Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line.

Interpretations of steroid hormone actions as slow, nuclear, transcriptional events have frequently been seen as competing against inferences of rapid membrane actions. We have discovered conditions where membrane-limited effects potentiate later transcriptional actions in a nerve cell line. Making use of a two-pulse hormonal schedule in a transfection system, early and brief administration of conjugated, membrane-limited estradiol was necessary but not sufficient for full transcriptional potency of the second estrogen pulse. Efficacy of the first pulse depended on intact signal transduction pathways. Surprisingly, the actions of both pulses were blocked by a classical estrogen receptor (ER) antagonist. Thus, two different modes of steroid hormone action can synergize.

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

On the actions that one nerve cell can have on another: Distinguishing “drivers” from “modulators”

When one nerve cell acts on another, its postsynaptic effect can vary greatly. In sensory systems, inputs from “drivers” can be differentiated from those of “modulators.” The driver can be identified as the transmitter of receptive field properties; the modulator can be identified as altering the probability of certain aspects of that transmission. Where receptive fields are not available, the distinction is more difficult and currently is undefined. We use the visual pathways, particularly the thalamic geniculate relay for which much relevant evidence is available, to explore ways in which drivers can be distinguished from modulators. The extent to which the distinction may apply first to other parts of the thalamus and then, possibly, to other parts of the brain is considered. We suggest the following distinctions: Cross-correlograms from driver inputs have sharper peaks than those from modulators; there are likely to be few drivers but many modulators for any one cell; and drivers are likely to act only through ionotropic receptors having a fast postsynaptic effect whereas modulators also are likely to activate metabotropic receptors having a slow and prolonged postsynaptic effect.

The nerve cell as a unit.

From Journal of Comparative Neurology.

Adipose-derived stem cells enhance peripheral nerve regeneration.

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Neuronal potential of cells in early postnatal rat sciatic nerve.

A population of undifferentiated cells with neuronal potentialities were revealed in rat sciatic nerve. Explant cultures of sciatic nerve were prepared from newborn or early postnatal rat. Cultures were growth in F14 medium supplemented with 10% of fetal calf serum, incubated in a humidified 3% CO2, 97% air atmosphere. Within 2 weeks, refractile cells exhibiting the morphology of neurons were observed in all examined cultures. These cells had ovoid or multipolar refractile cells bodies with extended cytoplasmic processes. The neuronal nature of these cells was confirmed by their immunostaining with specific neuronal markers: neurofilament triplets, neuron-specific enolase, peripherin, microtubule-associated proteins, and brain spectrin. This neuronal population displayed various phenotypes. The CO2 concentration in the incubator plays an important role, since the number of differentiated neurons was lower in cultures incubated in 5% CO2. Since the sciatic nerve is devoid of nerve cell bodies in vivo, we concluded that early postnatal sciatic nerve contains crest cells with neuronal potentialities differentiating into neurons in response to the culture's environmental cues.

Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts.

Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. © 2012 Elsevier Ltd.

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.

Neuronal ceroid-lipofuscinosis, a type of amaurotic family idiocy: clinical and pathological study of four cases

Neuronal ceroid-lipofuscinosis (NCL) is a recent term, proposed for acurate designation of the late-onset types of Amaurotic Family Idiocy (AFI). Histopathology shows ubiquitous intraneuronal accumulation of lipopigments, being the most important factor for characterization of the entity at present time. Biochemical changes and pathogenesis are obscure. NCL is in contrast to the infantile type of AFI (Tay-Sachs disease), in which intraneuronal accumulation of gangliosides (sphingolipids) is due to the well known deficiency of a lysosomal enzyme. The authors report on four cases of NCL, two brothers of the late infantile (Jansky-Bielschowsky) type and a brother and a sister of the juvenile (Spielmeyer-Sjögren) type. One autopsy and three cortical biopsies revealed moderate to severe distention of the neurons by lipopigment, with nerve cell loss, gliosis and cerebral atrophy. Lipopigment was also increased in liver, heart and spleen. The patients were the first in Brazilian literature in whom the storage material was identified as lipopigment by histochemical methods. A brief summary of the clinical features of NCL is presented, and relevant problems are discussed, concerning interpretation of the nature of the storage material, and significance of the disease for gerontological research.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus

AIM: To evaluate effects of pre- and postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. METHODS: Three groups of young Wistar rats (aged 42 d) were studied: normal-fed (N42), protein-deprived (D42), and protein-recovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)-diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy. RESULTS: The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the profile of nerve cell perikarya (ranging from 200 to 400 mu m(2)). CONCLUSION: Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons. (C) 2010 Baishideng. All rights reserved.

Early iron deficiency produces persistent damage to visual tracts in Wistar rats

In this study, morphological changes in the optic nerve were determined by light microscopy in Wistar rats on an iron-deficient diet for 32 days or for 21 days followed by 10 days on an iron-recovery diet. The morphometric findings showed significantly fewer blood vessels and oligodendrocytes in the iron-deficient rats and iron-recovery rats than in the control group, as well as more astrocytes in the iron-recovery rats. Serum iron levels of the iron-deficient rats were significantly lower than those of the controls. On the other hand, iron-recovery rats had normal serum iron levels, but no change in the abnormal morphology of the myelinated axons and morphometric parameters. Our data indicate that iron is necessary for maintenance of the optic nerve cell structure, and morphological damage from iron-deficiency is not easily reverted by iron reposition.

Dorsal root ganglionectomy for the diagnosis of sensory neuropathies. Surgical technique and results

Background: Inflammatory diseases stand out among sensory neuronopathies because, in their active phase, they can be treated with immunosuppressive agents. Immunosuppressive therapy may present severe adverse effects and requires previous inflammatory activity confirmation. Sensory neuronopathies are diagnosed based on clinical and EMG findings. Diagnostic confirmation and identification of inflammatory activity are based on sensory ganglion histopathological examination. We describe the surgical technique used for dorsal root ganglionectomy in patients with clinical/EMG diagnosis of sensory neuronopathies. Methods: The sensory ganglion was obtained from 15 patients through a small T7-T8 hemilaminectomy and foraminotomy to expose the C7 root from its origin to the spinal nerve bifurcation. In 6 patients, the dural cuff supposed to contain the ganglion was resected en bloc; and in 9 patients, the ganglion was obtained through a longitudinal incision of the dural cuff and microsurgical dissection from the ventral and dorsal roots and radicular arteries. All ganglia were histopathologically examined. Results: No ganglion was found in the dural cuff in 2 patients submitted to en bloc removal, and the ganglion was removed in all patients who underwent microsurgical dissection. All but 2 patients that had ganglion examination presented a neuronopathy of nerve cell loss, 3 with mononuclear inflammatory infiltrate. These patients underwent immunosuppressive therapy, and 2 of them presented clinical improvement. No surgical complications were observed. Conclusions: Microsurgical dorsal root ganglionectomy for diagnosing inflammatory sensory ganglionopathies was effective and safe. Although safe, en bloc resection of the proximal dural cuff was not effective for this purpose. (c) 2008 Published by Elsevier Inc.

Distribuição dos receptores da adenosina no plexo mioentérico – músculo longitudinal do íleo de rato: implicações funcionais

A acetilcolina (ACh) é o neurotransmissor mais importante no controlo da motilidade gastrointestinal. A libertação de ACh dos neurónios entéricos é regulada por receptores neuronais específicos (De Man et al., 2003). Estudos prévios demonstraram que a adenosina exerce um papel duplo na libertação de ACh dos neurónios entéricos através da activação dos receptores inibitórios A1 e facilitatórios A2A (Duarte-Araújo et al., 2004). O potencial terapêutico dos compostos relacionados com a adenosina no controlo da motilidade e da inflamação intestinal, levou-nos a investigar o papel dos receptores com baixa afinidade para a adenosina, A2B e A3, na libertação de acetilcolina induzida por estimulação eléctrica nos neurónios mioentéricos. Estudos de imunolocalização mostraram que os receptores A2B exibem um padrão de distribuição semelhante ao do marcador de células gliais (GFAP). No que respeita aos receptores A1 e A3, estes encontram-se distribuídos principalmente nos corpos celulares dos neurónios ganglionares mioentéricos, enquanto os receptores A2A estão localizados predominantemente nos terminais nervosos colinérgicos. Neste trabalho mostrou-se que a modulação da libertação de ACh-[3H] (usando os antagonistas selectivos DPCPX, ZM241385 e MRS1191) é balanceada através da activação tónica dos receptores inibitórios (A1) e facilitatórios (A2A e A3) pela adenosina endógena. O antagonista selectivo dos receptores A2B, PSB603, não foi capaz de modificar o efeito inibitório da NECA (análogo da adenosina com afinidade para receptores A2). O efeito facilitatório do agonista dos receptores A3, 2-Cl-IB MECA (1-10 nM), foi atenuado pelo MRS1191 e pelo ZM241385, os quais bloqueiam respectivamente os receptores A3 e A2A. Contrariamente à 2-Cl-IB MECA, a activação dos receptores A2A pelo CGS21680C, atenuou a facilitação da libertação de ACh induzida pela activação dos receptores nicotínicos numa situação em que a geração do potencial de acção neuronal foi bloqueada pela tetrodotoxina. A localização diferencial dos receptores excitatórios A3 e A2A ao longo dos neurónios mioentéricos explica porque razão a estimulação dos receptores A3 (com 2-Cl-IB MECA) localizados nos corpos celulares dos neurónios mioentéricos exerce um efeito sinérgico com os receptores facilitatórios A2A dos terminais nervosos no sentido de aumentarem a libertação de ACh. Os resultados apresentados consolidam e expandem a compreensão actual da distribuição e função dos receptores da adenosina no plexo mioentérico do íleo de rato, e devem ser tidos em consideração para a interpretação de dados relativos às implicações fisiopatológicas da adenosina nos transtornos da motilidade intestinal.