Nardilysin controls cardiac sympathetic innervation patterning through regulation of p75 neurotrophin receptor.

Cardiac sympathetic innervation is critically involved in the regulation of circulatory dynamics. However, the molecular mechanism for the innervation patterning has remained elusive. Here, we demonstrate that nardilysin (NRDC, Nrdc), an enhancer of ectodomain shedding, regulates cardiac sympathetic innervation. Nardilysin-deficient (Nrdc-/- ) mice show hypoplastic hearts, hypotension, bradycardia, and abnormal sympathetic innervation patterning. While the innervation of left ventricle (LV) of wild-type mice is denser in the subepicardium than in the subendocardium, Nrdc-/- LV lacks such a polarity and is uniformly and more abundantly innervated. At the molecular level, the full-length form of p75 neurotrophin receptor (p75NTR , Ngfr) is increased in Nrdc-/- LV due to the reduced ectodomain shedding of p75NTR . Importantly, the reduction of p75NTR rescued the abnormal innervation phenotype of Nrdc-/- mice. Moreover, sympathetic neuron-specific, but not cardiomyocyte-specific deletion of Nrdc recapitulated the abnormal innervation patterning of Nrdc-/- mice. In conclusion, neuronal nardilysin critically regulates cardiac sympathetic innervation and circulatory dynamics via modulation of p75NTR .

Nogo-66 Restricts Synaptic Strengthening via Lingo1 and the ROCK2-Cofilin Pathway to Control Actin Dynamics.

Nogo-A restricts long-term potentiation (LTP) at the Schaffer collateral-CA1 pathway in the adult hippocampus via 2 extracellular domains: Nogo-A-Δ20 and Nogo-66. Nogo-66 signals via Nogo Receptor 1 (NgR1) to regulate synaptic function. Whether the NgR1 coreceptors Lingo1 and p75NTR are involved in the signaling in this context is still not known. Moreover, the intracellular cascade mediating the activity of Nogo-66 in restricting LTP is unexplored. We combine electrophysiology and biochemistry in acute hippocampal slices and demonstrate that a loss of function for Lingo1 results in a significant increase in LTP levels at the Schaffer collateral-CA1 pathway, and that Lingo1 is the NgR1 coreceptor mediating the role of Nogo-66 in restricting LTP. Our data show that p75NTR is not involved in mediating the Nogo-66 effect on LTP. Moreover, loss of function for p75NTR and NgR1 equally attenuate LTD, suggesting that p75NTR might mediate the NgR1-dependent regulation of LTD, independently of Nogo-66. Finally, our results indicate that Nogo-66 signaling limits LTP via the ROCK2-Cofilin pathway to control the dynamics of the actin cytoskeleton. The present results elucidate the signaling pathway activated by Nogo-66 to control LTP and contribute to the understanding of how Nogo-A stabilizes the neural circuits to limit activity-dependent plasticity events in the mature hippocampus.

Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-ß signaling and astrocyte functions.

Astrocytes modulate neuronal activity and inhibit regeneration. We show that cleaved p75 neurotrophin receptor (p75(NTR)) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of transforming growth factor (TGF)-ß signaling. Cleaved p75(NTR) interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75(NTR) controls astrocyte-neuronal communication in response to profibrotic factors.

p75 neurotrophin receptor regulates glucose homeostasis and insulin sensitivity.

Insulin resistance is a key factor in the etiology of type 2 diabetes. Insulin-stimulated glucose uptake is mediated by the glucose transporter 4 (GLUT4), which is expressed mainly in skeletal muscle and adipose tissue. Insulin-stimulated translocation of GLUT4 from its intracellular compartment to the plasma membrane is regulated by small guanosine triphosphate hydrolases (GTPases) and is essential for the maintenance of normal glucose homeostasis. Here we show that the p75 neurotrophin receptor (p75(NTR)) is a regulator of glucose uptake and insulin resistance. p75(NTR) knockout mice show increased insulin sensitivity on normal chow diet, independent of changes in body weight. Euglycemic-hyperinsulinemic clamp studies demonstrate that deletion of the p75(NTR) gene increases the insulin-stimulated glucose disposal rate and suppression of hepatic glucose production. Genetic depletion or shRNA knockdown of p75(NTR) in adipocytes or myoblasts increases insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, overexpression of p75(NTR) in adipocytes decreases insulin-stimulated glucose transport. In adipocytes, p75(NTR) forms a complex with the Rab5 family GTPases Rab5 and Rab31 that regulate GLUT4 trafficking. Rab5 and Rab31 directly interact with p75(NTR) primarily via helix 4 of the p75(NTR) death domain. Adipocytes from p75(NTR) knockout mice show increased Rab5 and decreased Rab31 activities, and dominant negative Rab5 rescues the increase in glucose uptake seen in p75(NTR) knockout adipocytes. Our results identify p75(NTR) as a unique player in glucose metabolism and suggest that signaling from p75(NTR) to Rab5 family GTPases may represent a unique therapeutic target for insulin resistance and diabetes.

Long-distance control of synapse assembly by target-derived NGF.

We report a role for long-distance retrograde neurotrophin signaling in the establishment of synapses in the sympathetic nervous system. Target-derived NGF is both necessary and sufficient for formation of postsynaptic specializations on dendrites of sympathetic neurons. This, in turn, is a prerequisite for formation of presynaptic specializations, but not preganglionic axonal ingrowth from the spinal cord into sympathetic ganglia. We also find that NGF-TrkA signaling endosomes travel from distal axons to cell bodies and dendrites where they promote PSD clustering. Furthermore, the p75 neurotrophin receptor restricts PSD formation, suggesting an important role for antagonistic NGF-TrkA and p75 signaling pathways during retrograde control of synapse establishment. Thus, in addition to defining the appropriate number of sympathetic neurons that survive the period of developmental cell death, target-derived NGF also exerts control over the degree of connectivity between the spinal cord and sympathetic ganglia through retrograde control of synapse assembly.

The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo.

Oligomeric forms of amyloid-beta (Abeta) are thought to play a causal role in Alzheimer's disease (AD), and the p75 neurotrophin receptor (p75(NTR)) has been implicated in Abeta-induced neurodegeneration. To further define the functions of p75(NTR) in AD, we examined the interaction of oligomeric Abeta(1-42) with p75(NTR), and the effects of that interaction on neurite integrity in neuron cultures and in a chronic AD mouse model. Atomic force microscopy was used to ascertain the aggregated state of Abeta, and fluorescence resonance energy transfer analysis revealed that Abeta oligomers interact with the extracellular domain of p75(NTR). In vitro studies of Abeta-induced death in neuron cultures isolated from wild-type and p75(NTR-/-) mice, in which the p75(NTR) extracellular domain is deleted, showed reduced sensitivity of mutant cells to Abeta-induced cell death. Interestingly, Abeta-induced neuritic dystrophy and activation of c-Jun, a known mediator of Abeta-induced deleterious signaling, were completely prevented in p75(NTR-/-) neuron cultures. Thy1-hAPP(Lond/Swe) x p75(NTR-/-) mice exhibited significantly diminished hippocampal neuritic dystrophy and complete reversal of basal forebrain cholinergic neurite degeneration relative to those expressing wild-type p75(NTR). Abeta levels were not affected, suggesting that removal of p75(NTR) extracellular domain reduced the ability of excess Abeta to promote neuritic degeneration. These findings indicate that although p75(NTR) likely does not mediate all Abeta effects, it does play a significant role in enabling Abeta-induced neurodegeneration in vitro and in vivo, establishing p75(NTR) as an important therapeutic target for AD.

Developmental axon pruning mediated by BDNF-p75NTR-dependent axon degeneration.

The mechanisms that regulate the pruning of mammalian axons are just now being elucidated. Here, we describe a mechanism by which, during developmental sympathetic axon competition, winning axons secrete brain-derived neurotrophic factor (BDNF) in an activity-dependent fashion, which binds to the p75 neurotrophin receptor (p75NTR) on losing axons to cause their degeneration and, ultimately, axon pruning. Specifically, we found that pruning of rat and mouse sympathetic axons that project to the eye requires both activity-dependent BDNF and p75NTR. p75NTR and BDNF are also essential for activity-dependent axon pruning in culture, where they mediate pruning by directly causing axon degeneration. p75NTR, which is enriched in losing axons, causes axonal degeneration by suppressing TrkA-mediated signaling that is essential for axonal maintenance. These data provide a mechanism that explains how active axons can eliminate less-active, competing axons during developmental pruning by directly promoting p75NTR-mediated axonal degeneration.

Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor.

Alzheimer's disease is characterized by the accumulation of neurotoxic amyloidogenic peptide Abeta, degeneration of the cholinergic innervation to the hippocampus (the septohippocampal pathway), and progressive impairment of cognitive function, particularly memory. Abeta is a ligand for the p75 neurotrophin receptor (p75(NTR)), which is best known for mediating neuronal death and has been consistently linked to the pathology of Alzheimer's disease. Here we examined whether p75(NTR) is required for Abeta-mediated effects. Treatment of wild-type but not p75(NTR)-deficient embryonic mouse hippocampal neurons with human Abeta(1-42) peptide induced significant cell death. Furthermore, injection of Abeta(1-42) into the hippocampus of adult mice resulted in significant degeneration of wild-type but not p75(NTR)-deficient cholinergic basal forebrain neurons, indicating that the latter are resistant to Abeta-induced toxicity. We also found that neuronal death correlated with Abeta(1-42) peptide-stimulated accumulation of the death-inducing p75(NTR) C-terminal fragment generated by extracellular metalloprotease cleavage of full-length p75(NTR). Although neuronal death was prevented in the presence of the metalloprotease inhibitor TAPI-2 (tumor necrosis factor-alpha protease inhibitor-2), Abeta(1-42)-induced accumulation of the C-terminal fragment resulted from inhibition of gamma-secretase activity. These results provide a novel mechanism to explain the early and characteristic loss of cholinergic neurons in the septohippocampal pathway that occurs in Alzheimer's disease.

Regulation of sympathetic neuron differentiation by endogenous nerve growth factor and neurotrophin-3.

Nerve growth factor (NGF) and neurotrophin-3 (NT3) play distinctive roles in sympathetic axon growth and target field innervation and are required for sympathetic neuron survival in vivo. To ascertain if these neurotrophins selectively regulate the expression of genes that determine the functional characteristics of differentiated sympathetic neurons, we measured the mRNA levels for several such genes in the superior cervical ganglion of NGF(-/-), NT3(-/-) and wild type mouse embryos at a stage before excessive neuronal loss occurs in the absence of these neurotrophins. Despite the extensively documented ability of NGF to regulate the noradrenergic phenotype of sympathetic neurons, we found that tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) mRNA levels were normal in NGF(-/-) embryos, but significantly reduced in NT3(-/-) embryos. In contrast, the beta2 nicotinic acetylcholine receptor and PACAP receptor 1 mRNA levels were normal in NT3(-/-) embryos, but significantly reduced in NGF(-/-) embryos. Studies of mice lacking neurotrophin receptors suggested that the effects of NGF on gene expression require TrkA whereas those of NT3 require TrkA and p75(NTR). These findings demonstrate that endogenous NGF and NT3 have distinctive and separate effects on gene expression in early sympathetic neurons and that these selective effects on gene expression require a different combination of neurotrophin receptors.

Non-cell-autonomous regulation of GABAergic neuron development by neurotrophins and the p75 receptor.

Basal forebrain GABAergic and cholinergic circuits regulate the activity of cholinergic projections to the cortex and hippocampus. Because these projections influence cortical development and function, the development of basal forebrain excitatory and inhibitory neurons is critical for overall brain development. We show that the neurotransmitter phenotype of these neurons is developmentally regulated by neurotrophins and the p75 receptor. Neurotrophins (nerve growth factor and brain-derived neurotrophic factor) increased the number of both cholinergic and GABAergic neurons in neonatal basal forebrain neuron cultures from the region of the medial septum. However, the p75 receptor is required only for neurotrophin-dependent expansion of the GABAergic, not the cholinergic, population. Neurotrophin-induced GABAergic development can be rescued in p75-/- cultures by expression of a p75 rescue construct in neighboring cells or by treatment with medium collected from neurotrophin-treated wild-type cultures. Because p75 is not expressed in basal forebrain GABAergic neurons, this defines a new, non-cell-autonomous mechanism of p75 action in which ligand binding results in release of a soluble factor that modifies neurotrophin responses of nearby neurons. p75 is also required for the maintenance of basal forebrain GABAergic neurons in vivo, demonstrating that p75-mediated interactions between cholinergic and GABAergic neurons regulate the balance of excitatory and inhibitory components of basal forebrain circuits.

Modulation of semaphorin3A activity by p75 neurotrophin receptor influences peripheral axon patterning.

The p75 neurotrophin receptor (p75(NTR)) interacts with multiple ligands and coreceptors. It is thought to mediate myelin growth inhibition as part of the Nogo receptor complex, in addition to its other roles. Paradoxically, however, peripheral axons of p75(ExonIII-/-) mutant embryos are severely stunted. This inhibition of axon growth may be a result of neurite elongation defects in p75(NTR) mutant neurons. Here, we show that p75(ExonIII-/-) DRG neurons are hypersensitive to the repellent molecule Semaphorin3A (Sema3A). NGF modulates Sema3A activity equally well in both the p75(NTR) mutant and wild-type neurons, indicating that the hypersensitivity of p75(NTR) mutant neurons is probably not related to their NGF receptor activity. Neuropilin1 and p75(NTR) partially colocalize in DRG growth cones. After Sema3A stimulation, the degree of colocalization is dramatically increased, particularly in clusters associated with Sema3A receptor complex activation. Coimmunoprecipitation studies show that p75(NTR) interacts directly with the Sema3A receptors Neuropilin1 and PlexinA4. When coexpressed with both Neuropilin1 and PlexinA4, p75(NTR) reduces the interaction between these two receptor components. Finally, p75(NTR)/Sema3A double-mutant embryos show growth similar to that observed in Sema3A-null mice. These data indicate that p75(NTR) is an important functional modulator of Sema3A activity and that, in the absence of p75(NTR), oversensitivity to Sema3A leads to severe reduction in sensory innervation. Our results also suggest that while inhibition of p75(NTR) in CNS injury may enhance nerve regeneration resulting from the inhibition of myelin-associated protein, it may also inhibit nerve regeneration through its modulation of Sema3A.

Identification of human oral keratinocyte stem/progenitor cells by neurotrophin receptor p75 and the role of neurotrophin/p75 signaling.

This study was undertaken to determine whether human oral keratinocyte stem cells characteristically express higher levels of the low-affinity neurotrophin receptor p75 and to elucidate the function of p75 in oral keratinocytes. Examination of their expression patterns and cell-cycling status in vivo showed that p75 was exclusively expressed in the basal cell layer of both the tips of the papillae and the deep rete ridges. These immunostaining patterns suggest a cluster organization; most p75(+) cells did not actively cycle in vivo. Cell sorting showed that cells in the p75(+) subset were smaller and possessed higher in vitro proliferative capacity and clonal growth potential than the p75(-) subset. Clonal analysis revealed that holoclone-type (stem cell compartment), meroclone-type (intermediate compartment), and paraclone-type (transient amplifying cell compartment) cells, previously identified in skin and the ocular surface, were present in human oral mucosal epithelium. Holoclone-type cells showed stronger p75 expression at both the mRNA and protein level than did meroclone- and paraclone-type cells. Among the several neurotrophins, nerve growth factor (NGF) and neurotrophin-3 stimulated p75(+) oral keratinocyte cell proliferation, and only NGF protected them from apoptosis. Our in vivo and in vitro findings indicate that p75 is a potential marker of oral keratinocyte stem/progenitor cells and that some neurotrophin/p75 signaling affects cell growth and survival.

Progressive hearing loss in mice carrying a mutation in the p75 gene.

The neurotrophin receptor p75 (p75NTR) is expressed in the developmental stage of the cochlea. However, the role of the p75NTR in the inner ear remains to be established. In this study, we conducted electrophysiological and morphological analyses of the auditory function of mice carrying a mutation in the p75 gene at different longitudinal stages. The mice carrying a mutation in the p75 gene showed an age-related progressive hearing loss. At 1 month, there was no obvious morphological change in the cochlea of the mice carrying a mutation in the p75 gene compared to wild-type mice, except for a slight loss of spiral ganglion neurons (SGNs). Auditory function was not significantly different between both genotypes from 1 to up to 4 months of age. The mice carrying a mutation in the p75 gene started to show progressive hearing loss at 4 months, when both SGN degeneration and hair cell (HC) loss were observed at the basal turn. These results suggest that the neurotrophin receptor p75 may play a significant role in the maintenance of cochlear function, and that mice carrying a mutation in the p75 gene could be a good animal model of early onset progressive hearing loss.

Deficient p75 low-affinity neurotrophin receptor expression does alter the composition of cellular infiltrate in experimental autoimmune encephalomyelitis in C57BL/6 mice.

We have shown earlier that induction of experimental autoimmune encephalomyelitis (EAE)-a model for the human disease multiple sclerosis-in C57BL/6 wild-type mice resulted in the expression of the p75 low-affinity neurotrophin receptor (p75NTR) in endothelial cells in the CNS. In comparison to the clinical manifestation of EAE observed in wild-type C57BL/6 mice, C57BL/6 mice deficient for p75NTR (p75NTR knockout mice) developed a more severe or even lethal disease and concomitant increased levels of inflammation in the CNS. In order to elucidate the role of endothelial p75NTR in cellular infiltration under these pathological circumstances, we have performed a more detailed, quantitative examination of the composition of the cellular infiltrate invading the CNS in EAE wild-type and EAE p75NTR knockout mice. We compared spinal cords of EAE wild-type with those of EAE p75NTR knockout mice of the same clinical score (3.5) using immunohistochemical markers for the cell types present in the infiltratory cuffs in EAE: T-cells, B-cells, monocytes, microglia, resident and infiltrating macrophages and polymorphonuclear cells. Interestingly, we detected that the proportion of B-cells, cells of the monocyte-macrophage lineage and polymorphonuclear cells in the infiltratory cuff of EAE-p75NTR knockout mice was decreased at the account of the proportion of T-cells which appeared to be almost doubled in comparison to the EAE wild-type mice. The altered composition of the infiltrate in p75NTR deficient mice argues for an involvement of endothelial p75NTR in the interaction between the inflamed endothelium and the activated cells of the immune system, in particular the T-cells, in EAE.

Null mutations for exon III and exon IV of the p75 neurotrophin receptor gene enhance sympathetic sprouting in response to elevated levels of nerve growth factor in transgenic mice.

Under normal conditions, expression of the p75 neurotrophin receptor (p75NTR) by sympathetic neurons can increase the affinity of the signaling receptor, trkA, to target-derived nerve growth factor (NGF) at distal axons. We have previously reported that sprouting of sympathetic axons into NGF-rich target tissues is enhanced when p75NTR expression is perturbed, leading to the postulate that p75NTR may restrain sympathetic sprouting in response to elevated NGF levels. These observations were made using mice having a null mutation of the third p75NTR exon, a line that may express a hypomorphic form of this receptor. Since mice carrying a null mutation of the fourth p75NTR exon may not express a similar splice variant, we sought to determine whether these animals possess the same phenotype of enhanced sympathetic sprouting in response to elevated levels of NGF. Both lines of transgenic mice lacking p75NTR displayed similar degrees of sympathetic axonal sprouting into the cerebellum and trigeminal ganglia, two target tissues having elevated levels of NGF protein. Furthermore, the densities of sympathetic axons in both targets were significantly greater than those observed in age-matched NGF transgenic siblings expressing full-length p75NTR. Our new findings provide a comparative analysis of the phenotype in two independent mutations of the same neurotrophin receptor, revealing that p75NTR plays an important role in restricting sympathetic sprouting in response to higher NGF levels.

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice.

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons.

Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.

The p75 neurotrophin receptor negatively modulates dendrite complexity and spine density in hippocampal neurons.

The correlation between functional and structural neuronal plasticity is by now well documented. However, the molecular mechanisms translating patterns of neuronal activity into specific changes in the structure of neurons remain unclear. Neurotrophins can be released in an activity-dependent manner, and they are capable of controlling both neuronal morphology and functional synaptic changes. They are thus attractive molecules to be studied in the context of synaptic plasticity. In the CNS, most of the work so far has focused on the role of BDNF and of its tyrosine kinase B receptor (TrkB), but relatively little is known about the function of the pan-neurotrophin receptor p75NTR. In this study, we show in loss-of-function experiments that postnatal hippocampal pyramidal cells in two mutant lines of p75NTR have a higher spine density and greater dendritic complexity than wild-type (WT) mice. Conversely, in a gain-of-function approach, p75NTR overexpression in WT neurons significantly reduces dendritic complexity, as well as spine density in all dendritic compartments. These results show that p75NTR negatively modulates dendritic morphology in adult hippocampal pyramidal neurons and documents a new case of functional antagonism between Trk and p75NTR signaling.

Nerve growth factor rapidly increases muscarinic tone in mouse medial septum/diagonal band of Broca.

Nerve growth factor (NGF) has been implicated in maintaining and regulating normal functioning of the septohippocampal pathway. However, many aspects of its physiological actions and the underlying mechanisms await elucidation. In this study, we investigated the effect of acute NGF exposure on neurons in the mouse medial septum/diagonal band of Broca (MS/DB), focusing on the cholinergic neurons and the subpopulation of noncholinergic neurons that were identified to be putatively GABAergic. We report that MS/DB neurons in a thin slice preparation, when exposed to NGF via bath perfusion, rapidly and indiscriminately increased the rate of spontaneous firing in all MS/DB neurons. However, focal application of NGF to individual MS/DB neurons increased spontaneous firing in cholinergic, but not in the noncholinergic, subpopulation. The NGF-induced effect on cholinergic neurons was direct, requiring activation and signaling via TrkA receptors, which were immunohistochemically localized to the cholinergic neurons in the MS/DB. TrkA receptors were absent in putative GABAergic MS/DB neurons, and blockade of TrkA signaling in these and other noncholinergic neurons had no effect on their firing activity after exposure to NGF. Conversely, methyl scopolamine, blocked the increased firing activity of noncholinergic neurons during bath perfusion of NGF. We propose a cell type-specific mode of action for NGF in the MS/DB. The neurotrophin directly enhances cholinergic neuronal activity in the MS/DB through TrkA-mediated signaling, increasing acetylcholine release and, thus, muscarinic tone. This increase in muscarinic tone, in turn, results in heightened firing activity in noncholinergic MS/DB neurons.