Cyclic guanosine 3′,5′-monophosphate-mediated neuroprotection by nitric oxide in dissociated cultures of rat dorsal root ganglion neurones

In dissociated cultures of dorsal root ganglia (DRG) derived from 15-day-old rats, many neurones expressed nitric oxide synthase (NOS) and this expression was found to be reduced by nerve growth factor. The application of blockers of NOS caused selective death of those neurones expressing NOS. The soluble guanylate cyclase (sGC) blocker ODQ also caused neuronal death. The appearance of the neurones undergoing cell death was typical of apoptosis. This suggests that NO has a neuroprotective action in DRG neurones which is probably mediated by its activation of cyclic guanosine 3′,5′-monophosphate. These observations are discussed in relation to the developmental and neuropathic changes in NOS expression by DRG neurones.     

Role of CNPase in the oligodendrocytic extracellular 2′,3′‐cAMP‐adenosine pathway

Extracellular adenosine 3′,5′‐cyclic monophosphate (3′,5′‐cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2′,3′‐cAMP (positional isomer of 3′,5′‐cAMP) is also a source of adenosine, particularly in the brain in vivo post‐injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2′,3′‐cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2′,3′‐cAMP and their respective adenosine monophosphates (2′‐AMP and 3′‐AMP). Cells were also isolated from mice deficient in 2′,3′‐cyclic nucleotide‐3′‐phosphodiesterase (CNPase). Oligodendrocytes metabolized 2′,3′‐cAMP to 2′‐AMP with 10‐fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3′‐AMP was minimal in both oligodendrocytes and neurons. The production of 2′‐AMP from 2′,3′‐cAMP was reduced by 65% in CNPase ?/? versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2′‐AMP to adenosine, and this was also attenuated in CNPase ?/? oligodendrocytes. Inhibition of classic 3′,5′‐cAMP‐3′‐phosphodiesterases with 3‐isobutyl‐1‐methylxanthine did not block metabolism of 2′,3′‐cAMP to 2′‐AMP and inhibition of classic ecto‐5′‐nucleotidase (CD73) with α,β‐methylene‐adenosine‐5′‐diphosphate did not attenuate the conversion of 2′‐AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2′,3′‐cAMP‐adenosine pathway (2′,3′‐cAMP → 2′‐AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2′,3′‐cAMP to 2‐AMP in CNS cells. By reducing levels of 2′,3′‐cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. GLIA 2013;61:1595–1606     

Molecular cloning and characterization of rat brain 2′, 3′ -cyclic nucleotide 3′ -phosphodiesterase isoform 2

We have isolated a cDNA coding for the larger isoform of the rat brain 2′,3′ -cyclic nucleotide 3′ -phosphodiesterase (CNP2), a protein associated with myelination in the central nervous system (CNS). The complete 420 amino acid sequence was deduced from the nucleotide sequence of the cDNA. Sequence comparisons show that rat CNP shares 96% homology with mouse, 84% with bovine, and 86% with human CNP. Errors in the published sequence of rat CNP1 have now been corrected. Comparisons with other proteins reveal several interesting conserved motifs, including two leucine repeat heptads, and two consensus motifs for phosphorylation in the N-terminal domain of CNP2.© 1994 Wiley-Liss, Inc.     

Immunofluorescence demonstration of 2′:3′-cyclic-nucleotide 3′-phosphodiesterase in cultured oligodendrocytes of mouse, rat, calf and human

Previous biochemical studies have shown that high enzyme activity of 2′:3′-cyclic-nucleotide 3′-phosphodiesterase (CNP) is found in isolated myelin and oligodendrocytes. We report here the specific and intense immunofluorescence staining of cultured oligodendrocytes obtained from the brains of mouse, rat, calf and human by rabbit antiserum specific for purified bovine CNP. Astrocytes and fibroblasts present in the cultures were negative for the immunostaining. The specificity of the CNP immunoreactivity was confirmed by blocking the reaction by prior absorption of the antiserum by purified CNP.     

Structural correlates of physiological abnormalities in β,β′-Iminodipropionitrile

β,β′-Iminodipropionitrile (IDPN) produces neurofilamentous giant axonal swellings in proximal internodes of large myelinated axons. Secondary demyelinative changes result from the production of these axonal enlargments. Electrophysiological studies have demonstrated profound alterations in the electrical properties of motor neurons (MN) within the spinal cord. On the basis of intracellular recordings, it has been suggested that electrical contacts may exist between swollen axons and neighboring MN. In addition, the possibility remained that synaptic contacts develop on demyelinated axonal swellings. In the present study, we report the lack of either synapses on demyelinated axonal swellings or direct electrical contacts between neighboring MN. Axonal swellings are surrounded by attenuated processes of glial cells (probably fibrillary astrocytes), a finding discussed in terms of its possible role in the production of ephaptic transmission. There was considerable variation in the degree of axonal enlargements and in the extent of secondary (passive and active) demyelination. It is suggested that these morphological changes may represent structural correlates of some electrophysiological alterations observed in IDPN neuropathy.     

Localization of 2′,3′-cyclic nucleotide-3′-phosphohydrolase within the vertebrate retina

Histochemical and immunochemical techniques are used to locate 2',3'-cyclic nucleotide-3'-phosphohydrolase (2',3'-cNMP-3'ase) within cells of the vertebrate retina. A new histochemical method is described which links the hydrolysis of 2',3'-cNADP to the formation of a reduced, insoluble tetrazolium formazan. Photoreceptors from fish, bovine, and rat retinas are stained by this procedure. The reaction is blocked by 2'-AMP, a known inhibitor of 2',3'-cNMP-3'ase. Rabbit antibodies prepared against 2',3'-cNMP-3'ase from bovine brain are found to cross-react with bovine and rat retinal enzymes. Peroxidase-labeled antibody shows by light microscopy the greatest staining along the inner segments of the photoreceptors. Electron microscopy of the same preparations confirms binding to the plasma membrane of the inner segments of both rods and cones. Retinal 2',3'-cNMP-3'ase is thus predominantly associated with the photoreceptors, suggesting some role for 2',3'-cyclic nucleotides as substrates in visual function.     

Immunohistochemical localization of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase in the central nervous system

We describe the immunohistochemical localization for brain 2′, 3′-cyclic nucleotide 3′-phospho diesterase (EC 3.1.4.37) by the peroxidase-antiperoxidase method. Myelinated fibre tracts were immunohistochemically stained in 15-day-old rat cerebrum and spinal cord; at high magnification, myelinated nerve fibres and immature glial cells with their processes were shown to be stained.     

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca²⁺]_i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca²⁺]_i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca²⁺]_i increases which were abolished in Ca²⁺-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10⁻⁶ M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd³⁺; 250 μM) and amiloride (100 μM) abolished the [Ca²⁺]_i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca²⁺]_i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca²⁺]_i transients but had little effect on capsaicin-induced [Ca²⁺]_i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca²⁺]_i transients in small diameter DRG somas are mediated by influx of Ca²⁺ through a Gd³⁺- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca²⁺ transients are sensitive to κ-opioid agonists.     

Selective interruption of axonal transport of neurofilament proteins in the visual system by β,β′-iminodipropionitrile (IDPN) intoxication

β,β′-iminodipropionitrile (IDPN) is an agent that produces a marked impairment in the transport of neurofilaments. Its effect on other slowly transported cytoskeletal components sucas tubulin and actin is variable. Previous studies have evaluated transport of neurofilaments after IDPN intoxication in a neurofilament-ricsystem (sciatic motor nerves) and in a system devoid of neurofilaments (axons of the dorsal motor nucleus of the vagus). In the former, IDPN impairs the transport of tubulin and actin but to a lesser degree than it does neurofilament proteins. In the latter, tubulin and actin transport were not impaired, and neurofilament proteins were not present. In this study we evaluated the transport of the cytoskeletal components in a system witan intermediate amount of neurofilaments (the visual system). In the visual system, there is a selective and marked (50%) impairment in the transport of neurofilaments witno impairment in transport of tubulin or microtubule-associated proteins (tau group). We conclude that these different patterns of impairment in transport reflect the differences in pre-intoxication neurofilament content of the nerves examined, the effect of IDPN on the transport of the other components of slow transport being secondary to the presence of stagnated neurofilaments. This model also suggests that transport of neurofilaments can be selectively impaired without producing an effect on other major slow transport components.     

Immunocytochemical study of the GABA system in chicken vestibular endorgans and the vestibular ganglion

The immunocytochemical distribution of gamma-aminobutyric acid (GABA), GABA synthesizing enzyme; L-glutamate decarboxylase (GAD) and degradative enzyme; GABA transaminase (GABA-T) in the chicken vestibular endorgans and the vestibular ganglion was investigated. GABA and GAD-like immunoreactivity were confined to the sensory hair cell cytoplasm, suggesting that GAD synthesizes GABA in the hair cell. GABA-T-like immunoreactivity, indicative of GABA degradation, was found around hair cells, along nerve fibers running through the stroma and within the ganglion cell. These immunocytochemical findings indicate that the GABAergic system exists in the chicken vestibular endorgans and that GABA may function as an afferent neurotransmitter at the level of hair cells.     

Gamma-Diketone central neuropathy: quantitative analyses of cytoskeletal components in myelinated axons of the rat rubrospinal tract

Loss of axon caliber is a primary component of gamma-diketone neuropathy [LoPachin RM, DeCaprio AP. gamma-Diketone central neuropathy: axon atrophy and the role of cytoskeletal protein adduction. Toxicol Appl Pharmacol 2004;199:20-34]. It is possible that this effect is mediated by changes in the density of cytoskeletal components and corresponding spatial relationships. To examine this possibility, morphometric methods were used to quantify the effects of 2,5-hexanedione (HD) intoxication on neurofilament-microtubule densities and nearest neighbor distances in myelinated rubrospinal axons. Rats were exposed to HD at one of two daily dose-rates (175 or 400 mg/kg per day, gavage) until a moderate level of neurotoxicity was achieved (99 or 21 days of intoxication, respectively) as determined by gait analysis and measurements of hindlimb grip strength. Results indicate that, regardless of dose-rate, HD intoxication did not cause changes in axonal neurofilament (NF) density, but did significantly increase microtubule (MT) density. No consistent alterations in interneurofilament or NF-MT distances were detected by ultrastructural morphometric analyses. These data suggest that the axon atrophy induced by HD was not mediated by major disruptions of stationary cytoskeletal organization. Recent biochemical studies of spinal cord from HD intoxicated rats showed that, although the NF protein content in the stationary cytoskeleton (polymer fraction) was not affected, the mobile subunit pool was depleted substantially [LoPachin RM, He D, Reid ML, Opanashuk LA. 2,5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions. Toxicol Appl Pharmacol 2004;198:61-73]. The stability of the polymer fraction during HD intoxication is consistent with the absence of significant ultrastructural modifications noted in the present study. Together, these findings implicate loss of mobile NF proteins as the primary mechanism of axon atrophy.     

Intracellular localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in rat oligodendrocytes and C6 glioma cells, and effect of cell maturation and enzyme induction on localization

We investigated whether the membrane-associated myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), is localized primarily inside the cell or exposed on the cell surface of rat oligodendrocytes and rat C6 glioma cells. Determinations were made by enzyme assays of intact, viable cells vs cells broken by freezing and thawing. Assay of both oligodendrocytes and C6 cells showed that the great majority of the CNP activity was localized inside the cells. Oligodendrocytes were also tested by immunofluorescence staining of unfixed, living cells whose membranes had been made permeable to antibody by fixation. Fixed oligodendrocytes showed intense fluorescence when incubated with rabbit anti-CNP antiserum and fluorescein-conjugated second antibody whereas unfixed cells were not stained. We then tested the possible influence on CNP localization of 3 conditions known to increase CNP specific activity: maturation of oligodendrocytes in vitro during a period when CNP specific activity increases 8-fold or more; growth of C6 cultures to high cell density; and induction of CNP activity in oligodendrocytes and C6 cells by dibutyryl cyclic AMP. Under all conditions, most CNP activity was intracellular. These results show that both the catalytic and major antigenic sites of CNP are localized primarily inside the cell, and suggest an intracellular role for CNP in oligodendrocytes. The results with C6 cells also show that these cells resemble oligodendrocytes with respect to CNP localization.     

Circadian rhythm of adenosine-3′,5′-monophosphate content in suprachiasmatic nucleus (SCN) and ventromedial hypothalamus (VMH) in the rat

Twenty-four hour patterns of adenosine-3',5'-monophosphate (cAMP) contents in the suprachiasmatic nucleus (SCN) and the ventromedial hypothalamus (VMH) were determined at 3 h intervals under various conditions in adult male rats. The cAMP levels in both the nuclei showed a circadian variation with a clear peak time, which shifted under constant conditions (blinded and constant illumination). Under both entrained and free-running conditions, a consistent phase-angle difference of 9 h in peak time was observed between cAMP rhythms in the SCN and VMH. Under the LD cycle, the peak time of cAMP rhythms in the SCN and VMH appeared at 16.00 h and 07.00 h, respectively. The cAMP rhythm in the VMH synchronized with a restricted feeding time, when the rats were subjected to restriction of access to food under constant light. On the contrary, that in SCN did not synchronize with the restricted feeding time. These results indicate that the 24-h patterns of cAMP contents in SCN and VMH are the manifestation of endogenous circadian rhythms. However, the generating mechanism of cAMP rhythm may be different between SCN and VMH, as the restricted feeding regime affected the cAMP patterns of both the nuclei in different ways.     

Effect of N,N-diethyldithiocarbamate on ionomycin-induced increase in oxidation of cellular 2′,7′-dichlorofluorescin in dissociated cerebellar neurons

Effect of N,N-diethyldithiocarbamate (DDC), an inhibitor for cytosolic superoxide dismutase, on an ionomycin-induced increase in oxidative metabolism was examined in cerebellar neurons dissociated from the rats, using a flow cytometer and 2′,7′-dichlorofluorescin diacetate and fluo-3-AM, fluorescent dyes for intracellular hydrogen peroxide and Ca²⁺, respectively. DDC reduced the ionomycin-induced augmentation of 2′-7′-dichlorofluorescin fluorescence in a dose-dependent manner. DDC did not affect cellular content of 2′,7′-dichlorofluorescin and ionomycin-induced increase in intracellular Ca²⁺ concentration. Results indicate that ionomycin increases the formation of superoxide anion in brain neuron.     

Immunohistochemical localization of γ-aminobutyric acid- and aspartate-containing neurons in the guinea pig vestibular nuclei

The immunohistochemical distributions of γ-aminobutyric acid (GABA)- and aspartate-containing neurons were studied in the guinea pig vestibular nuclei using purified antisera to GABA and aspartate, respectively. Most GABA-containing neurons had small cell bodies and were scattered throughout all regions of the vesticular nuclei. The largest number of these cells was found in the medial nucleus. Intraventricular injection of colchicine markedly increased GABA-like immunoreactivity in these cell bodies. GABA-containing terminals were distributed throughout all 4 subdivisions of the nuclei, with the richest localization found around the floor of the fourth ventricle. Various sized aspartate-containing neurons were noted in the vestibular nuclei and small cells were present in the superior, medial and lateral nucleus. Medium-sized cells were observed throughout the vestubular nuclei. Giant cells in the lateral nucleus also contained aspartate and were surrounded by GABA-like immunoreactive terminals, thereby suggesting the modulation of aspartate-containing neurons by GABAergic fibers from Purkinje cells.     

Plasticity of developing and adult dorsal root ganglion neurons as revealed in vitro

We review recent data on the plasticity of dorsal root ganglion (DRG) neurons as revealed during cultivation in vitro. Some experiments on cultured developing DRG neurons and on adult DRG neurons in vivo are also mentioned. Cultured developing and adult DRG neurons can be switched from an apolar to a multipolar phenotype by fetal calf serum or fibronectin. The effect is concentration dependent and occurs through an early modification of cell-substratum interaction. Adult DRG neurons synthesize and release within hours after injury TGFβ-1, which is a mitogen and a differentiation factor for Schwann cells. Finally, adult DRG neurons express in vitro neurotransmitters that are not expressed in vivo. This neurotransmitter plasticity can be modulated in vitro by some growth factors and in vivo by distal or proximal axotomy.     

3,5,3′-l-Triidothyronine promotes survival and axon elongation of embryonic rat septal neurons

The effect of 3,5,3′-l-triiodothyronine (T₃) on survival and morphology of primary cultured neurons of the fetal rat brain was studied. In defined conditions of serum-free culture media we found the death preventing effect of T₃ in all tested neuronal populations cultivated at high initial densities of plating (10⁵ cells/cm²). While the survival of cerebrocortical neurons was improved very slightly, the number of surviving hippocampal and septal neurons reached 127.2 ± 2.0% or 134.8 ± 12.3% of their respective controls. The septal neurons responded at normal physiological concentration of T₃ (1nM) in high density as well as in low density cultures (5 × 10³ cells/cm²). Moreover the treatment with 10 nM of T₃ caused significant extension of the axon elongation of septal neurons (194.5 ± 15.7%). These findings suggest the direct positive effect of T₃ on pure cell population of septal neurons derived from embryonic rat brain and support the evidence for the role of this peripheral hormone during neuritogenesis.     

Immunocytochemical demonstration of β-endorphin and β-lipotropin in cultured human spinal ganglion neurons

Human fetal spinal ganglion neurons isolated and cultured in vitro were found to contain immunoreactive β-endorphin and β-lipotropin as demonstrated by immunoperoxidase and immunofluorescence techniques. The specificity of the immunoreactions was confirmed by the negative staining by prior absorption of the specific antisera with added peptides. The culture system described may provide a valuable model system in which cellular mechanisms underlying the functions of opioid peptides can be investigated.     

Electrophysiological investigation of β, β′-iminodipropionitrile neuropathy: Intracellular recordings in spinal cord

β, β′-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN).Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested.Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from3.3 ± 0.1to5.8 ± 0.5ms. IS-SD portions of the action potentials could not be fractionated in 49% of cells regardless of interpulse interval. Many MN failed to follow frequencies as low as 10 Hz. Delayed depolarizations were observed in 14% of MN recordings. Repetitive action potentials were elicited by single stimuli in 14% of MN and more frequently by orthodromic than antidromic stimulation. Action potentials could often be elicited in the same MN by stimulation of more than one ventral root filament. The incidence of this ephaptic transmission or crosstalk was estimated to be 12%. The findings are discussed in terms of the influence of proximal axon swellings on action potential generation in MN, propagation along non-homogeneous regions of axons and functional chromatolysis.