basal forebrain cholinergic neurons in P2w culture, CNTF had little effect on the survival of choline acetyltransferase (ChAT)-positive neurons and did not increase ChAT activity in the culture. However, CNTF enhanced BDNF-mediated promotion of cell survival of cholinergic neurons when added concomitantly. BDNF alone induced only a three-fold increase in ChAT activity in control cultures, but the concomitant addition of CNTF resulted in an eight-fold increase. CNTF did not enhance BDNF-mediated cell survival of total neurons from the basal forebrain, hippocampus or cerebellum, suggesting that the synergistic effects of CNTF on the BDNF-mediated increase of viability might be strong in basal forebrain cholinergic neurons. CNTF also enhanced the neurotrophin-4/5-mediated increase of ChAT activity, but not the nerve growth factor (NGF)-mediated one. Furthermore, the BDNF-mediated increase was also enhanced by leukemia inhibitory factor but not by interleukin-6. Similar synergistic pattern between neurotrophins and cytokines were also observed in the induction of ChAT activity in embryonic basal forebrain culture. These results suggest that TrkB, a functional high-affinity receptor of BDNF and NT-4/5, and LIFR beta, a receptor component contained in CNTF and LIF receptor complex, might be involved in the observed synergistic effects.     

The NeurotroDhins BDNF, NT-3 and NT-4/5 Promote Survival and Morphological and Biochemical Differentiation of Striatal Neurons In Vitro

The neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT-4/5) and nerve growth factor (NGF), were compared for their effects on the survival and differentiation of embryonic rat striatal neurons grown in low-density cultures. Treatment with BDNF for 8 days resulted in a 40% increase in overall neuronal survival, a 3- to 5-fold increase in the number of calbindin-immunoreactive neurons, and an 80% increase in GABA-positive neurons. Treatment with NT-3 or NT-4/5 produced a 2- to 3-fold increase in the number of calbindin-positive neurons and an increase in GABA-positive cell number similar to that induced by BDNF. BDNF treatment produced a striking morphological differentiation of striatal GABAergic neurons, which was characterized by a doubling of the number of neurite branch points, the total area of arborization and the perikaryal area compared to control cultures. All three of these factors increased high-affinity GABA uptake 2-fold. NGF had no effect on any of the parameters examined. Our results show that BDNF, NT-3 and NT-4/5 promote the survival and/or differentiation of calbindin-immunopositive and GABAergic striatal neurons.     

Differential regulation of neurotrophin expression in basal forebrain astrocytes by neuronal signals

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3) promote the function and/or survival of basal forebrain (BF) cholinergic neurons in vivo and in culture. The neurotrophin source is commonly thought to be targets of cholinergic neurons and the possibility that local glial sources support cholinergic neurons has not been well examined. These sources, however, may be critical to BF neurons before or even after they reach their targets. We investigated neurotrophin expression in BF astrocytes and its regulation by neural signals. Solution hybridization and immunocytochemical assays revealed that NGF, BDNF, and NT(3) mRNA and proteins were expressed in cultured BF astrocytes. To investigate roles of neuronal signals in neurotrophin regulation, effects of K(+), glutamate, and the cholinergic agonist carbachol were examined. These stimuli affected neurotrophin expression differentially. KCl increased BDNF mRNA but did not alter NGF or NT(3) mRNA. The effect was blocked by nifedipine, suggesting that it was mediated by L-type voltage-dependent calcium currents. Carbachol also increased BDNF mRNA levels without changing NGF or NT(3). Effects were blocked by the muscarinic antagonist, atropine. In contrast, glutamate increased both NGF and BDNF mRNA. NT(3) mRNA again was unaffected. The metabotropic agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) reproduced glutamate effects, whereas kainate or N-methyl-D-aspartate (NMDA) plus glycine did not. Lack of antagonism by ionotropic antagonists and blockade of glutamate effects by metabotropic antagonists confirmed metabotropic mediation. We suggest that BF astrocytes are local sources of neurotrophins for BF cholinergic neurons during development and are regulated differentially by specific neuronal signals. Critical neuronal-glial interactions may underlie basal forebrain function.     

Region-specific neurotrophin imbalances in Alzheimer disease: decreased levels of brain-derived neurotrophic factor and increased levels of nerve growth factor in hippocampus and cortical areas

BACKGROUND: Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) are members of the neurotrophin gene family that support the survival of specific neuronal populations, including those that are affected by neurodegeneration in Alzheimer disease (AD). OBJECTIVE: To determine whether neurotrophin protein levels are altered in the AD-affected brain compared with control brains. METHODS: We quantitated protein levels of NGF, BDNF, NT-3, and NT-4/5, and calculated neurotrophin/NT-3 ratios in AD-affected postmortem hippocampus, frontal and parietal cortex, and cerebellum, and compared them with age-matched control tissue (patients with AD/controls: hippocampus, 9/9 cases; frontal cortex, 19/9; parietal cortex, 8/5; and cerebellum, 5/7, respectively). We applied highly sensitive and specific enzyme-linked immunosorbent assays in rapid-autopsy-derived brain tissue (mean+/-SD postmortem interval, 2. 57+/-1.75 h, n=71) to minimize postmortem proteolytic activity. RESULTS: Levels of BDNF were significantly reduced in hippocampus and parietal cortex (P<.001, and P<.01) as well as BDNF/NT-3 ratios in frontal and parietal cortices (P<.05, and P<.01) in the group with AD compared with the control group. Levels of NGF and NGF/NT-3 ratio were significantly elevated in the group with AD compared with the control group in the hippocampus and frontal cortex (P<.001). Levels of NT-4/5 and the NT-4/NT-3 ratio were slightly reduced in hippocampus and cerebellum in the group with AD compared with the control group (P<.05). In contrast, the levels of NT-3 were unchanged in all brain regions investigated. CONCLUSION: Decreased levels of BDNF may constitute a lack of trophic support and, thus, may contribute to the degeneration of specific neuronal populations in the AD-affected brain, including the basal forebrain cholinergic system. Arch Neurol. 2000.     

Synergistic Trophic Actions on Rat Basal Forebrain Neurons Revealed by a Synthetic NGF/BDNF Chimaeric Molecule

Basal forebrain cholinergic neurons, which degenerate in Alzheimer's disease, respond to multiple trophic factors, including the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). This dual responsiveness prompted us to investigate the effects of a synthetic chimaeric molecule, containing the active domains of both NGF and BDNF. The NGF/BDNF chimaeric factor exhibited synergistic actions, and was 100-fold more potent than wild-type BDNF in enhancing survival of cultured dissociated basal forebrain cholinergic neurons. This effect was apparently due to true BDNF/NGF synergy, since addition of the two wild-type trophins simultaneously reproduced the effect of the chimaera. Synergy was selective for neurons which respond to both factors; substantia nigra dopaminergic neurons, which respond to BDNF but not NGF, exhibited no potentiation. The chimaeric factor thus revealed a synergy that may normally occur in the brain, and constitutes a potentially novel therapeutic agent with greater potency than naturally occurring individual trophins.     

Neurotrophin gene expression by cell lines derived from human gliomas

The expression of neurotrophin (NGF, BDNF, and NT-3) mRNAs in 24 cell lines derived from human malignant gliomas was studied by Northern analysis. Widespread expression of neurotrophin genes was found with BDNF being the most abundantly expressed. Nearly all cell lines expressed BDNF, and about two-thirds of the cell lines expressed NGF and NT-3. Half of the cell lines analyzed expressed all three neurotrophins. Secretion of NGF into the medium of several cell lines could be detected by ELISA and a PC12 neurite outgrowth assay. Immuno- and bioactive NGF was isolated from conditioned medium of one cell line. No evidence of expression of the neurotrophin receptors trk and trkB by Northern analysis was found. Receptor crosslinking with radiolabeled cognate ligands failed to detect functional receptors in all but one cell line. In this cell line a receptor complex for BDNF was found that corresponded to truncated trkB receptors that lack the signal transducing tyrosine kinase domain. Neurotrophins did not stimulate mitosis of the glioma cultures. The findings suggest that production of neurotrophins by glioma cells is a general phenomenon, although neurotrophins made by gliomas lacking their receptors may not play an autocrine but rather a paracrine role. © 1993 Wiley-Liss, Inc.     

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 role 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.     

Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro

Different subpopulations of adult primary sensory neurons in the dorsal root ganglia express receptors for different trophic factors, and are therefore potentially responsive to distinct trophic signals. We have compared the effect of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and NT-3, and of glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth in dissociated cultures of sensory neurons from the lumbar ganglia of young adult rats, and attempted to establish subset-specific effects of these trophic factors. We analysed three parameters of neurite growth (percentage of process-bearing neurons, length of longest neurite and total neurite length), which may correlate with particular types of axon growth in vivo, and may therefore respond differently to trophic factor presence. Our results showed that percentage of process-bearing neurons and total neurite length were influenced by trophic factors, whilst the length of the longest neurite was trophic factor independent. Only NGF and GDNF were found to enhance significantly the proportion of process-bearing neurons in vitro. GDNF was more effective than NGF on small, IB4- neurons, which are known to develop GDNF responsiveness early in postnatal development. NGF, and to a much lesser extent GDNF, enhanced the total length of the neurites produced by neurons in culture. BDNF exerted an inhibitory effect on growth, and both BDNF and NT-3 could partially block some of the growth-promoting effects of NGF on specific neuronal subpopulations.     

Distribution of neurotrophin receptors in human palatine tonsils: an immunohistochemical study

Increasing evidence indicates that nerve growth factor (NGF) exerts effects on cells of the immune system, but the possible immunomodulatory effect of other neurotrophins (brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3; and NT-4/5) has not been studied. Neurotrophins act on responsive cells by binding a low-affinity pan-neurotrophin receptor (p75), and more specific high-affinity receptors (gp140trkAA, gp145trkB and gp145trkC considered as preferred signaling transduction receptors for NGF, BDNF and NT-3, respectively). The expression of neurotrophin receptor proteins may be considered, therefore, as a potential indication of neurotrophin activity. In the present study we investigated the distribution of both types of neurotrophin receptors in the human palatine tonsils using immunohistochemical methods. In the follicular germinal centers both lymphocytes and follicular dendritic cells (FDC) displayed gp75 IR, but not IR for trk neurotrophin receptor proteins. gp140trkA-like IR and gp145trkC-like IR were encountered on paracortical interdigitating cells (PIC), and in the high endothelial venule cells. gp145trkB-like IR was found in a cell subpopulation which probably represented macrophages. Present results suggest that NGF, NT-3 and NT-4/5 may act in PIC and indirectly in lymphocytes, whereas BDNF and NT-4/5 could control macrophages. The role of p75 on lymphocytes and FDC and whether trk neurotrophin receptor proteins present in lymphoid tissues are functional receptors for neurotrophins remains to be elucidated.     

Monoamine-activated α2-macroglobulin inhibits choline acetyltransferase of embryonic basal forebrain neurons and reversal of the inhibition by NGF and BDNF but not NT-3

Monoamine-activated α₂-macroglobulin (α₂M) has recently been shown to inhibit the growth and survival of cholinergic neurons of the basal forebrain (Liebl and Koo: J Neurosci Res 35:170–182, 1993). The mechanism of this inhibitory effect is believed to involve the regulation of growth factor activities by α₂M. The objectives of this study are to determine whether monoamine-activated α₂M can inhibit choline acetyltransferase (ChAT) activity of cholinergic basal forebrain neurons, and whether some common neurotrophins in the CNS can reverse the inhibition. This study demonstrates that both methylamine-activated α₂M (MA-α₂M) and serotonin-activated α₂M (5HT-α₂M) can dose-dependently suppress the expression of normal basal levels of ChAT activity in embryonic rat basal forebrain cells in vitro, while normal α₂M has little or no effect. As little as 0.35 μM monoamine-activated α₂M can suppress the ChAT activity, whereas either nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), but not neurotrophin-3 (NT-3), stimulates ChAT expression of these cells. The addition of either NGF or BDNF to the α₂M-suppressed cells can increase ChAT activity back to its normal levels, while NT-3 can not. These results demonstrate that (1) monoamine-activated α₂M is a potent non-cytotoxic inhibitor of the ChAT activity in cholinergic basal forebrain neurons, and (2) NGF and BDNF are capable of not only stimulating the ChAT activity but can also specifically reverse the α₂M inhibition. The potential physiological role of monoamine-activated α₂M and neurotrophins in the degeneration and regeneration of cholinergic neurons is discussed. In addition, we propose that α₂M may serve as an important tool for evaluating the roles of growth factors in the nervous system. © 1994 Wiley-Liss, Inc.     

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.     

Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses,enhanced morphological features,and decreased dependence on nerve growth factor

Prior studies examining the dependence of basal forebrain cholinergic neurons (BFCNs) on nerve growth factor (NGF) for survival have reached differing conclusions depending on the experimental paradigm employed, suggesting the importance of environmental and developmental variables. The present study examined the NGF dependence of BFCNs and modulatory effects of target (cortical) neurons under the controlled conditions of dissociated cell cultures. Initial experiments found BFCNs (identified by using choline acetyltransferase immunocytochemistry) in pure basal forebrain (BF) cultures to be dependent on NGF between the 2nd and 4th week in vitro. During that developmental period, NGF deprivation for 3 days, induced by application of anti-NGF antibody, resulted in degeneration of over 80% of BFCNs, whereas at earlier or later times, BFCNs were largely resistant to NGF deprivation. When BF neurons were plated together with cortical neurons (as dissociated co-cultures), the BFCNs grew neuritic processes (labeled with acetylcholinesterase histochemistry) that appeared to specifically target cortical neurons; electron microscopy revealed that synapses formed between these cells. BFCNs in co-cultures were more resistant to NGF deprivation, were larger, and had much more extensive neuritic growth than BFCNs in pure BF cultures. The resistance of BFCNs to NGF deprivation provided by cortical neurons could be largely reproduced by addition of other trophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; neurotrophin 4/5, NT4/5; or glial-derived neurotrophic factor, GDNF) during NGF deprivation in pure BF cultures. These results suggest that developing BFCNs undergo a critical period requiring trophic influences that may be provided by NGF or other trophic factors, as well as unknown factors derived from cortical neurons. © 1996 Wiley-Liss, Inc.