Trophic effect of angiotensin II, vasopressin and oxytocin on the ventral spinal cord of rat embryo

We studied trophic effects of angiotensin II, vasopressin and oxytocin on explanted ventral spinal cord cultures derived from 13 to 14-old day rat embryos. There was a significant neurite promoting effect in angiotensin II and vasopressin-treated cultures. Angiotensin II had the most potent effect at any concentrations. It became clear that minimum effective concentration was 10(-8)M in both angiotensin II and vasopressin. However, oxytocin had no neurotrophic effect at any concentrations. Our results demonstrated that angiotensin II and vasopressin have a neurotrophic effect on ventral spinal cord in cultures, and may contribute to therapeutic strategy of amyotrophic lateral sclerosis.     

Neurotrophic effect of angiotensin II, vasopressin and oxytocin on the ventral spinal cord of rat embryo

We studied trophic effects of angiotensin II, vasopressin and oxytocin on explanted ventral spinal cord cultures from 13-14-old day rat embryos. There was a significant neurite promoting effect in angiotensin II and vasopressin-treated cultures. Angiotensin II had the most potent effect at any concentrations. It became clear that minimum effective concentration was 10(-8) M in angiotensin II and vasopressin respectively. Effect of these two neuropeptides was concentration-dependent. However, oxytocin had no neurotrophic effect at any concentrations. Our results demonstrated that angiotensin II and vasopressin have a neurotrophic effect on ventral spinal cord in cultures, and may contribute to therapeutic strategy of amyotrophic lateral sclerosis.     

Neurotrophic action of autopsied ventral spinal cord extracts in patients with amyotrophic lateral sclerosis on the ventral spinal cord of rat embryo

The effects of ventral spinal cord extracts from normal controls and patients with amyotrophic lateral sclerosis (ALS) on neurite appearance in ventral spinal cord explant of 13-day-old Sprague-Dawley rats were investigated. Ventral spinal cord extracts from normal controls and ALS were significantly more effective in stimulating growth than control medium only. There was no statistically significant difference between normal and ALS ventral spinal cord extracts in growth of neurites. Our results may be an important for the consideration of the hypothesis that ALS may be a disorder of motor neuron growth factors.     

Electrophysiological effects of angiotensin II on cultured mouse spinal cord neurons

The effects of angiotensin II (AII) on the membrane properties of cultured spinal neurons were investigated using electrophysiological methods. In 26% of neurons tested AII induced changes in membrane potential and input resistance which varied according to the concentration of applied peptide. At low concentrations (10(-6) M), AII increased input resistance by an ionic mechanism which appears to involve a reduction in Cl- conductance. At higher concentration (10(-4) M), AII evoked depolarization associated with a decrease in input resistance. This response appears to depend on an increase in Na+ conductance. Our observations indicate that AII can have multiple effects on neuronal membrane properties dependent on the concentration of applied peptide.     

Trophic effects of enkephalin, beta-endorphine and dynorphine on ventral spinal cord in culture

We studied trophic effects of enkephalin (ENK), beta-endorphine (END) and dynorphine (DYN) on explanted cultures of ventral spinal cord from 13-14 day old rat embryo. The addition of each of these three neuropeptides to the growth medium caused no changes in neurite extension and in increased number of glial cells compared to control samples. The results indicate that neurite appearance is neither prompted nor inhibited by addition of ENK, END and DYN. It is considered that ENK, END and DYN are not growth factor of cultured ventral spinal cord of rat embryo.     

Trophic effects of cholecystokinin and calcitonin gene-related peptide on ventral spinal cord in culture

We studied trophic effects of cholecystokinin (CCK) and calcitonin gene-related peptide (CGRP) on explanted cultures of ventral spinal cord from 13-14 old day rat embryo. There was a significant neurite promoting effect (NPE) in CCK-treated culture. The neuritic extension of ventral spinal cord exceeds control values 3.5-6.5 times. Otherwise, CGRP and NPE at any concentration. The possible mechanism of trophic effect of CCK on ventral spinal cord was briefly discussed. Our results may contribute to a therapeutic strategy for amyotrophic lateral sclerosis.     

Characterization of two angiotensin II binding sites in cultured mouse spinal cord neurones

Characteristics of angiotensin II (AII) binding have been determined in cultured mouse spinal cord neurones using [125I]AII and [3H]AII. The Scatchard plot of equilibrium binding was curvilinear and could be described by postulating the existence of two different classes of independent binding sites (Kd1 = 0.43 nM, Bmax1 = 12.5 fmol/1.5 X 10(6) cells; Kd2 = 25.6 nM, Bmax2 = 220 fmol/1.5 X 10(6) cells). These values are in close agreement with the Kd values obtained from kinetic studies. The high affinity binding sites appeared to be similar to the single class of sites described in other studies. The relative inhibition potency of AII-related peptides was studied. Sar1,-Leu8-AII was the most potent in inhibiting specific AII binding. The characteristics of the two AII binding sites suggest that they correspond to two receptors as described in a previous electrophysiological approach using this model in our laboratory. Taken together, these data confirm that this model of neurones in primary culture is a unique and very attractive model of receptor studies. The classical criteria necessary for positive identification of a ligand-receptor have been satisfied: saturability, reversibility, specificity and most importantly correlation of the binding parameters and biological effects of AII.     

Solubilization and characterization of covalently labeled angiotensin II receptors in cultured mouse spinal cord cells

To investigate the physicochemical characteristics of angiotensin II receptors in nervous tissue, [125I]-labeled AII was covalently linked to its binding sites in cultured mouse spinal cord cells, using the chemical crosslinking agent disuccinimidyl suberate. The liganded complexes were analyzed by SDS/polyacrylamide gel electrophoresis. Autoradiograms revealed a single band which corresponding to a component with a calculated Mr value of 68,000 +/- 5,000. The same radioactive band was observed under reducing or non-reducing conditions, although in the presence of beta-mercaptoethanol the band was less intense. This suggests that the AII binding unit may contain essential disulfide bonds close to the AII binding site. After solubilization with 1% Triton X-100, extracts were analyzed by HPLC gel filtration. Three radioactive species were identified with apparent molecular weights of 65,000; 115,000 and 185,000. Results obtained in the presence or absence of 10 nM unlabeled AII showed that these three units were common to both the high and the low affinity binding sites. The results also demonstrate that AII receptors in nervous tissue have similar physicochemical properties to those from other tissues, and that the 68,000 subunit probably contains the AII binding site.     

The ventral spinothalamic tract and other ascending systems of the ventral funiculus of the spinal cord

The ascending degeneration resulting from experimental lesions of the ventral funiculus of the spinal cord of Macaca mulatta has been studied using the Nauta technique and its variants. The ventral spinothalamic tract is shown to be independent entity with respect to the lateral spinothalamic tract; its fibers are widely distributed in the ventral funiculus and it establishes connections with the brain stem and thalamus which are analogous but not identical to those of the latter. Its role in the relay of nociceptive input is discussed in view of the similarity in hodology of the two systems and it is proposed that it may be responsible for the failure of anterolateral cordotomy to control pain on a long term basis. Other ascending systems in the ventral funiculus include the spino-olivery and spino-reticular tracts, as well as minor connections to the N. of Edinger-Westphal, the red nucleus and the superior colliculus. The projections from the ventral quadrant of the spinal cord to the brain stem are almost entirely ipsilateral until the rostral mesencephalon is reached, at which level the N. of Darkschewitz receives both ipsilateral and crossed input; the magnocellular nuclues of the medial geniculate body receives a small contribution which is mainly ipsilateral. In the thalamus the VPL receives predominantly ipsilateral projections while the input to the paralaminar nuclei is only slightly less pronounced contralaterally than ipsilaterally.     

Growth inhibitory effects of a mu opioid on cultured cholinergic neurons from fetal rat ventral forebrain, brainstem, and spinal cord

Cholinergic pathways play a role in respiration in the mammalian brain, and agents that affect respiratory function such as opioid peptides might have positive or negative neurotrophic effects during the development of these cholinergic connections. Rat fetal nerve cell cultures from developmental stages E14-E18 were established in 96-well plates from ventral forebrain (VFB), an area rich in cholinergic neurons, and from brainstem and rostral spinal cord, areas where respiratory control systems and cholinergic neurons co-exist. High affinity 3H-choline uptake was highest in E14 VFB cultures and decreased to 20% of this value by E16 and E18. Choline uptakes in E14 brainstem and spinal cord were only 20% and 13%, respectively, of E14 VFB uptake. A mu opioid receptor agonist, d-ala2-mePhe4-gly(ol)5]-enkephalin (DAMGO), was tested for its effect on somal area and neurite outgrowth in E16 cultures. Cholinergic neurons were identified by immunostaining with choline acetyltransferase antibody. DAMGO (10(-8) M) significantly decreased somal area in VFB cultures and spinal cord, but had no effect on somal area in brainstem. Naltrexone (10(-6) M) reversed this inhibition. Spinal cord cell neurite outgrowth was inhibited by DAMGO, and this inhibition was reversed by naltrexone. DAMGO had no significant effect on neurite length in VFB. Brainstem neurite length was paradoxically increased by both DAMGO and naltrexone. It was concluded that mu-selective opioid peptides inhibit growth of cultured cholinergic neurons in VFB and spinal cord, but not in the brainstem. There was no evidence for endogenous opioid activity in either VFB or spinal cord cultures.     

Neurotrophic effect of cholecystokinin on ventral spinal cord in culture

Studies of the effect of cholecystokinin on organotypic cultures of rat ventral spinal cord revealed that concentrations of 10(-4) to 10(-14) M exert a marked growth-stimulating effect. In the presence of cholecystokinin, the neuritic extension of the ventral spinal cord exceeds control values 3.5-6.5 times. The growth zone of spinal cord explants increases in cholecystokinin-treated cultures. The possible mechanism of the trophic effect of cholecystokinin on the ventral spinal cord is discussed briefly.     

Neurotrophic effect of ceruletide and LH-RH on ventral spinal cord in culture

This experiment was undertaken in order to study the probable trophic effect of ceruletide and LH-RH on cultured ventral spinal cord. The ventral spinal cord from 13-14-gestational day rat embryos were explanted, following a conventional culturing method. Explants in the culture medium were fed ceruletide and LH-RH at different concentrations. An equal volume of PBS was administered to the control explants. For quantitative analysis of the trophic effect of ceruletide and LH-RH, a numerical score from 0 to 4 was determined at the 7th culture day, based on the length and extension of neurite growth estimated. The presence of ceruletide, the neuritic extension of ventral spinal cord exceeds control values 3.5-6.3 times. The growth zone of the spinal cord explants increases in the ceruletide treated culture, LH-RH treated culture, on the other hand, had no promoting effect on neurite growth. The possible mechanism of trophic effect of ceruletide on cultured ventral spinal cord was briefly discussed. These experimental observation have important implications regarding to potential therapeutic effects of ceruletide n amyotrophic lateral sclerosis.     

Distribution and characterization of VIP-related peptides in the rat spinal cord

The possible existence in the rat spinal cord of a peptide related to VIP, VIP(22–28), has been evaluated. VIP contains paired basic aminoacid residues at which postrranslational cleavage of these peptides might occur. The lumbo-sacral region of rat spinal cord had the most VIP(22–28)-like immunoreactivity (ir-VIP(22–28)). Chromatographic analysis of spinal extracts showed that ir-VIP(22–28) consisted of two major peaks, one eluting as authentic VIP(1–28) and the other as VIP(22–28). HPLC confirmed these results, revealing the presence of intact VIP(1–28) and two or more less hydrophobic peptides, one of which corresponded to authentic VIP(22–28). The other two components found have not yet been identified. Further studies are necessary to provide information on the biological significance of VIP(22–28).     

Stimulatory effect of opioid peptides and naloxone on rat spinal cord cells in primary dissociated culture

Opioid peptides leu-enkephalin, a synthetic analog of enkephalin dalargin and an opiate receptor blocker naloxone were studied for their morphological effect on the cells of dissociated cultures of rat spinal cord.Low density seeding of cells (3.10⁵; 6.10⁵ cells/ml) on collagen substrate was performed to document that opioid peptides increase the number of cultured cells and neurite outgrowth and lead to the activation of the initiated processes of aggregate formation. Upon higher density of plating (5.10⁶ cells/ml) with poly-l-lysine as a substrate, activation of the aggregate formation process was demonstrated, both opioid peptides and naloxone leading to an increase in the size of aggregates. Statistical treatment of the results obtained in this set of experiments documented that leu-enkephalin, dalargin and naloxone increased 2.2-, 2.2–2.6-, 2.4-fold, respectively, the size of aggregates compared to the control, i.e. the reaction of spinal cord cells to opioid peptides and opiate receptor blocker naloxon was unidirected. The total effect of opioid peptides and naloxon resulted in a 3.6-fold increase in the size of the aggregates compared to the control. The data obtained in this study allow the assumption that opioid peptides and naloxone, while activating spinal cord cells via receptors of a different type, manifest the properties of factors thus increasing survival and adhesion of spinal cord cells in culture.     

Metabolic activity of cultured rat brainstem, hippocampal and spinal cord slices

The use of cultured brain slices has become an accepted technique for the ex vivo analysis of neural mechanisms, yet the viability of this preparation is not routinely measured. The tetrazolium dye 3-(4, 5-dimethlythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by active mitochondria to an insoluble purple precipitate which accumulates within living cells and is easily visualized with bright field or phase contrast microscopy. In this study, the MTT assay was used to assess the viability of cultured brainstem, hippocampal and spinal cord slices (150-300 micrometer) from 0 to 22 day-old neonatal rats at post-explant time points ranging from 2 to 29 days. After 2 weeks, 180-300 micrometer cultured slices from 4-13 day old rats remained 90-100% viable. Those from 0-1 day old rats had similar viability but displayed peripheral tissue outgrowth. Slices from older 18-22 day rats were no longer viable after 10-14 days. After 4 weeks, the thicker (300 micrometer) slices of hippocampus and spinal cord retained 75-89% viability, in contrast to the 50-74% viability of the brainstem. Thinner brainstem and hippocampal slices (150-220 micrometer) slices were less than 50% viable at 4 weeks. Morphologic characteristics of the brain regions gradually degenerated over the 4-week culture period. Slice viability was markedly influenced by tissue thickness, donor age and brain region. Use of the MTT assay provides an inexpensive and expeditious means to assess a significant functional parameter of regional slice viability under variable conditions and enhances the feasibility of this preparation for functional studies, such as those concerned with genetic and protein expression within circumscribed areas of the brain.     

Location of angiotensin II binding sites on neuronal and glial cells of cultured mouse spinal cord: an autoradiographic study

Cultures of mouse spinal cord were used to visualize binding sites for [125I]angiotensin II (AII) by autoradiography. Visualization by light microscopy shows that neurones, but also glial cells possess angiotensin II binding sites which are located both on soma and processes. These findings open a new field of investigation for the understanding of the physiological significance of AII in the CNS.     

Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [¹²⁵I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [¹²⁵I]Sar¹–Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [¹²⁵I]Sar¹–Ang II binding was displaced by the AT₁ receptor antagonist losartan, indicating the presence of AT₁ receptors. In the paraventricular nucleus [¹²⁵I]Sar¹–Ang II binding was displaced by Ang II (K_i=7.6×10⁻⁹) and losartan (K_i=1.4×10⁻⁷) but also by the AT₂ receptor ligand PD 123319 (K_i=5.0×10⁻⁷). In addition, a low amount of AT₂ receptor binding was detected in the paraventricular nucleus using [¹²⁵I]CGP 42112 as radioligand, and the binding was displaced by Ang II (K_i=2.4×10⁻⁹), CGP 42112 (K_i=7.9×10⁻¹⁰), and PD 123319 (K_i=2.2×10⁻⁷). ACE, Ang II, and AT₁ as well as AT₂ receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.     

Release of immunoassayable neurohypophyseal peptides from rat spinal cord, in vivo

The subarachnoid space of the spinal cord was perfused in vivo in urethane-anesthetized rats and perfusates were assayed for arginine-vasopressin (AVP) and oxytocin immunoreactivity. In control perfusates, oxytocin concentrations were 3 times those of AVP. Electrical stimulation of the paraventricular nucleus (PVN) of the hypothalamus, but not of other hypothalamic areas, yielded increased amounts of immunoassayable peptides in the spinal cord perfusates. Intravenous infusion of AVP did not elevate AVP concentrations in the cord perfusates. These data suggest that electrical stimulation of PVN neurons caused release of AVP and oxytocin from spinal cord terminals and support the concept that these peptides are neurotransmitters in the cord.     

Evidence for the ventral origin of oligodendrocyte precursors in the rat spinal cord

The neuroepithelial cells of the mammalian neural tube are thought to give rise to all classes of differentiated neurons and macroglial cells in the adult CNS. In most cases, the regulation and timing of commitment of neuroepithelial cells to specific differentiative pathways are unknown. It has been proposed that in developing spinal cord, the macroglial cells--astrocytes and oligodendrocytes--arise either by the direct transformation of radial glial cells in the developing cord or, alternatively, by the differentiation of distinct precursor cells which migrate to presumptive white matter from the region of the central canal during development. In this study, the timing of oligodendrocyte differentiation in different levels of the spinal cord and the capacity of specific regions of the spinal cord to give rise to oligodendrocytes at various ages was tested in vitro. At embryonic day 14, all complete segments, as well as all ventral regions along the rostral-caudal axis of the spinal cord, have the capacity for oligodendrogenesis. By contrast, dorsal regions of the thoracic and lumbar spinal cord do not develop the capacity for oligodendrogenesis until later in development. The capacity of dorsal rat spinal cord to give rise to oligodendrocytes appears to be associated with the ventral-to-dorsal migration of oligodendrocyte precursors. These observations suggest that commitment to an oligodendrocyte differentiative pathway appears to occur in a distinct population of ventrally located glial precursors in the embryonic rat spinal cord.