M. Egle De Stefano

Subpopulations of rat dorsal root ganglion neurons express active vesicular acetylcholine transporter

The vesicular acetylcholine transporter (VAChT) is a transmembrane protein required, in cholinergic neurons, for selective storage of acetylcholine into synaptic vesicles. Although dorsal root ganglion (DRG) neurons utilize neuropeptides and amino acids for neurotransmission, we have previously demonstrated the presence of a cholinergic system. To investigate whether, in sensory neurons, the vesicular accumulation of acetylcholine relies on the same mechanisms active in classical cholinergic neurons, we investigated VAChT presence, subcellular distribution, and activity. RT‐PCR and Western blot analysis demonstrated the presence of VAChT mRNA and protein product in DRG neurons and in the striatum and cortex, used as positive controls. Moreover, in situ hybridization and immunocytochemistry showed VAChT staining located mainly in the medium/large‐sized subpopulation of the sensory neurons. A few small neurons were also faintly labeled by immunocytochemistry. In the electron microscope, immunolabeling was associated with vesicle‐like elements distributed in the neuronal cytoplasm and in both myelinated and unmyelinated intraganglionic nerve fibers. Finally, [3H]acetylcholine active transport, evaluated either in the presence or in the absence of ATP, also demonstrated that, as previously reported, the uptake of acetylcholine by VAChT is ATP dependent. This study suggests that DRG neurons not only are able to synthesize and degrade ACh and to convey cholinergic stimuli but also are capable of accumulating and, possibly, releasing acetylcholine by the same mechanism used by the better known cholinergic neurons.

Dystrophin Stabilizes α3- But Not α7-Containing Nicotinic Acetylcholine Receptor Subtypes at the Postsynaptic Apparatus in the Mouse Superior Cervical Ganglion

The nicotinic acetylcholine receptor (nAChR) subtypes were characterized in the superior cervical ganglion (SCG) of wild-type and dystrophin-lacking mdx mice. The binding of Epibatidine and alphaBungarotoxin, ligands for alpha3- and alpha7-containing receptors, respectively, revealed, for each ligand, a single class of high-affinity binding sites, with similar affinity in both wild-type and mdx mice. The Epibatidine-labeled receptors were immunoprecipitated by antibodies against the alpha3, beta2, and beta4 subunits. Immunocytochemistry showed that the percentage of alpha3-, beta2-, and beta4- but not of alpha7-immunopositive postsynaptic specializations was significantly lower in mdx than in wild-type mouse SCG. These observations suggest that the mouse SCG contains nAChRs, stabilized by dystrophin, in which the alpha3 subunit is associated with the beta2 and/or beta4 subunits. Conversely, dystrophin is not involved in the stabilization of the alpha7-containing nAChRs, as the percentage of alpha7-immunopositive synapses is similar in both wild-type and mdx mouse SCG.

Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics

Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

Lack of dystrophin leads to the selective loss of superior cervical ganglion neurons projecting to muscular targets in genetically dystrophic mdx mice

Autonomic imbalance is a pathological aspect of Duchenne muscular dystrophy. Here, we show that the sympathetic superior cervical ganglion (SCG) of mdx mice, which lack dystrophin (Dp427), has 36% fewer neurons than that of wild-type animals. Cell loss occurs around P10 and affects those neurons innervating muscular targets (heart and iris), which, differently from the submandibular gland (non-muscular target), are precociously damaged by the lack of Dp427. In addition, although we reveal altered axonal defasciculation in the submandibular gland and reduced terminal sprouting in all SCG target organs, poor adrenergic innervation is observed only in the heart and iris. These alterations, detected as early as P5, when neuronal loss has not yet occurred, suggest that in mdx mice the absence of Dp427 directly impairs the axonal growth and terminal sprouting of sympathetic neurons. However, when these intrinsic alterations combine with structural and/or functional damages of muscular targets, neuronal death occurs.

Somatostatin immunoreactivity in quail pterygopalatine ganglion

In the ciliary ganglion of the chicken and quail, somatostatin (SOM) is an exclusive marker for parasympathetic postganglionic neurons innervating the choroid. A second parasympathetic pathway projecting to the choroid originates from the pterygopalatine ganglion. The aim of this study was to investigate SOM immunoreactivity in the pterygopalatine ganglion of the Japanese quail (Coturnix coturnix japonica) and on neurons within the choroid, the intrinsic choroidal neurons (ICN). We did so using immunohistochemistry and subsequent light, electron and confocal laser scanning microscopy. Pterygopalatine neurons were characterized by nNOS‐immunohistochemistry or NADPH‐diaphorase cytochemistry. SOM immunoreactivity was absent in the perikarya, but neurons were densely surrounded by SOM‐positive nerve fibres. Electron microscopy revealed that these fibres formed contacts with and without membrane specializations on pterygopalatine neurons. In the choroid, neuronal nitric‐oxide synthase (nNOS)‐immunoreactive ICN were likewise closely apposed by SOM‐immunoreactive nerve fibres, as revealed by confocal microscopy. There was no detectable co‐localization of the markers. In the absence of tracing studies, it is open to speculation whether SOM immunoreactivity originates from preganglionic fibres of the superior salivatory nucleus, postganglionic fibres of the ciliary ganglion or fibres of the brainstem via as yet unknown pathways. SOM may regulate the production of NO in pterygopalatine neurons and ICN, respectively, and is therefore involved in neuronal circuits regulating ocular homeostasis.

M2 muscarinic receptor activation regulates schwann cell differentiation and myelin organization

Glial cells express acetylcholine receptors. In particular, rat Schwann cells express different muscarinic receptor subtypes, the most abundant of which is the M2 subtype. M2 receptor activation causes a reversible arrest of the cell cycle. This negative effect on Schwann cell proliferation suggests that these cells may possibly progress into a differentiating program. In this study we analyzed the in vitro modulation, by the M2 agonist arecaidine, of transcription factors and specific signaling pathways involved in Schwann cell differentiation. The arecaidine‐induced M2 receptor activation significantly upregulates transcription factors involved in the promyelinating phase (e.g., Sox10 and Krox20) and downregulates proteins involved in the maintenance of the undifferentiated state (e.g., c‐jun, Notch‐1, and Jagged‐1). Furthermore, arecaidine stimulation significantly increases the expression of myelin proteins, which is accompanied by evident changes in cell morphology, as indicated by electron microscopy analysis, and by substantial cellular re‐distribution of actin and cell adhesion molecules. Moreover, ultrastructural and morphometric analyses on sciatic nerves of M2/M4 knockout mice show numerous degenerating axons and clear alterations in myelin organization compared with wild‐type mice. Therefore, our data demonstrate that acetylcholine mediates axon‐glia cross talk, favoring Schwann cell progression into a differentiated myelinating phenotype and contributing to compact myelin organization.

Components of the NGF signaling complex are altered in mdx mouse superior cervical ganglion and its target organs.

We previously reported that in the superior cervical ganglion (SCG) of dystrophic mdx mice, which lack full-length dystrophin, there is a loss of neurons projecting to SCG muscular targets, like the iris. Nonetheless, surviving neurons, innervating either iris or submandibular gland (SuGl), a SCG non-muscular target, underwent reduced axon defasciculation and terminal branching. Here we report that, during early post-natal development, levels of pro-apoptotic proNGF in mdx mouse iris, but not in the SuGl, are higher than in the wild-type. This increase, along with reduced levels of NGF receptors (TrkA and p75NTR) in SCG, may be partly responsible for the observed loss of neurons projecting to the iris. These alterations, combined with a reduction in polysialylated-NCAM and neurofilament protein levels in SCG, may also account for reduced axon defasciculation and terminal branching in mdx mouse SCG targets.

Involvement of the plasminogen enzymatic cascade in the reaction to axotomy of rat sympathetic neurons

Axotomy of superior cervical ganglion (SCG) neurons is characterized by peripheral regeneration of injured axons and temporary disassembly of the intraganglionic synapses, necessary for synaptic silencing. Both events require remodeling of the extracellular matrix achieved through controlled proteolysis of its components by different enzymatic systems. In this study, we investigate the involvement of the plasminogen enzymatic cascade in the response to axotomy of rat SCG neurons. All components of this proteolytic pathway, tissue plasminogen activator (tPA), plasminogen, membrane receptor annexin II and tPA inhibitor (PAI-1), are constitutively expressed in uninjured SCG and increase significantly after SCG neuron axotomy. Immunolocalization of plasminogen, the key protein converted into the enzymatically active plasmin by tPA, in both neurons and non-neuronal cells indicates that all cell types are involved in the response to axotomy. The time course of activation of tPA/plasmin enzymatic pathway suggests its involvement in both intraganglionic synapse remodeling and axonal regeneration.

The rise in cytoplasmic ubiquitin levels is an early step in the response of parasympathetic ganglionic neurons to axonal injury followed by regeneration

We investigated the involvement of ubiquitin in the neuronal response to axonal injury in the quail parasympathetic ciliary ganglion by immuno-light and electron microscopy. Image analysis of immunoreacted cryosections shows that ubiquitin- immunoreactivity in the ciliary neurons increases significantly 6 hours after postganglionic nerve crush. The immunolabeling reaches a peak 1 day after injury and begins to decrease between days 3 and 6 when, in contrast to the cytoplasm, numerous highly eccentric nuclei are strongly immunolabeled. Electron microscopy shows ubiquitin-immunorcactivity associated with cytoplasmic organdies and with several postsynaptic densities of the numerous synapses established by the preganglionic boutons on the soma of the ciliary neurons. The number of immunopositive postsynaptic densities increases significantly 1 day after axonal damage, followed by temporary detachment of the preganglionic boutons from the injured neurons between days 3 and 6. The early increase in cytoplasmic ubiquitin-immunoreactivity suggests a prompt ubiquitination of damaged proteins addressed to degradation, while the nuclear immunolabeling may reflect high histone ubiquitination, a process involved in keeping chromatin transcriptionally active. The possible ubiquitin-mediated removal of postsynaptic apparatus constituents such as ACh receptors, proteins involved in their clustering and stabilization, and/or adhesion molecules may be a crucial step for the detachment of the preganglionic boutons, thus favoring injury-induced synaptic plasticity.