Thyrotropin-releasing-hormone (TRH) and its physiological metabolite TRH-OH inhibit Na+ channel activity in mammalian septal neurons

The interaction of thyrotropin-releasing hormone (TRH) and its physiological metabolite TRH-OH with Na+ channels was studied in enzymatically dissociated guinea pig septal neurons by using the whole-cell variant of the patch-clamp technique. In about 60% of the cells tested, the neuropeptides at concentrations between 0.01 and 2.5 ,uM produced a dose-dependent reversible attenuation of Na+ currents. With 2 ,uM TRH-OH, peak Na+ current amplitude was reduced by 20-50% (27 ± 8%, mean ± SD; n = 16), whereas at the same concentration TRH was approximately half as effective as TRH-OH. In the presence of the tripeptides, the voltage-dependent parameters of the Na+ current were unaltered. TRH-induced reduction of Na+ current amplitude was transient and recovered almost completely during maintained exposure to the peptides. In addition, the response to either TRH-OH or TRH decreased with repeated treatment. Our results demonstrate that neuronal Na+ channels can be modulated by naturally occurring neuropeptides

Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections

X-gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. However, this procedure does not permit the direct confocal acquisition of images, thus preventing the identification of labelled cells on the depth (Z) axis of tissue sections and leading sometimes to erroneous conclusions in co-localization and gene expression studies. Here we report a technique, based on X-gal fluorescence emission and mathematically-based optical correction, to obtain high quality fluorescence confocal images. This method, combined with immunofluorescence, makes it possible to unequivocally identify X-gal-labelled cells in tissue sections, emerging as a valuable tool in gene expression and cell tracing analysis

Rat Adrenal Chromaffin Cells Are Neonatal CO2 Sensors

We studied the participation of adrenal medulla (AM) chromaffin cells in hypercapnic chemotransduction. Using amperometric recordings, we measured catecholamine (CAT) secretion from cells inAMslices of neonatal and adult rats perfused with solutions bubbled with different concentrations of CO2. The secretory activity augmented from 1.74_0.19 pC/min at 5% CO2 to 6.36_0.77 pC/min at 10% CO2. This response to CO2 was dose dependent and appeared without changes in extracellular pH, although it was paralleled by a drop in intracellular pH. Responsiveness to hypercapnia was higher in neonatal than in adult slices. The secretory response to hypercapnia required extracellular Ca2_ influx. Both the CO2-induced internal pH drop and increase in CAT secretion were markedly diminished by methazolamide (2_M), a membrane-permeant carbonic anhydrase (CA) inhibitor.Wedetected the presence of twoCAisoforms (CAI and CAII) in neonatalAMslices by in situ hybridization and real-time PCR. The expression of these enzymes decreased in adultAMtogether with the disappearance of responsiveness to CO2. In patch-clamped chromaffin cells, hypercapnia elicited a depolarizing receptor potential, which led to action potential firing, extracellular Ca2_ influx, and CAT secretion. This receptor potential (inhibited by methazolamide) was primarily attributable to activation of a resting cationic conductance. In addition, voltage-gated K_ current amplitude was also decreased by high CO2. The CO2-sensing properties of chromaffin cells may be of physiologic relevance, particularly for the adaptation of neonates to extrauterine life, before complete maturation of peripheral and central chemoreceptors

Effects of Rho Kinase Inhibitors on Grafts of Dopaminergic Cell Precursors in a Rat Model of Parkinson's Disease.

In models of Parkinson’s disease (PD), Rho kinase (ROCK) inhibitors have antiapoptotic and axonstabilizing effects on damaged neurons, decrease the neuroinflammatory response, and protect against dopaminergic neuron death and axonal retraction. ROCK inhibitors have also shown protective effects against apoptosis induced by handling and dissociation of several types of stem cells. However, the effect of ROCK inhibitors on dopaminergic cell grafts has not been investigated. In the present study, treatment of dopaminergic cell suspension with ROCK inhibitors yielded significant decreases in the number of surviving dopaminergic neurons, in the density of graft-derived dopaminergic fibers, and in graft vascularization. Dopaminergic neuron death also markedly increased in primary mesencephalic cultures when the cell suspension was treated with ROCK inhibitors before plating, which suggests that decreased angiogenesis is not the only factor leading to cell death in grafts. Interestingly, treatment of the host 6-hydroxydopamine-lesioned rats with ROCK inhibitors induced a slight, nonsignificant increase in the number of surviving neurons, as well as marked increases in the density of graft-derived dopaminergic fibers and the size of the striatal reinnervated area. The study findings discourage treatment of cell suspensions before grafting. However, treatment of the host induces a marked increase in graft-derived striatal reinnervation. Because ROCK inhibitors have also exerted neuroprotective effects in several models of PD, treatment of the host with ROCK inhibitors, currently used against vascular diseases in clinical practice, before and after grafting may be a useful adjuvant to cell therapy in PDMinisterio de Salud PI12/00798; RD12/0019/0020Ministerio de Economía y Competitividad BFU2012-370

Trophic restoration of the nigrostriatal dopaminergic pathway in long-term carotid body-grafted parkinsonian rats

We studied the mechanisms underlying long-term functional recovery of hemiparkinsonian rats grafted intrastriatally with carotid body (CB) cell aggregates. Amelioration of their motor syndrome is a result of the trophic actions of these grafts on the remaining ipsilateral substantia nigra neurons rather than of the release of dopamine from the CB grafts. The grafts maintain a stable morphological appearance and differentiated cell phenotype for the duration of the life of the host. Adult CB expresses high levels of glial cell line-derived neurotrophic factor (GDNF) and the multicomponent GDNF receptor complex. These properties may contribute to the trophic actions of the CB transplants on nigrostriatal neurons and to their extraordinary longevity. We show that CB glomus cells, although highly dopaminergic, are protected from dopamine-mediated oxidative damage because of the absence of the high-affinity dopamine transporter. Thus, intrastriatal CB grafts are uniquely suited for long-term delivery of trophic factors capable of promoting restoration of the nigrostriatal pathway

Carotid Body Transplants as a Therapy for Parkinson’s Disease

Affecting over 1.5 million people across the world, Parkinson's disease is a progressive neurological condition characterized, in part, by the loss of dopaminergic neurons in the substantia nigra pars compacta. It affects 1.5% of the global population over 65 years of age. As life expectancy is increasing, over the next few years the number of patients with Parkinson´s disease will grow exponentially. To date, there are no available treatments that are capable of curing Parkinson´s disease, and the current goal of therapy, dopamine replacement strategies, is to reduce symptoms. After several years of disease progression, treatment is complicated by the onset of motor fluctuations and dyskinesias. This information reveals the great importance and social need of finding an effective therapeutic intervention for Parkinson´s disease. This exemplary new book reviews some of the most outstanding examples of new drugs currently in pharmaceutical development or new targets currently undergoing the validation process to try to reach the Parkinson´s drug market in the next few years as potential disease modifying drugs. Providing up to date and comprehensive coverage, this book will be essential reading for researchers working in academia and industry in any aspect of medicinal chemistry or drug discovery

Selective Glial Cell Line-Derived Neurotrophic Factor Production in Adult Dopaminergic Carotid Body Cells In Situ and after Intrastriatal Transplantation

Glial cell line-derived neurotrophic factor (GDNF) exerts a notable protective effect on dopaminergic neurons in rodent and primate models of Parkinson’s disease (PD). The clinical applicability of this therapy is, however, hampered by the need of a durable and stable GDNF source allowing the safe and continuous delivery of the trophic factor into the brain parenchyma. Intrastriatal carotid body (CB) autografting is a neuroprotective therapy potentially useful in PD. It induces long-term recovery of parkinsonian animals through a trophic effect on nigrostriatal neurons and causes amelioration of symptoms insomePDpatients. Moreover, the adult rodentCBhas been shown to express GDNF. Here we show, using heterozygous GDNF/lacZ knock-out mice, that unexpectedly CB dopaminergic glomus, or type I, cells are the source of CB GDNF. Among the neural or paraneural cells tested, glomus cells are those that synthesize and release the highest amount ofGDNFin the adult rodent (as measured by standard and in situ ELISA). Furthermore,GDNFexpression by glomus cells is maintained after intrastriatal grafting and in CB of aged and parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated animals. Thus, glomus cells appear to be prototypical abundant sources of GDNF, ideally suited to be used as biological pumps for the endogenous delivery of trophic factors in PD and other neurodegenerative diseases

Effects of Rho kinase inhibitors on grafts of dopaminergic cell precursors in a rat model of Parkinson's Disease

In models of Parkinson’s disease (PD), Rho kinase (ROCK) inhibitors have antiapoptotic and axonstabilizing effects on damaged neurons, decrease the neuroinflammatory response, and protect against dopaminergic neuron death and axonal retraction. ROCK inhibitors have also shown protective effects against apoptosis induced by handling and dissociation of several types of stem cells. However, the effect of ROCK inhibitors on dopaminergic cell grafts has not been investigated. In the present study, treatment of dopaminergic cell suspension with ROCK inhibitors yielded significant decreases in the number of surviving dopaminergic neurons, in the density of graft-derived dopaminergic fibers, and in graft vascularization. Dopaminergic neuron death also markedly increased in primary mesencephalic cultures when the cell suspension was treated with ROCK inhibitors before plating, which suggests that decreased angiogenesis is not the only factor leading to cell death in grafts. Interestingly, treatment of the host 6-hydroxydopamine-lesioned rats with ROCK inhibitors induced a slight, nonsignificant increase in the number of surviving neurons, as well as marked increases in the density of graft-derived dopaminergic fibers and the size of the striatal reinnervated area. The study findings discourage treatment of cell suspensions before grafting. However, treatment of the host induces a marked increase in graft-derived striatal reinnervation. Because ROCK inhibitors have also exerted neuroprotective effects in several models of PD, treatment of the host with ROCK inhibitors, currently used against vascular diseases in clinical practice, before and after grafting may be a useful adjuvant to cell therapy in PDThis work was supported by Spanish Ministry of Health (PI12/00798 and RD12/0019/0020) and Spanish Ministry of Economy and Competitiveness (BFU2012-3708)S

An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab

BACKGROUND: Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment. METHODS: Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum. RESULTS: Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression. CONCLUSIONS: Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies