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An increasing number of rare mutations linked to autism spectrum disorders have been reported in genes encoding for proteins involved in synapse formation and maintenance, such as the post-synaptic cell adhesion proteins neuroligins. Most of the autism-linked mutations in the neuroligin genes map on the extracellular protein domain. The autism-linked substitution R451C in Neuroligin3 (NLGN3) induces a local misfolding of the extracellular domain, causing defective trafficking and retention of the mutant protein in the endoplasmic reticulum (ER). The activation of the unfolded protein response (UPR), due to misfolded proteins accumulating in the ER, has been implicated in pathological and physiological conditions of the nervous system. It was previously shown that the over-expression of R451C NLGN3 in a cellular system leads to the activation of the UPR. Here, we have investigated whether this protective cellular response is detectable in the knock-in mouse model of autism endogenously expressing R451C NLGN3. Our data showed up-regulation of UPR markers uniquely in the cerebellum of the R451C mice compared to WT littermates, at both embryonic and adult stages, but not in other brain regions. Miniature excitatory currents in the Purkinje cells of the R451C mice showed higher frequency than in the WT, which was rescued inhibiting the PERK branch of UPR. Taken together, our data indicate that the R451C mutation in neuroligin3 elicits UPR in vivo, which appears to trigger alterations of synaptic function in the cerebellum of a mouse model expressing the R451C autism-linked mutation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.

We demonstrated previously in the quail ciliary ganglion, that the immunoreactivity for the neural cell adhesion molecule labeling the postsynaptic specializations of intraganglionic synapses decreases when synaptic remodeling is induced by crushing the postganglionic ciliary nerves. Here we show, in the same experimental conditions, that the immunolabeling for its polysialylated non-stabilizing isoform, which promotes cell plasticity, increases at these subcellular compartments. In control ganglia, poor immunolabeling for the polysialylated neural cell adhesion molecule was occasionally observed surrounding the soma of the ciliary neurons, in correspondence with the calyciform presynaptic ending and the perineuronal satellite cells sheath. At the electron microscope, several neuronal compartments, including some postsynaptic specializations, somatic spines and multivesicular bodies, were immunopositive. Three to six days after ciliary nerve crush, both the number of ciliary neurons labeled for the polysialylated neural cell adhesion molecule and the intensity of their immunolabeling increased markedly. Electron microscopy revealed that, in parallel to the injury-induced detachment of the preganglionic boutons, numerous postsynaptic specializations were found to be immunopositive. Twenty days later, when intraganglionic connections were re-established, polysialylated neural cell adhesion molecule immunoreactivity was comparable to that observed in control ganglia. The increase in immunolabeling also involved the other neuronal compartments mentioned above, the perineuronal satellite cells and the intercellular space between these and the ciliary neurons. From these results we suggest that the switch, at the postsynaptic specializations, between the neural cell adhesion molecule and its polysialylated form may be among the molecular changes occurring in axotomized neurons leading to injury-induced synaptic remodeling. Moreover, from the increase in polysialylated neural cell adhesion molecule immunolabeling, observed at the somatic spines and at the interface between neurons and perineuronal satellite cells, we suggest that this molecule may be involved not only in synaptic remodeling, but also in other more general aspects of injury-induced neuronal plasticity.

Our previous data suggested that in mouse sympathetic superior cervical ganglion (SCG) the dystrophin-dystroglycan complex may be involved in the stabilization of the nicotinic acetylcholine receptor (nAChR) clusters. Here we used SCG of dystrophic mdx mice, which express only the shorter isoforms of dystrophin (Dys), to investigate whether the lack of the full-length dystrophin (Dp427) could affect the localization of the dystroglycan and the alpha3 nAChR subunit (alpha3AChR) at the postsynaptic apparatus. We found a selective reduction in intraganglionic postsynaptic specializations immunopositive for alpha3AChR and for alpha- and beta-dystroglycan compared with the wild-type. Moreover, in mdx mice, unlike the wild-type, the disassembly of intraganglionic synapses induced by postganglionic nerve crush occurred at the slower rate and was not preceded by the loss of immunoreactivity for Dys isoforms, beta-dystroglycan, and alpha3AChR. These data indicate that the absence of Dp427 at the intraganglionic postsynaptic apparatus of mdx mouse SCG interferes with the presence of both dystroglycan and nAChR clusters at these sites and affects the rate of synapse disassembly induced by postganglionic nerve crush. Moreover, they suggest that the decrease in ganglionic nAChR may be one of the factors responsible for autonomic imbalance described in Duchenne muscular dystrophy patients.

Postganglionic nerve crush of the avian ciliary ganglion induces detachment of preganglionic terminals from the soma of the injured ciliary neurons, followed by reattachment at about the same time that the postganglionic axons regenerate to their targets. In order to determine the role played by cell adhesion molecules in this response, we have studied injury-induced changes in the amount and distribution of N-cadherin and neural cell adhesion molecule, together with modifications in the expression of their messenger RNAs. Both N-cadherin and neural cell adhesion molecule immunoreactivities associated with postsynaptic specializations decreased between one and three days following postganglionic nerve crush, preceding the detachment of the preganglionic boutons. Immunoreactivities subsequently increased between 13 and 20 days, in parallel with restoration of synaptic contacts on the ganglion cells and the progressive reinnervation of the peripheral targets. In contrast to the rapid decrease in immunoreactivity, the messenger RNA levels of N-cadherin and neural cell adhesion molecule both increased after crush, and remained elevated throughout the 20-day period of the experiment. These results are consistent with roles for N-cadherin and neural cell adhesion molecule in the maintenance of synaptic contacts. The rapid regulation of these proteins in injury-induced synaptic plasticity occurs at the post-transcriptional level, whereas longer term regulation associated with the re-establishment of synapses may be promoted by the increased levels of gene expression.

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-immunoreactivity associated with cytoplasmic organelles 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.

In mouse sympathetic superior cervical ganglion (SCG), cortical cytoskeletal proteins such as dystrophin (Dys) and beta1sigma2 spectrin colocalize with beta-dystroglycan (beta-DG), a transmembrane dystrophin-associated protein, and the acetylcholine receptor (AChR) at the postsynaptic specialization. The function of the dystrophin-dystroglycan complex in the organization of the neuronal cholinergic postsynaptic apparatus was studied following changes in the immunoreactivity of these proteins during the disassembly and subsequent reassembly of the postsynaptic specializations induced by axotomy of the ganglionic neurons. After axotomy, a decrease in the number of intraganglionic synapses was observed (t1/2 8 h 45'), preceded by a rapid decline of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3 AChR subunit (alpha3AChR) (t1/2 3 h 45', 4 h 30' and 6 h, respectively). In contrast, the percentage of postsynaptic densities immunopositive for beta1sigma2 spectrin remained unaltered. When the axotomized neurons began to regenerate their axons, the number of intraganglionic synapses increased, as did that of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3AChR. The latter number increased more slowly than that of Dys and beta-DG. These observations suggest that in SCG neurons, the dystrophin-dystroglycan complex might play a role in the assembly-disassembly of the postsynaptic apparatus, and is probably involved in the stabilization of AChR clusters.

The distribution of muscarinic acetylcholine receptors was investigated by immuno-light and electron microscopy in the chick dorsal root ganglion during embryonic development (E12 and E18) and after hatching. The monoclonal antibody we used recognizes the acetylcholine binding site shared by all five muscarinic acetylcholine receptor subtypes. At E12, light microscopy reveals several immunopositive neurons with variable degrees of immunolabeling, heterogeneously distributed throughout the ganglion. Later in development and after hatching, the intensity of immunolabeling seems to decrease and the immunopositive neurons, of the small-medium-sized type, are located mostly in the medio-dorsal region of the ganglion. Under the electron microscope, the immunoreaction is associated with the Nissl bodies, budding Golgi cisterns and, especially at E12, with discrete loci along the neuronal plasma membrane. Unmyelinated nerve fibers, in both central and peripheral branches, are also immunopositive, suggesting that muscarinic acetylcholine receptors are transported towards the spinal cord and the periphery, respectively. A large number of perineuronal satellite cells and both myelinating and unmyelinating Schwann cells are intensely labeled. These observations, combined with previous data on the pharmacological and functional characterization of muscarinic acetylcholine receptors in the avian dorsal root ganglion, suggest that both sensory neurons and non-neuronal cells are able to respond to acetylcholine stimuli. Since muscarinic acetylcholine receptor-immunoreactivity is restricted to the small-medium-sized neurons and their unmyelinated fibers, of the nociceptive type, we suggest that these receptors are involved in modulating the transduction of noxious stimuli from the periphery.

In normal mouse superior cervical ganglion, dystrophin immunoreactivity is present in ganglionic neurons, satellite cells and Schwann cells. It is associated with several cytoplasmic organelles and specialized plasma membrane domains, including two types of structurally and functionally different intercellular junctions: synapses, where it is located at postsynaptic densities, and adherens junctions. Dystrophin immunostaining can be ascribed to the 427,000 mol. wt full-length dystrophin, as well as to the several dystrophin isoforms present in superior cervical ganglion, as revealed by western immunoblots. In mdx mouse superior cervical ganglion, which lacks the 427,000 mol. wt dystrophin, the unchanged pattern of dystrophin immunolabelling observed at several subcellular structures indicates the presence of dystrophin isoforms at these sites. Moreover, the absence of labelled adherens junctions indicates the presence of full-length dystrophin at this type of junction in the normal mouse superior cervical ganglion. The lower number of immunopositive postsynaptic densities in mdx mouse superior cervical ganglion than in normal mouse ganglion suggests the presence, in the latter, of postsynaptic densities with differently organized dystrophin cytoskeleton: some containing dystrophin isoforms alone or together with 427,000 mol. wt dystrophin, and others containing 427,000 mol. wt dystrophin alone.

Purpose: To clarify the fine structure of the avian choroid and thus help explain the mechanisms for normal and abnormal eye function and growth., Methods: Eyes from normal chickens and from experimental chickens subjected to unilateral paracentesis were fixed either by perfusion or in situ, with or without post-fixation by microwave irradiation, and then processed for light and electron microscopic analysis., Results: The avian choroid contains thin-walled lacunae, whose fine structure is identical to that of lymphatic vessels. The lacunae are much smaller toward the anterior chamber and the Schlemm's canal than posteriorly in the eye bulb. Large lacunae are situated primarily in the suprachoroidea, and their blind-ended capillary branches enter the choriocapillaris and the walls of large veins. The walls of the large veins contain villous structures that protrude into their lumina and are penetrated by thin lacunar branches and by side lines of the venous lumen. In normal chickens, the lacunae usually are devoid of blood cells. After paracentesis of the anterior eye chamber, the lacunae become filled with erythrocytes on the side that was operated on, but not on the contralateral side., Conclusions: The authors propose that the lacunae of the avian choroid represent a system of posterior short lymphatic vessels, which drain intraocular fluids directly into the eye's venous system, and that the villous structures are sites of communication between lacunae and veins. The demonstration of a choroidal lymphatic system opens new insights into the processes of fluid removal, control of intraocular pressure, and regulation of choroidal thickness in the avian eye under normal and experimental conditions.

The response to postganglionic nerve crush and Ricinus toxin administration by the ciliary neurons of the quail ciliary ganglion was investigated at the ultrastructural level. The toxin was either applied at the crush site on the postganglionic nerves or injected into the anterior eye chamber without any other operative intervention. Crush of postganglionic nerves without toxin administration and saline injection into the anterior eye chamber served as controls for the two toxin administration procedures. Postganglionic nerve crush caused a distinct chromatolytic reaction, accompanied by massive detachment of the preganglionic axon terminals from the ciliary neurons and loss of most of the synapses, both chemical and electrical. This process does not induce cell death and is reversible. Saline injection in the anterior eye chamber caused a moderate retrograde reaction in some of the ciliary neurons, presumably as a consequence of paracentesis. The changes consisted mainly of an increase of perikaryal neurofilaments with, at most, a minor detachment of the preganglionic boutons from a small portion of the cell body at the nuclear pole. Ricinus toxin administration induced neuronal degeneration following a pattern common to both delivery modes. The degenerative process consisted of disruption and detachment of polyribosomes from the rough endoplasmic reticulum, an increase of smooth cisterns and tubules, a dramatic increase of neurofilament bundles, compartmentalization of the cytoplasmic organelles and, finally, karyorrhexis and cell lysis. The final stages of Ricinus toxin degeneration involve a progressive accumulation of extracellular flocculo-filamentous material and cell lysis. After administration of Ricinus toxin to the crush site, ricin-affected neurons showed withdrawal of the preganglionic boutons from a portion of the ciliary neuron, especially at the nuclear pole. After Ricinus toxin injection into the anterior eye chamber, however, the bouton shell surrounding the affected ciliary neurons remained intact in the early stages of degeneration. Detachment of the preganglionic terminals and disruption of the cell junctions, therefore, is the consequence of nerve crush and not of the toxin itself. This study demonstrated that quail ciliary neurons are a suitable model for experimental neuropathology and neurotoxicology.

The effects of Ricinus toxin on the neurons of the ciliary ganglia were investigated in the quail. The neuronal death and the morphological alterations of the ganglionic cells were assessed following injection of the toxin in the anterior chamber of the eye or after application of the toxin on the postganglionic nerves at a crush site. A 45% loss of choroid neurons without loss of ciliary neurons was observed after postganglionic nerve crush alone. Injection of the toxin in the anterior chamber of the eye led to a selective loss of ciliary neurons (38%). Application of the toxin to the crushed postganglionic nerves led to a loss from both neuronal populations (40% of total neurons). This work indicates that different procedures result in selective lesion of the different neuronal populations in the ciliary ganglion.

The distribution of acetylcholinesterase and of two neuropeptide (substance P and calcitonin gene-related peptide) immunoreactivities has been investigated in sensory neurons of lumbosacral dorsal root ganglia during chick embryo development, combining immunolocalization of neuropeptides with simultaneous histochemical detection of acetylcholinesterase, in order to study co-localization of the two peptides and their relations with acetylcholinesterase. Acetylcholinesterase at E7 of development appears in only a few neurons, usually the larger ones located in the lateroventral region of the ganglia. As development proceeds the number of neurons and intensity of staining increase. Until E12-13 acetylcholinesterase positivity is limited to the region of the ganglion containing larger neurons. At later stages (E20) it spreads progressively, leading to staining of cells over the whole ganglion. Substance P-like immunoreactivity appears at E6 and for calcitonin gene-related peptide at E7. These immunoreactivities progressively increase with development, remaining limited to the small neuron compartment of the dorsomedial region of the ganglion. Immunoreactivity for both neuropeptides reaches a maximum around E10-13 and then declines. Using simultaneous double immunostaining, calcitonin gene-related peptide and substance P-like immunoreactivities are largely co-localized, although their distribution is not completely coincident. Neuropeptide-positive cells are usually devoid of any acetylcholinesterase activity until E15. They become positive for the enzyme at later stages. The significance of acetylcholinesterase expression in sensory neurons and the possible relation of its appearance and neuron size is discussed.