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Biomedical subjects

V H Gilad

Publications and source records attributed to V H Gilad.

At least 37 records · Page 2Linked to original sources
PubMed1992

Polyamines modulate the binding of GABAA-benzodiazepine receptor ligands in membranes from the rat forebrain.

The effects of spermine, spermidine and putrescine on the binding of the GABAA-benzodiazepine receptor complex were examined in the hippocampus and frontal cortex membranes of the rat. The results demonstrated modulatory effects of polyamines on the binding of diazepam and flunitrazepam but not on that of GABA, muscimol and Ro 15-1788. When membranes were prepared without detergent, the polyamines enhanced the binding of diazepam. However, while the binding capacity increased after homogenization in the presence of the non-ionic detergent Triton X-100, the polyamines did not enhance the binding but inhibited the binding of diazepam and flunitrazepam at greater concentrations. Considered together with other studies, the present findings indicate that polyamines can modulate the binding characteristics of several different neurotransmitter receptor-ionophore complexes.

Animals↗
PubMed1992

Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia.

The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria dishes have a modified plastic surface with a net positive charge). On both polylysine and Primaria substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.

Animals↗
PubMed1992

Brain filament proteins in primary cultures derived from chick embryos early in development.

Brain filament expression and neurofilament post-translational modifications (phosphorylations) were studied in primary cultures derived from whole 3-4 day chick embryos. After 2-3 days in culture, neurofilament-positive cells formed neuronal aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. Aggregates and neuritic bundles were stained with several monoclonal antibodies reacting with phosphorylated neurofilament epitopes. With two monoclonal antibodies reacting with phosphorylated forms of the high molecular weight neurofilament subunit, staining was only observed after 8 and 10 days in vitro. There was a major difference between rat and chicken with respect to astrocyte differentiation in culture. In chicken, the flat cells surrounding the neuronal aggregates remained constantly GFAP-negative throughout the whole experimental period (10 days). GFAP-positive cells were first observed within the neuronal aggregates on day 8 in vitro.

Animals↗
PubMed1991

Polyamine uptake, binding and release in rat brain.

The uptake, binding and release of the polyamines, spermidine and spermine, and of their diamine precursor, putrescine, were examined in synaptosomal preparations from rat hippocampus. The specific and relatively high-affinity uptake by synaptosomes was found only with putrescine (Vmax = 21.6 pmol/mg protein per h; Km = 28.6 nM) and not with the other polyamines. In contrast, specific binding to membranes was found for spermidine (Bmax = 28.6 pmol/mg protein; Kd = 42.9 nM) and for spermine (Bmax = 156.3 pmol/mg protein; Kd = 83.3 nM), but not for putrescine. High potassium concentrations (35 mM) both induced the release of accumulated polyamines from synaptosomes and inhibited their binding. Specific polyamine binding evidently occurs selectively on the inner but not on the outer synaptosomal membranes.

Animals↗
PubMed1991

Polyamines can protect against ischemia-induced nerve cell death in gerbil forebrain.

We have previously demonstrated that administration of the polyamines putrescine, spermidine, or spermine can prevent neuronal degeneration in rats during naturally occurring cell death or after injurious treatments such as nerve injury or monosodium glutamate neurotoxicity. The present study demonstrates that also in adult gerbils polyamine treatment can protect forebrain neurons from degeneration after ischemia. Neurons in the hippocampus and striatum were rescued from delayed cell death after brief (5 min) global ischemia in gerbils which were treated with daily injections (10 mg/kg) of polyamines. The evidence accrued, so far, indicates that systemic polyamines can protect a wide variety of central and peripheral neurons from natural or induced degeneration.

Analysis of Variance↗
PubMed1990

Region-selective stress-induced increase of glutamate uptake and release in rat forebrain.

The study describes stress-induced changes in high-affinity uptake and release of glutamate by synaptosomal preparations from several regions of rat brain. The results demonstrate that restraint stress can lead to increased glutamate uptake and release in limbic forebrain regions (frontal cortex, hippocampus and septum) but not in the striatum. The increase in glutamate uptake was evident after 30 min of stress. A plateau (140-150% of unhandled controls) was reached after 1 h and was maintained after 4 h of continuous stress. The stress-induced increase in glutamate uptake was observed with glutamate concentrations of up to 10 microM, but not with 500 microM. the results indicate that forebrain glutamatergic terminals are activated by stressful stimuli in a regionally selective manner, and suggest that enhanced high-affinity uptake is important in clearing increased levels of released glutamate.

Animals↗
PubMed1990

Dibutyryl cyclic AMP-induced changes in neuron-astroglia interactions and fibronectin immunocytochemistry in dissociated rat cerebellar cultures.

In mixed primary embryonic CNS cultures flat astroglia grow exclusively underneath the initially formed neuronal network. This invasive under-growth results in neuronal detachment and degeneration. The present study sought to find out whether or not morphological differentiation of astroglia, from flat to process-bearing cells, could alter astroglial-neuronal growth relationships in rat cerebellar cultures. Morphological differentiation of astroglia was induced by treatment with dibutyryl cyclic AMP. The results demonstrate that in contrast to flat astroglia, large stellate astroglia can grow over the neurite bundles, and that in these dibutyryl cyclic AMP-treated cultures neurons can persist. Immunocytochemical studies show that the extracellular matrix protein fibronectin is present in these cultures and appears to be associated with flat astroglia rather than with stellate astroglia. The study indicates that in the presence of dibutyryl cyclic AMP transformed stellate astroglia interact differently with neurons and with the growth substratum as compared with flat astroglia.

Animals↗
PubMed1990

Aging and stress-induced changes in choline and glutamate uptake in hippocampus and septum of two rat strains differing in longevity and reactivity to stressors.

Stress induced changes in neurochemical indices of neurotransmission are more pronounced in the septohippocampal cholinergic system of Wistar Kyoto rats, which are behaviorally more reactive to stressors and have a shorter life span, than in Brown Norway rats. Moreover, pronounced degeneration of septohippocampal cholinergic neurons occurs earlier in life in Wistar Kyoto rats. In the present study the high affinity synaptosomal uptakes of choline and glutamate were used as indices for cholinergic and glutamatergic systems respectively. Following 2 hr of mild restrain stress increases in both uptake systems were observed in all regions examined (hippocampus, septum and frontal cortex). The stress-induced increases were generally similar in young (3 months) and aged (20 months) rats of both strains. The noted exception was that choline uptake levels, which were reduced in the hippocampus of unhandled aged WKY rats, remained unchanged after stress. The results confirm the involvement of the septohippocampal cholinergic system in the response to acute stress and extend the findings to include the hippocamposeptal glutamatergic system activation as well. It is suggested that in spite of neuronal degeneration during aging, these responses to stress can be maintained by compensatory efforts of neurons that remain intact.

Aging↗
PubMed1989

Effects of chronic stressors or corticosterone treatment on the septohippocampal cholinergic system of the rat.

The effects of prolonged (2 months) corticosterone (CORT) treatment on several cholinergic markers of various brain areas were compared to the effects of prolonged intermittent exposure to stress. CORT, but not stress, caused a significant reduction in the number of acetylcholinesterase-stained neurons in the medial septal area. Neither treatment resulted in any hippocampal pyramidal cell loss. It is concluded that a time-dependent degeneration of the septohippocampal cholinergic system follows 2 months of CORT administration but not chronic intermittent stress of this duration.

Anesthesia↗
PubMed1989

Expression of neurofilament immunoreactivity in developing rat cerebellum in vitro and in vivo.

The developmental expression of neurofilaments immunoreactivity was examined in frozen sections and in primary cultures of rat cerebellum by immunocytochemistry with a series of monoclonal antibodies and with a polyclonal antibody. In tissue sections immunocytochemical staining with all the antibodies used was observed in basket cells where adult-like appearance could be detected by 14 days of age and adult-level intensity was achieved by about 25 days. Granule cells remained unstained. Intense staining appeared in cerebellar white matter as early as 7 days after birth. In contrast, neurofilaments immunoreactivity was detected in cultured granule cells from 7-day-old cerebellum. Only polyclonal antibodies reacting with the highly conserved middle alpha-helical domain of the neurofilament subunits were reactive in culture. Staining could be detected in the nerve cell bodies from the first day after plating; thereafter staining intensity increased and was also distributed in neurite extensions. We conclude that unlike their counterparts in vivo cultured embryonic granule cells can express certain neurofilaments immunoreactivity.

Animals↗
PubMed1989

Treatment with polyamines can prevent monosodium glutamate neurotoxicity in the rat retina.

It has been previously shown that treatment of newborn rats with the polyamines putrescine, spermidine and spermine can rescue sympathetic neurons from naturally occurring cell death and from induced death after axotomy or immunosympathectomy. The present study demonstrates that polyamine treatment can also prevent the neurodegenerative effects in the retina and the loss of body weight caused by monosodium glutamate. The findings indicate that polyamine treatment may have a rather general beneficial effect on neuron survival.

Animals↗
PubMed1989

Polyamines induce precocious development in rats. Possible interaction with growth factors.

The study reports the effects of daily subcutaneous injections of the biogenic polyamines putrescine, spermidine and spermine (10 mg/kg each) given for a short postnatal period, on growth and development of rats. Polyamine treatment, while only slightly enhancing normal body weight gain, prevented the weight loss caused by surgical injury of 5-day-old animals. The treatment resulted in earlier eyelid and ear opening and in earlier maturation of righting and gripping responses. Increased number of neurons in the superior cervical ganglion that is caused by polyamine treatment, could not be prevented by castration of newborn rats, thus excluding the testes as a site through which polyamines may exert their action. An apparent increase in immunohistochemically detectable nerve growth factor was evident in iris and submaxillary salivary gland of polyamine-treated animals, but no change in epidermal growth factor immunohistochemistry was detected in the salivary gland. We conclude: (1) treatment of newborn rats with polyamines can accelerate somatic and neurobehavioral development; (2) further studies are required in order to verify and quantitate the effects of polyamines on growth factors, and (3) the results imply that exogenous polyamines may exert their growth-promoting effects on a number of cell types when these cells experience periods of polyamine dependence.

Animals↗
PubMed1989

Effects of glycosaminoglycans and proteinase inhibitors on astroglia-induced detachment of cultured rat cerebellar neurons.

Neurons in mixed primary embryonic CNS cultures degenerate secondary to their detachment from the substratum. The present study demonstrates that in primary cultures of postnatal cerebellum, detachment of neurons can be prevented by antiproliferative drugs which inhibit the growth of astroglia. Several types of proteinase inhibitors did not affect the process of detachment. However, among several types of glycosaminoglycans, heparan sulfate and to a lesser degree heparin, could reversibly inhibit neuron detachment without causing morphological changes of astroglia. The enzymes heparitinase and heparinase caused neuron detachment but only within the first 24-48 hr after plating and not in older cultures. We conclude: (1) cerebellar interneurons in culture are not dependent on astroglia for their survival; (2) astroglia are most probably responsible for neuron detachment via a membrane associated activity and (3) heparan sulfate-like glycosaminoglycans are important in neuron-substratum attachment.

Animals↗
PubMed1988

Astroglia-induced detachment of central neurons but astroglia-dependent growth of peripheral neurons in rat embryonic spinal cord primary cultures.

In mixed primary cultures, intrinsic neurons from embryonic mammalian brains degenerate secondary to their detachment from the substratum and this is caused by the under-growing co-cultured astroglia. In the present study we sought to find out whether or not peripheral neurons, sensory and motor neurons which reside and/or only project outside the CNS respectively, interact with astroglia similarly as their central counterparts do. Mixed primary cultures prepared from dissociated embryonic rat spinal cord and dorsal root ganglia were examined by phase and immunofluorescence microscopy after labeling with antibodies to neurofilaments (neuronal markers) and to glial fibrillary acidic protein and vimentin (astroglia markers). Acetylcholinesterase staining served as a marker for motor neurons. In this system astroglia grew exclusively under intrinsic neurons of the spinal cord and with time (about 8 days) all these neurons detached and disappeared. In contrast, astroglia were intimately associated with perikarya of peripheral neurons, sometimes growing over them. Furthermore, the neuritic processes of these neurons were attached to the undergrowing astroglia. Central neurons could be rescued by treatment of cultures with the antimitotic drug cytosine arabinoside which led to the elimination of astroglia. However, this treatment resulted in death of all peripheral neurons. We conclude: (1) survival of intrinsic CNS neurons in culture is independent of astroglia; (2) astroglia are responsible for the detachment of these neurons from their growth substratum; (3) survival of peripheral sensory and motoneurons is dependent on co-cultured astroglia and (4) the differences in neuron-astroglia interactions between central and peripheral neurons are membrane-associated and probably independent of soluble factors.

Animals↗
PubMed1988

Retrograde changes in transglutaminase activity after peripheral nerve injuries.

It has been previously demonstrated that transglutaminase activity in rat superior cervical ganglion is rapidly and transiently increased minutes after nerve injury. The present series of experiments sought to determine: (1) whether or not similar changes are expressed by other peripheral neuronal systems, and (2) if injury-induced changes in the enzyme activity can be detected along the injured nerve, and if so do they occur in axons or in non-neuronal cells. In the nodose ganglion transglutaminase activity increased (approximately 40%) 48 h after the vagus nerve was crushed 25 mm from the ganglion. In the vagus nerve the activity was transiently increased (approximately 100%) within 1 h, followed by a second increase (approximately 140%) after 3 h. This occurred only in the proximal nerve stump close to the injury site and not in the section of nerve closer to the ganglion. Comparable enzyme activity was found in unoperated vagus nerve and in distal stumps of previously ligated vagus nerves. In dorsal root ganglia no changes were found for up to 24 h after the sciatic nerve was crushed 40 mm from the ganglion. In the facial nucleus a transient increase was observed after the facial nerve was crushed about 14 mm distally with a peak (approximately 300) at 3 days and a decline within 14 days. A second lesion of the facial nerve made 12 days following a conditioning lesion led to a rebound of enzyme activity in the facial nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Early polyamine treatment enhances survival of sympathetic neurons after postnatal axonal injury or immunosympathectomy.

We have recently demonstrated that following injury of their axon, sympathetic neurons of the rat superior cervical ganglion become dependent on polyamine synthesis for their survival. In addition we have observed that the treatment of newborn rats with biogenic polyamines can prevent the naturally occurring reduction in the number of neurons in the ganglion. In the present study groups of newborn rats were subjected to either postganglionic nerve crush (axotomy) or to treatment with antiserum to nerve growth factor (immunosympathectomy), two treatments which result in a massive loss of neurons in the ganglion. Daily injections of the polyamines putrescine, spermidine and spermine (10 mg/kg each), for 7 days after the operation to the axotomized group, and for 9 days starting with the first antiserum injection to the immunosympathectomized group, attenuated the nerve cell loss. The polyamine treatment also attenuated the reduction in the activity of the neurotransmitter-synthesizing enzyme tyrosine hydroxylase observed after both axotomy and immunosympathectomy in the ganglion. These results further indicate that polyamines are important for the survival of sympathetic neurons and, while their mechanism of action is unknown, an interaction with nerve growth factor regulation cannot be excluded. In the iris, the reduction observed in [3H]norepinephrine uptake after the two noxious treatments was unproportionately small when compared to the large drop in the number of parent neurons in the ganglion. This suggests that compensatory mechanisms exist which act to adjust the number of functional axon terminals per neuron so that the number of terminals innervating the target remains relatively constant.

Animals↗
PubMed1987

Age-dependent loss and compensatory changes of septohippocampal cholinergic neurons in two rat strains differing in longevity and response to stress.

Inbred Wistar-Kyoto rats which are behaviorally more reactive to stress have a shorter life span than Brown-Norway rats. This is paralleled by higher basal activity and more pronounced changes in the septohippocampal cholinergic system of Wistar-Kyotos after stress. Age- and strain-dependent differences were therefore characterized in the septohippocampal system of 3- and 24-month-old (aged) Wistar-Kyotos and Brown-Norways, and in 30-month-old Brown-Norways. High affinity [3H]choline uptake and newly synthesized [3H]acetylcholine release served as markers for cholinergic terminals in the hippocampus. [3H]Quinuclidinylbenzilate binding served as a marker of muscarinic receptors in the hippocampus. Choline acetyltransferase activity served as a marker for cholinergic neurons and their terminals in the septum and hippocampus respectively. Acetylcholinesterase histochemical staining served to localize cholinergic neurons and their terminals in the septum and hippocampus respectively. In the hippocampus of aged Wistar-Kyotos choline uptake and acetylcholine release were reduced by approximately 50% compared to their young counterparts, but remained unchanged in aged Brown-Norways. Hippocampal choline acetyltransferase activity, acetylcholinesterase staining and muscarinic binding were unchanged in aged rats of both strains. Pyramidal cell loss (observed in Cresyl violet stained sections) was detected in hippocampus of 24-month-old Wistar-Kyotos and 30-month-old, but not younger Brown-Norways. Numbers of acetylcholinesterase-stained cells in the septum were reduced by 45 and 25% in 24-month-old Wistar-Kyotos and Brown-Norways respectively, and by 50% in 30-month-old Brown-Norways. Mean diameter of these cells was increased only in aged Wistar-Kyotos (approximately 46%) and in 30-month-old Brown-Norways (40%). The results indicate: (1) there is an ongoing age-dependent degeneration of septohippocampal cholinergic neurons which is associated with two principal compensatory changes in remaining cholinergic neurons: (a) hypertrophy of perikarya and (b) relative increase in activity of presynaptic markers in terminals with unchanged regional distribution, suggesting possible collateral sprouting; (2) age-dependent loss of septal cholinergic neurons precedes loss of hippocampal pyramidal neurons and (3) loss of pyramidal neurons in the hippocampus is associated with a compensatory increased muscarinic binding by remaining target hippocampal neurons. The results imply that higher basal and stress-induced activity of septohippocampal cholinergic neurons may be correlated with an accelerated and more pronounced age-dependent degeneration of this cholinergic system.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗