Cross-talk between neurons and glia: highlights on soluble factors

The development of the nervous system is guided by a balanced action between intrinsic factors represented by the genetic program and epigenetic factors characterized by cell-cell interactions which neural cells might perform throughout nervous system morphogenesis. Highly relevant among them are neuron-glia interactions. Several soluble factors secreted by either glial or neuronal cells have been implicated in the mutual influence these cells exert on each other. In this review, we will focus our attention on recent advances in the understanding of the role of glial and neuronal trophic factors in nervous system development. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership.

The 9-O-acetyl GD3 gangliosides are expressed by migrating chains of subventricular zone neurons in vitro

Neurons from the anterior subventricular zone (SVZ) of the cerebral cortex migrate tangentially to become interneurons in the olfactory bulb during development and in adult rodents. This migration was defined as neuronophilic, independent of a radial glial substrate. The cortical SVZ and the rostral migratory stream to the olfactory bulb were shown to be rich in 9-O-acetyl GD3 gangliosides (9-O-acGD3), which have been previously shown to be implicated in gliophilic migration in the rodent cerebral cortex and cerebellum. In the present study, we performed SVZ explant cultures using rats during their first postnatal week to analyze the expression of these gangliosides in chain migration of neuronal precursors. We characterized migrating chains of these neuroblasts through morphological analysis and immunocytochemistry for the neural cell adhesion molecule. By using the Jones monoclonal antibody which binds specifically to 9-O-acGD3 we showed that migrating chains from the SVZ explants express 9-O-acGD3 which is distributed in a punctate manner in individual cells. 9-O-acGD3 is also present in migrating chains that form in the absence of radial glia, typical of the neuronophilic chain migration of the SVZ. Our data indicate that 9-O-acetylated gangliosides may participate in neuronophilic as well as gliophilic migration.

Behavioral correlates of the activity of serotonergic and non-serotonergic neurons in caudal raphe nuclei

We investigated the behavioral correlates of the activity of serotonergic and non-serotonergic neurons in the nucleus raphe pallidus (NRP) and nucleus raphe obscurus (NRO) of unanesthetized and unrestrained cats. The animals were implanted with electrodes for recording single unit activity, parietal oscillographic activity, and splenius, digastric and masseter electromyographic activities. They were tested along the waking-sleep cycle, during sensory stimulation and during drinking behavior. The discharge of the serotonergic neurons decreased progressively from quiet waking to slow wave sleep and to fast wave sleep. Ten different patterns of relative discharge across the three states were observed for the non-serotonergic neurons. Several non-serotonergic neurons showed cyclic discharge fluctuations related to respiration during one, two or all three states. While serotonergic neurons were usually unresponsive to the sensory stimuli used, many non-serotonergic neurons responded to these stimuli. Several non-serotonergic neurons showed a phasic relationship with splenius muscle activity during auditory stimulation. One serotonergic neuron showed a tonic relationship with digastric muscle activity during drinking behavior. A few non-serotonergic neurons exhibited a tonic relationship with digastric and/or masseter muscle activity during this behavior. Many non-serotonergic neurons exhibited a phasic relationship with these muscle activities, also during this behavior. These results suggest that the serotonergic neurons in the NRP and NRO constitute a relatively homogeneous population from a functional point of view, while the non-serotonergic neurons form groups with considerable functional specificity. The data support the idea that the NRP and NRO are implicated in the control of somatic motor output.

Frequent occurrence of nicotinic acetylcholine receptors in GABAergic neurons of the chick visual system

Double-labeling immunohistochemical methods were used to investigate the occurrence of the alpha8 and alpha5 nicotinic receptor subunits in presumptive GABAergic neurons of the chick nervous system. Nicotinic receptor immunoreactivity was often found in cells exhibiting GABA-like immunoreactivity, especially in the visual system. The alpha8 subunit appeared to be present in presumptive GABAergic cells of the ventral lateral geniculate nucleus, nucleus of the basal optic root of the accessory optic system, and the optic tectum, among several other structures. The alpha5 subunit was also found in GABA-positive neurons, as observed in the lentiform nucleus of the mesencephalon and other pretectal nuclei. The numbers of alpha8- and alpha5-positive neurons that were also GABA-positive represented high percentages of the total number of neurons containing nicotinic receptor labeling in several brain areas, which indicates that most of the alpha8 and alpha5 nicotinic receptor subunits are present in GABAergic cells. Taken together with data from other studies, our results indicate an important role of the nicotinic acetylcholine receptors in the functional organization of GABAergic circuits in the visual system.

Effects of lipopolysaccharide on low- and high-density cultured rabbit vascular smooth muscle cells: differential modulation of nitric oxide release, ERK1/ERK2 MAP kinase activity, protein tyrosine phosphatase activity, and DNA synthesis

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 µM) and low (K0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß)2 association.

The origin of cortical neurons

Neurons of the mammalian cerebral cortex comprise two broad classes: pyramidal neurons, which project to distant targets, and the inhibitory nonpyramidal cells, the cortical interneurons. Pyramidal neurons are generated in the germinal ventricular zone, which lines the lateral ventricles, and migrate along the processes of radial glial cells to their positions in the developing cortex in an `inside-out' sequence. The GABA-containing nonpyramidal cells originate for the most part in the ganglionic eminence, the primordium of the basal ganglia in the ventral telencephalon. These cells follow tangential migratory routes to enter the cortex and are in close association with the corticofugal axonal system. Once they enter the cortex, they move towards the ventricular zone, possibly to obtain positional information, before they migrate radially in the direction of the pial surface to take up their positions in the developing cortex. The mechanisms that guide interneurons throughout these long and complex migratory routes are currently under investigation.

JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.

High concentrations of KCl release noradrenaline from noradrenergic neurons in the rat anococcygeus muscle

The aim of the present study was to investigate the effects of high concentrations of KCl in releasing noradrenaline from sympathetic nerves and its actions on postsynaptic alpha-adrenoceptors. We measured the isotonic contractions induced by KCl in the isolated rat anococcygeus muscle under different experimental conditions. The contractile responses induced by KCl were inhibited by alpha-adrenoceptor antagonists in 2.5 mM Ca2+ solution. Prazosin reduced the maximum effect from 100 to 53.9 ± 10.2% (P<0.05) while the pD2 values were not changed. The contractile responses induced by KCl were abolished by prazosin in Ca2+-free solution (P<0.05). Treatment of the rats with reserpine reduced the maximum effect induced by KCl as compared to the contractile responses induced by acetylcholine from 339.5 ± 157.8 to 167.3 ± 65.5% (P<0.05), and increased the pD2 from 1.57 ± 0.01 to 1.65 ± 0.006 (P<0.05), but abolished the inhibitory effect of prazosin (P<0.05). In contrast, L-NAME increased the contractile responses induced by 120 mM KCl by 6.2 ± 2.3% (P<0.05), indicating that KCl could stimulate the neurons that release nitric oxide, an inhibitory component of the contractile response induced by KCl. Our results indicate that high concentrations of KCl induce the release of noradrenaline from noradrenergic neurons, which interacts with alpha1-adrenoceptors in smooth muscle cells, producing a contractile response in 2.5 mM Ca2+ (100%) and in Ca2+-free solution, part of which is due to a direct effect of KCl on the rat anococcygeus muscle.

A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body.

Tacrolimus (FK506) reduces ischemia-induced hippocampal damage in rats: a 7- and 30-day study

The neuroprotective effect of the immunosuppressant agent FK506 was evaluated in rats after brain ischemia induced for 15 min in the 4-vessel occlusion model. In the first experimental series, single doses of 1.0, 3.0 or 6.0 mg FK506/kg were given intravenously (iv) immediately after ischemia. In the second series, FK506 (1.0 mg/kg) was given iv at the beginning of reperfusion, followed by doses applied intraperitoneally (ip) 6, 24, 48, and 72 h post-ischemia. The same protocol was used in the third series except that all 5 doses were given iv. Damage to the hippocampal field CA1 was assessed 7 or 30 days post-ischemia on three different stereotaxic planes along the septotemporal axis of the hippocampus. Ischemia caused marked neurodegeneration on all planes (P<0.001). FK506 failed to provide neuroprotection to CA1 both when applied iv as a single dose of 1.0, 3.0 or 6.0 mg/kg (experiment 1), and after five iv injections of 1.0 mg/kg (experiment 3). In contrast, the repeated administration of FK506 combining iv plus ip administration reduced CA1 cell death on all stereotaxic planes both 7 and 30 days post-ischemia (experiment 2; P<=0.01). Compared to vehicle alone, FK506 reduced rectal temperature in a dose-dependent manner (P<=0.05); however, this effect did not alter normothermia (37ºC). FK506 reduced ischemic brain damage, an effect sustained over time and apparently dependent on repeated doses and on delivery route. The present data extend previous findings on the rat 4-vessel occlusion model, further supporting the possible use of FK506 in the treatment of ischemic brain damage.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Radiography, computed tomography and magnetic resonance imaging at 0.5 Tesla of mechanically inducedosteoarthritis in rabbit knees

In the present experimental study we assessed induced osteoarthritis data in rabbits, compared three diagnostic methods, i.e., radiography (XR), computed tomography (CT) and magnetic resonance imaging (MRI), and correlated the imaging findings with those obtained by macroscopic evaluation. Ten young female rabbits of the Norfolk breed were used. Seven rabbits had the right knee immobilized in extension for a period of 12 weeks (immobilized group), and three others did not have a limb immobilized and were maintained under the same conditions (control group). Alterations observed by XR, CT and MRI after the period of immobilization were osteophytes, osteochondral lesions, increase and decrease of joint space, all of them present both in the immobilized and non-immobilized contralateral limbs. However, a significantly higher score was obtained for the immobilized limbs (XT: P = 0.016, CT: P = 0.031, MRI: P = 0.0156). All imaging methods were able to detect osteoarthritis changes after the 12 weeks of immobilization. Macroscopic evaluation identified increased thickening of joint capsule, proliferative and connective tissue in the femoropatellar joint, and irregularities of articular cartilage, especially in immobilized knees. The differences among XR, CT and MRI were not statistically significant for the immobilized knees. However, MRI using a 0.5 Tesla scanner was statistically different from CT and XR for the non-immobilized contralateral knees. We conclude that the three methods detected osteoarthritis lesions in rabbit knees, but MRI was less sensitive than XR and CT in detecting lesions compatible with initial osteoarthritis. Since none of the techniques revealed all the lesions, it is important to use all methods to establish an accurate diagnosis.

Abnormalities of hippocampal signal intensity in patients with familial mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (MTLE) is associated with hippocampal atrophy and hippocampal signal abnormalities. In our series of familial MTLE (FMTLE), we found a high proportion of hippocampal abnormalities. To quantify signal abnormalities in patients with FMTLE we studied 152 individuals (46 of them asymptomatic) with FMTLE. We used NIH-Image® for volumetry and signal quantification in coronal T1 inversion recovery and T2 for all cross-sections of the hippocampus. Values diverging by 2 or more SD from the control mean were considered abnormal. T2 hippocampal signal abnormalities were found in 52% of all individuals: 54% of affected subjects and 48% of asymptomatic subjects. T1 hippocampal signal changes were found in 34% of all individuals: 42.5% of affected subjects and 15% of asymptomatic subjects. Analysis of the hippocampal head (first three slices) revealed T2 abnormalities in 73% of all individuals (74% of affected subjects and 72% of asymptomatic subjects) and T1 abnormalities in 59% (67% of affected subjects and 41% of asymptomatic subjects). Affected individuals had smaller volumes than controls (P < 0.0001). There was no difference in hippocampal volumes between asymptomatic subjects and controls, although 39% of asymptomatic patients had hippocampal atrophy. Patients with an abnormal hippocampal signal (133 individuals) had smaller ipsilateral volume, but no linear correlation could be determined. Hippocampal signal abnormalities in FMTLE were more frequently found in the hippocampal head in both affected and asymptomatic family members, including those with normal volumes. These results indicate that subtle abnormalities leading to an abnormal hippocampal signal in FMTLE are not necessarily related to seizures and may be determined by genetic factors.