Varda H. Gilad

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.

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.

Polyamines in neurotrauma: Ubiquitous molecules in search of a function

In spite of their abundance, the function of PAs in the adult nervous system remains enigmatic. It is postulated that after trauma, the induction of polyamine metabolism (i.e. the polyamine response), which is inherently transient, is an integral part of a protective biochemical program that is essential for neuronal survival. Several functions ascribed to PAs may assume importance in cellular defense. Thus, regulation of the ionic environment, modulation of signal pathways, control of cellular Ca2+ homeostasis, inhibition of lipid peroxidation, and interaction with nucleic acids are all putative sites for PA action. During maturation, the CNS, unlike the peripheral nervous system, undergoes changes which result in the expression of an incomplete polyamine response after trauma. This may be due to an altered pattern of gene expression, and/or restrictive compartmentalization of the PAs and their metabolizing enzymes. Induction of this partial polyamine response after injury results in a sustained accumulation of putrescine, which by itself may be harmful, without the concomitant increase in spermidine and spermine. Administration of exogenous PAs after trauma exerts a neuroprotective effect. Exogenous PAs are postulated to gain access into cells via an induced uptake system after trauma, and function similarly to newly synthesized PAs. Besides the injured neurons themselves, tissues which are connected or associated with these neurons may be potential targets where PAs could act to stimulate neurotrophic factor production. Based on the neuroprotective effects of PAs in laboratory animals and on their proposed role in mechanisms of neuronal survival, the development of PA-based compounds as therapeutic neuroprotective agents should be pursued.

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.

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.

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.

Chronic lithium treatment prevents the dexamethasone-induced increase of brain polymine metabolizing enzymes

The paper describes the effects of various regimens of lithium chloride treatment on dexamethasone-induced increases in brain polyamine metabolizing enzymes. In contrast to peripheral tissues where acute lithium treatment suppresses the increase in ornithine decarboxylase activity, in the brain only chronic treatment was effective in preventing this increase and also the increases in the activities of S-adenosylmethionine decarboxylase and spermidine/spermine N1-acetyltransferase. This findings indicate a novel brain target for lithiums action and in turn provide new avenues for exploring polyamine function in the brain.

Accumulation of exogenous polyamines in gerbil brain after ischemia

Regionally selective delayed neuronal degeneration is a characteristic sequel of cerebral ischemia. Recent evidence indicates that changes in brain polyamine metabolism may be critical for nerve cell survival after ischemia. Within hours after ischemia, intracellular putrescine levels are greatly increased and remain elevated for days, whereas only minor changes are noted in the levels of the polyamines spermine and spermidine. In contrast, the extracellular levels of all polyamines are low after ischemia. Injections of polyamines following ischemia, however, can protect neurons in the gerbil brain from delayed cell death, with spermine being the most potent of the polyamines. In the present study, therefore, we sought to determine if increased polyamine uptake occurs in the brain after ischemia. In the hippocampal slice preparation, temperature-dependent uptake was unique for spermine, but not for spermidine or putrescine. Uptake of [14C]spermine was transiently increased after ischemia, peaking at 150% of control by 12-13 h and subsiding by 24 h. Intravenous injections of [3H]spermidine resulted in a postischemic accumulation of this polyamine throughout the forebrain parenchyma. We conclude that: 1. Active cellular uptake of spermine is transiently increased early after ischemia; 2. A nonspecific accumulation of exogenous polyamines occurs early after ischemia probably owing to a compromised blood-brain barrier, and 3. The findings indicate that exogenous polyamines can exert their effect directly in the brain after ischemia.

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 restraint 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.

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.