Protection of DAergic neurons mediates treadmill running attenuated olfactory deficits and olfactory neurogenesis promotion in depression model

In this study, we aimed to test the effects of treadmill running on depression induced olfactory functions and OB neurogenesis in depression model. Depression model was created with chronic unpredictable mild stress (CUMS) and treadmill running was performed as the antidepressant treatment. Behavioral results showed that treadmill running not only attenuated the depression mood but also improved the olfactory discrimination and sensitivity in CUMS depression model. Immune-staining further indicates treadmill running promoted neurogenesis in hippocampal OB region. Moreover, treadmill running prevented the loss of DAergic neurons in glomerular layer of OB region, indicating the critical role of DAergic neuronal functions in regulating treadmill running mediated olfactory functions. In depression model, inhibiting DAergic neurons by intra-OB injection of 6-OHDA resulted in the compromised improving effects of treadmill running olfactory discrimination. In conclusion, treadmill running could attenuate depression associated olfactory deficits by promoting olfactory neurogenesis and improve DAergic neural functions.     

Characteristics of odorant elicited calcium fluxes in acutely-isolated chick olfactory neurons

To understand avian olfaction, it is important to characterize the peripheral olfactory system of a representative bird species. This study determined the functional properties of olfactory receptor neurons of the chicken olfactory epithelium. Individual neurons were acutely isolated from embryonic day-18 to newborn chicks by dissection and enzymatic dissociation. We tested single olfactory neurons with behaviorally relevant odorant mixtures and measured their responses using ratiometric calcium imaging; techniques used in this study were identical to those used in other studies of olfaction in other vertebrate species. Chick olfactory neurons displayed properties similar to those found in other vertebrates: they responded to odorant stimuli with either decreases or increases in intracellular calcium, calcium increases were mediated by a calcium influx, and responses were reversibly inhibited by 100 M L–cis–diltiazem, 1 mM Neomycin, and 20 M U73122, which are biochemical inhibitors of second messenger signaling. In addition, some cells showed a complex pattern of responses, with different odorant mixtures eliciting increases or decreases in calcium in the same cell. It appears that there are common features of odorant signaling shared by a variety of vertebrate species, as well as features that may be peculiar to chickens.     

Dexmedetomidine protects against degeneration of dopaminergic neurons and improves motor activity in Parkinson's disease mice model

Parkinson’s disease (PD) is the result of dopaminergic (DA) neuronal death in the substantianigra pars compacta (SNc). Current treatments for PD such as L-dopa are limited in effectiveness and fail to address the cause. Targeted anti-inflammatory therapies, particularly directed at nuclear factor kappa B (NF‐κB) activity in alleviating degeneration of DA-neurons is of evolving interest. In the present study, we hypothesised that dexmedetomidine (DEX), an alpha-2 receptor adrenergic agonist, suppress the inflammatory responses associated with PD and restores dopaminergic levels by alleviating substantia nigral degeneration. Male mice (C57Bl/10, 8–11 months old and of 34–40 g of weight) were divided into: the control, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and MPTP + dexmedetomidine (MPTP + DEX) (n = 26 each group). Dex restored dopamine levels in SNpc of MPTP-induced PD mice model. Results of immunohisto staining revealed that Dex treatment post-MPTP induction restored TH-positive cells, with only 12.37% increase (^##p < 0.01 vs MPTP) on the third day and a steep 55% increase (^###p < 0.001 vs MPTP) following the seventh day of Dex treatment. Moreover, the expressions of proinflammatory markers regulated by NF-κB were diminished in Dex + MPTP group. In addition, cylinder test revealed that Dex treatment improved asymmetric limb usage pattern in MPTP induced mice over the course of 7 days. Hence, in this study, we provided insight on the effect of Dex in the inhibition of NF-κB1 regulated proinflammatory mediators to improve dopamine levels and reduce SNpc dopaminergic neuronal degeneration.     

Adenosine prevents the death of mesencephalic dopaminergic neurons by a mechanism that involves astrocytes

The purinergic nucleoside adenosine effectively prevented the death of dopaminergic neurons that occurs spontaneously and progressively in cultures of rat mesencephalon. Adenosine also significantly increased dopamine uptake, attesting to the state of differentiation and functional integrity of the neurons that were rescued. The effects of adenosine were (a) specific to the dopaminergic neurons in these cultures, (b) long-lived, (c) still observed when the treatment was delayed after plating, (d) potentiated by inhibition of adenosine deaminase, and (e) abolished when this enzyme was added in excess to the culture medium. The action of adenosine was mimicked by 5'-(N-ethylcarboxamido)adenosine and dibutyryl-cyclic AMP, but not by CGS-21680, suggesting the possible involvement of A2B subtype purinergic receptors. ATP was also neuroprotective, but its action resulted principally from conversion to adenosine by ectonucleotidases. Several anticancer drugs, including cytosine arabinoside, have been shown previously to prevent apoptosis in cultured dopaminergic neurons by a mechanism that requires the inhibition of proliferating astrocytes. In the presence of adenosine, astrocytes were more differentiated, and their proliferation rate was significantly reduced, suggesting that the neurotrophic effect of the adenine nucleoside resulted also from the repression of the astroglial cells. We did not find evidence of a trophic intermediary in adenosine-treated cultures, however, leading to the hypothesis that limitation of astrocyte replication in itself and/or ensuing changes in the glial phenotype were crucial. Our results suggest that molecules that modulate adenine nucleotide/nucleoside release may be useful for the treatment of chronic neurodegenerative conditions affecting dopaminergic neurons, such as Parkinson's disease.     

Isolation,Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.     

Vasonatrin peptide,a novel protector of dopaminergic neurons against the injuries induced by n-methyl-4-phenylpyridiniums

Vasonatrin peptide (VNP), a novel manmade natriuretic peptide, is known as a cardiovascular active substance. However, its neuroeffects are largely unknown. Here, cultured dopaminergic neurons from ventral mesencephalon of mouse were exposed to N-methyl-4-phenylpyridinium (MPP⁺), and the effects of VNP on the neurotoxicity of MPP⁺ were investigated. As a result, MPP⁺ caused injuries in the dopaminergic neurons. VNP significantly reduced the cytotoxicity of MPP⁺ by increasing axon number and length of dopaminergic neurons, and by enhancing the cell viability. Also, the MPP⁺-induced depolymerization of β-Tubulin III was attenuated by the treatment of VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-br-cGMP (a cell-permeable analog of cGMP), whereas inhibited by HS-142-1 (the antagonist of the particulate guanylyl cyclase-coupled natriuretic peptide receptors), or KT-5823 (a cGMP-dependent protein kinase inhibitor). Taken together, VNP attenuates the neurotoxicity of MPP⁺ via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of neuron degeneration of dopaminergic neurons in Parkinson's disease (PD).     

Alpha-synuclein up-regulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP

Mutations in alpha-synuclein cause a form of familial Parkinson's disease (PD), and wild-type alpha-synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for alpha-synuclein in PD. Thus far, however, little is known about the importance of alpha-synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein-1, the rodent homologue of human alpha-synuclein, in both normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein-positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein-positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP-induced dopaminergic neurodegeneration. In these MPTP-injected mice, there was also a dramatic increase in the number of synuclein-immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein-positive neurons were tyrosine hydroxylase-positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up-regulated following MPTP-induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.     

Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2

An interaction of odorants with olfactory receptors is thought to be the initial step in odorant detection. However, ligands have been reported for only 6 out of 380 human olfactory receptors, with their structural determinants of odorant recognition just beginning to emerge. Guided by the notion that amino acid positions that interact with specific odorants would be conserved in orthologs, but variable in paralogs, and based on the prediction of a set of 22 of such amino acid positions, we have combined site-directed mutagenesis, rhodopsin-based homology modelling, and functional expression in HeLa/Olf cells of receptors OR1A1 and OR1A2. We found that (i) their odorant profiles are centred around citronellic terpenoid structures, (ii) two evolutionary conserved amino acid residues in transmembrane domain 3 are necessary for the responsiveness of OR1A1 and the mouse ortholog Olfr43 to (S)-(-)-citronellol, (iii) changes at these two positions are sufficient to account for the differential (S)-(-)-citronellol responsiveness of the paralogs OR1A1 and OR1A2, and (iv) the interaction sites for (S)-(-)-citronellal and (S)-(-)-citronellol differ in both human receptors. Our results show that the orientation of odorants within a homology modelling-derived binding pocket of olfactory receptor orthologs is defined by evolutionary conserved amino acid positions.     

Chronic Activation of the Cyclic AMP Signaling Pathway Promotes Development and Long-Term Survival of Mesencephalic Dopaminergic Neurons

Abstract: Dibutyryl cyclic AMP (dbcAMP), a permeant analogue of cyclic AMP (cAMP), prevented, for at least 3 weeks, the death of tyrosine hydroxylase (TH)-immunopositive dopaminergic neurons, which occurred spontaneously by apoptosis in mesencephalic cultures. Treatment with the cyclic nucleotide analogue also led to a significant increase in the uptake of [³H]dopamine, attesting that the rescued TH⁺ neurons were fully functional and differentiated. dbcAMP was most effective when added immediately after plating, but delayed treatment could still arrest the ongoing degenerative process. Trophic/survival effects were long-lasting, declining only progressively after withdrawal of dbcAMP from the culture medium. They were independent of cell density and still detectable in the absence of serum proteins. The effects of dbcAMP were mimicked by depolarizing concentrations of potassium and by agents that increase endogenous production of cAMP, such as forskolin or 3-isobutyl-1-methylxanthine, but not by native cAMP, which cannot cross cell membranes. Elimination of glial cells by arabinoside-C did not reduce the activity of dbcAMP. GABAergic neurons, also present in these cultures, were much less dependent on the cyclic nucleotide analogue for their survival, and serotoninergic cells were not dependent at all. Therefore, cAMP-dependent signaling may be particularly crucial for the maturation and long-term survival of mesencephalic dopaminergic neurons.     

Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20-50 mM). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK-801 and GYKI-52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK-801 or GYKI-52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK-801 were mimicked by BAY K-8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca(2+) influx through L-type calcium channels. Measurement of intracellular calcium revealed that MK-801 or GYKI-52466 were required to maintain Ca(2+) levels within a trophic range, thus preventing K+-induced excitotoxic stress and Ca(2+) overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.     

Identification of neurons that express 5-hydroxytryptamine4 receptors in intestine

5-Hydroxytryptamine (5-HT) is an endogenous stimulant of intestinal propulsive reflexes. It exerts its effects partly through 5-HT₄ receptors; 5-HT₄ receptor agonists that are stimulants of intestinal transit are in clinical use. Both pharmacological and recent immunohistochemical studies indicate that 5-HT₄ receptors are present on enteric neurons but the specific neurons that express the receptors have not been determined. In the present work, we describe the characterization of an anti-5-HT₄ receptor antiserum that reveals immunoreactivity for enteric neurons and other cell types in the gastrointestinal tract. With this antiserum, 5-HT₄ receptor immunoreactivity has been found in the muscularis mucosae of the rat oesophagus, a standard assay tissue for 5-HT₄ receptors. It is also present in the muscularis mucosae of the guinea-pig and mouse oesophagus. In guinea-pig small intestine and rat and mouse colon, 5-HT₄ receptor immunoreactivity occurs in subpopulations of enteric neurons, including prominent large neurons. Double-staining has shown that these large neurons in the guinea-pig small intestine are also immunoreactive for two markers of intrinsic primary afferent neurons, cytoplasmic NeuN and calbindin. Some muscle motor neurons in the myenteric ganglia are immunoreactive for this receptor, whereas it is rarely expressed by secretomotor neurons. Immunoreactivity also occurs in the interstitial cells of Cajal but is faint in the external muscle. Expression of the protein and mRNA has been confirmed in extracts containing enteric neurons. The observations suggest that one site of action of 5-HT₄ receptor agonists is the intrinsic primary afferent neurons.This work was supported by the National Health and Medical Research Council of Australia and Pfizer Pharmaceuticals, Japan.     

Characterization of adenosine receptors in a model of cultured neurons from rat forebrain

The neuromodulator adenosine is acting through specific receptors coupled to adenylate cyclase via G-proteins. The expression of both adenosine receptors A₁ and A₂ as well as forkolin binding sites was investigated by radioligand binding techniques in 8-day-old neurons isolated from fetal rat forebrain and cultured in chemically-defined medium. Adenosine A₁ receptors were specifically labeled with [³H]chloro-N⁶-cyclopentyladenosine (CCPA), whereas [³H]CGS 21680 was used for the analysis of A₂ receptors. Cultured neurons exhibited high affinity binding sites for CCPA (Bmax=160 fmol/mg protein; Kd=2.9 nM), and for CGS 21680 (Bmax=14 fmol/mg protein; Kd=1.7 nM). These data correlate well with those obtained in crude membranes isolated from the newborn rat forebrain. The incubation of culture membranes in the additional presence of guanylyl-5-imidodiphosphate (Gpp(NH)p, a GTP analogue) led to significantly increased Kd-values, suggesting the association of adenosine receptors with G-proteins. Finally, cultured neurons also bound specifically [³H]forskolin with characteristics close to those found in the newborn brain, indicating that cultured neurons appear as an appropriate model for studying the neuromodulatory properties of adenosine.     

Stimulation of olfactory receptors alters regulation of [Cai] in olfactory neurons of the catfish (Ictalurus punctatus)

Summary Intracellular calcium was measured in single olfactory neurons from the channel catfish (Icatalurus punctatus) using the fluorescent Ca²⁺ indicator fura 2. In 5% of the cells, olfactory stimuli (amino acids) elicited an influx of calcium through the plasma membrane which led to a rapid transient increase in intracellular calcium concentration. Amino acids did not induce release of calcium from internal stores in these cells. Some cells responded specifically to one stimulus (l-alanine,l-arginine,l-norleucine andl-glutamate) while one cell responded to all stimuli. An increase in intracellular calcium could also be elicited in 50% of the cells by direct G-protein stimulation using aluminum fluoride. Because the fraction of cells which respond to direct G-protein stimulation is substantially larger than the fraction of cells responding to amino acids, we tested for possible damage of receptor proteins due to exposure of the olfactory neurons to papain during cell isolation. We find that pretreatment with papain does not alter specific binding ofl-alanine andl-arginine to olfactory receptor sites in isolated olfactory cilia. The results are discussed in terms of their relevance to olfactory transduction.     

Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neuronsin developing zebrafish

BACKGROUND: Recent data have demonstrated that treatment with sodium benzoate (SB) leads to significant developmental defects in motor neuron axons and neuromuscular junctions in zebrafish larvae, thereby implying that SB can be neurotoxic. This study examined whether SB affects the development of dopaminergic neurons in the zebrafish brain. METHODS: Zebrafish embryos were exposed to different concentrations of SB for various durations, during which the survival rates were recorded, the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the neurons in the ventral diencephalon were detected by in situ hybridization and immunofluorescence, and the locomotor activity of larval zebrafish was measured. RESULTS: The survival rates were significantly decreased with the increase of duration and dose of SB-treatment. Compared to untreated clutch mates (untreated controls), treatment with SB significantly downregulated expression of TH and DAT in neurons in the ventral diencephalon of 3-day post-fertilization (dpf) zebrafish embryos in a dose-dependent manner. Furthermore, there was a marked decrease in locomotor activity in zebrafish larvae at 6dpf in response to SB treatment. CONCLUSIONS: The results suggest that SB exposure can cause significantly decreased survival rates of zebrafish embryos in a time- and dose-dependent manner and downregulated expression of TH and DAT in dopaminergic neurons in the zebrafish ventral diencephalon, which results in decreased locomotor activity of zebrafish larvae. This study may provide some important information for further elucidating the mechanism underlying SB-induced developmental neurotoxicity. Birth Defects Res (Part B)86: 85-91, 2009. © 2009 Wiley-Liss, Inc.     

Impaired neurite development associated with mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of children with autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restrictive interests, and repetitive stereotypic behaviors. Among the various mechanisms underlying the pathogenesis of ASD, dysfunctions of dopaminergic signaling and mitochondria have been hypothesized to explain the core symptoms of children with ASD. However, only a few studies focusing on the pathological association between dopaminergic neurons (DN) and mitochondria in ASD have been performed using patient-derived stem cells and in vitro differentiated neurons. Stem cells from human exfoliated deciduous teeth (SHED) are neural crest-derived mesenchymal stem cells present in the dental pulp of exfoliated deciduous teeth; these cells can differentiate into dopaminergic neurons (DN) in vitro. This study aimed to investigate the pathological association between development of DN and mitochondria in ASD by using SHED as a disease- or patient-specific cellular model. The SHED obtained from three children with ASD and three typically developing children were differentiated into DN, and the neurobiology of these cells was examined. The DN derived from children with ASD showed impaired neurite outgrowth and branching, associated with decreased mitochondrial membrane potential, ATP production, number of mitochondria within the neurites, amount of mitochondria per cell area and intracellular calcium level. In addition, impaired neurite outgrowth and branching of ASD-derived DN were not improved by brain-derived neurotrophic factor (BDNF), suggesting impairment of the BDNF signaling pathway in ASD. These results imply that intracerebral dopamine production may have decreased in these children. The earliest age at which deciduous teeth spontaneously exfoliate in humans, and SHED can be noninvasively collected, is approximately 6 years. Our results suggest that in vitro analysis of SHED-derived DN obtained from children with ASD provides neurobiological information that may be useful in determining treatment strategies in the early stages of ASD.     

Factors promoting survival of mesencephalic dopaminergic neurons

Growth factors promoting survival of mesencephalic dopaminergic neurons are discussed in the context of their requirement during development and adulthood. The expression of growth factors should be detectable in the nigrostriatal system during critical periods of development, i.e., during the period of ontogenetic cell death and synaptogenesis and during neurite extension and neurotransmitter synthesis. Growth factors discussed include members of the family of glial-cell-line-derived neurotrophic factors (GDNF), neurotrophins, transforming growth factors beta, and low molecular compounds mimicking growth factor activities. To date, the available data support the notion that GDNF is a highly promising candidate, although GDNF-null mice lack a dopaminergic phenotype. There remains a possibility that endogenous dopaminotrophic factors remain to be discovered.The authors work is supported by grants from the Deutsche Forschungsgemeinschaft through the DFG-Research Center for Molecular Physiology of the Brain