Up-regulation of glutamate receptors is associated with LTP defects in the early stages of diabetes mellitus.

AIMS/HYPOTHESIS: Recent studies involving electrophysiology and immunolabelling indicate that short-term insulin treatment of hippocampal neurons in culture induces changes in glutamate receptor function, suggesting that this receptor system can be altered on a relatively rapid time scale during diabetic conditions. To investigate this hypothesis, we examined whether brain glutamate receptors and long-term potentiation are altered in the early stages of diabetes mellitus in non-obese diabetic mice, a genetic model of Type I (insulin-dependent) diabetes mellitus. METHODS: In vitro receptor autoradiography and immunoblotting were used to study the impact of diabetes on brain glutamate receptors. From an electrophysiological point of view, field potential recordings were also examined in area CA1 of hippocampal slices to determine the influence of diabetes on long-term potentiation. RESULTS: Quantitative autoradiographic analysis revealed enhanced 3H-glutamate binding to several brain regions of diabetes mice, with maximal increases in the cerebral cortex and hippocampus. Saturation kinetics within the cerebral cortex disclosed that this change of 3H-glutamate was possibly due to an increase in the maximal number of N-methyl- D-aspartate binding sites, an interpretation that was corroborated by Western blot analysis of N-methyl- D-aspartate 2A subunits. Impairment in the expression of hippocampal long-term potentiation was also observed in diabetic mice, while the failure to elicit synaptic potentiation was prevented by insulin treatment. CONCLUSION/INTERPRETATION: Because glutamate receptors are thought to be involved in several degenerative processes, our results suggest that up-regulation of these receptors in the early stages of diabetes could represent an important mechanism underlying neurological complications within the brain of diabetic patients.

AMPA receptor regulation and LTP in the hippocampus of young and aged apolipoprotein E-deficient mice.

In the present study, modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors by phosphatidylserine (PS) and synaptic plasticity were investigated in the hippocampus of young (4-month-old) and aged (18-month-old) apolipoprotein E (apoE)-deficient mice. Qualitative as well as quantitative analysis of brain sections in both young and aged apoE-deficient mice did not reveal any substantial changes of AMPA receptor binding in the various hippocampal regions, compared to age-matched controls. Nevertheless, enhancement of AMPA receptor binding elicited by PS treatment was found to be abolished in most hippocampal regions of young apoE-deficient mice, while modulation of AMPA receptors by this phospholipid was not significantly altered in the hippocampal formation of aged apoE-deficient animals. At the electrophysiological level, long-term potentiation (LTP) induced by theta burst stimulation was lower in area CA1 of the hippocampus of young, but not aged, apoE-deficient mice compared to age-matched controls. These results confirm that apoE is important for AMPA receptor regulation and LTP expression in the hippocampal formation. However, the presence of LTP in aged apoE-deficient animals, together with apparent recovery of the PS action on AMPA receptors, suggests that aged apoE-knockout mice possess compensatory mechanisms that reduce biochemical and electrophysiological alterations of glutamatergic neurons.

Increased density of glucagon receptors in liver from endurance-trained rats.

The binding properties of glucagon receptors were determined in plasma membranes isolated from liver of untrained (n = 6) and swimming endurance-trained Sprague-Dawley male rats (n = 7; 3 h/day, 5 days/wk, for 8 wk). Plasma membranes were purified from liver by aqueous two-phase affinity partitioning, and saturation kinetics were obtained by incubation of plasma membranes (10 microg of proteins/150 microl) with (125)I-labeled glucagon at concentrations ranging from 0.15 to 3.0 nM for 30 min at 30 degrees C. Saturating curve analysis indicated no difference in the affinity of glucagon receptors (0.57 +/- 0.06 and 0.77 +/- 0.09 nM in untrained and trained groups, respectively) but a significant higher glucagon receptor density in liver from untrained vs. trained rats (3.09 +/- 0.12 vs. 4.28 +/- 0.19 pmol/mg proteins). These results suggest that the reported increase in liver glucagon sensitivity in endurance-trained subjects (Drouin R, Lavoie C, Bourque J, Ducros F, Poisson D, and Chiasson J-L. Am J Physiol Endocrinol Metab 274: E23-E28, 1998) could be partly due to an increased glucagon receptor density in response to training.

Inositol hexakisphosphate-mediated regulation of glutamate receptors in rat brain sections.

D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), one of the most abundant inositol phosphates within cells, has been proposed to play a key role in vesicle trafficking and receptor compartmentalization. In the present study, we used in vitro receptor autoradiography, subcellular fractionation, and immunoblotting to investigate its effects on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Qualitative and quantitative analysis of 3H-AMPA binding indicated that incubation of frozen-thawed brain sections with InsP6 at 35 degrees C enhanced AMPA receptor binding in several brain regions, with maximal increases in the hippocampus and cerebellum. Moreover, saturation kinetics demonstrated that InsP6-induced augmentation of AMPA binding was due to an increment in the maximal number of AMPA binding sites. At the immunological level, Western blots performed on crude mitochondrial/synaptic (P2) fractions revealed that InsP6 (but not InsP5 and InsP3) treatment increased glutamate receptor (GluR)1 and GluR2 subunits of AMPA receptors, an effect that was associated with concomitant reductions in microsomal (P3) fractions. Interestingly, the InsP6-induced modulation of AMPA receptor binding was blocked at room temperature, and pretreatment with heparin also dampered its action on both AMPA receptor binding and GluR subunits. These effects of InsP6 appear to be specific to AMPA receptors, as neither 3H-glutamate binding to NMDA receptors nor levels of NR1 and NR2A subunits in P2 and P3 fractions were affected. Taken together, our data strongly suggest that InsP6 specifically regulates AMPA receptor distribution, possibly through a clathrin-dependent process.

Modification of glutamate receptors by phospholipase A2: its role in adaptive neural plasticity.

Long-term potentiation (LTP) and long-term depression (LTD) are two electrophysiological models that have been studied extensively in recent years as they may represent basic mechanisms in many neuronal networks to store certain types of information. In several brain regions, it has been shown that these two forms of synaptic plasticity require sufficient dendritic depolarization, with the amplitude of the calcium signal being crucial for the generation of either LTP or LTD. The rise in calcium concentration mediated by the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has been proposed to stimulate various calcium-dependent enzymatic processes that could convert the induction signal into long-lasting changes in synaptic structure; protein kinases and phosphatases have so far been considered predominantly with regard to LTP and LTD formation. According to several lines of experimental evidence, changes in synaptic function observed with LTP and LTD are thought to be the result of modifications of postsynaptic currents mediated by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors. Moreover, it has become apparent recently that activation of the calcium-dependent enzyme phospholipase A2 (PLA2) could be part of the molecular mechanisms involved in alterations of AMPA receptor properties during long-term changes in synaptic operation. In the present review, we will first describe the results that indicate a critical role of the phospholipases in regulating synaptic function. Next, sections will be devoted to the effects of PLA2 and phospholipids on the binding properties of glutamate receptors, and a revised biochemical model will be presented as an attempt to integrate the PLA2 enzyme into the mechanisms ( in particular kinases and phosphatases) that participate in adaptive neural plasticity. Finally, we will review data relevant to the issue of selective changes in AMPA binding after environmental enrichment and LTP.

Postnatal development of thalamocortical projections upon striate and extrastriate visual cortical areas in the cat.

The development of visual thalamocortical projections was analyzed quantitatively by comparing, in cresyl violet-stained brain sections of early postnatal (10-17 days) and adult cats, the cell body dimensions and total cell packing density (CPD) of neuronal populations in different laminae (A, A1 and C) of the dorsal lateral geniculate (dLGN), medial interlaminar nucleus (MIN), and in lateral (LPl), intermediate (LPi) and medial (LPm) subdivisions of the lateral posterior complex. Following injections of different fluorescent tracers (FB, NY, EB, RITC) into cortical visual areas 17/18, posterior medial (PMLS) and posterior lateral (PLLS) lateral suprasylvian and anterior ectosylvian (AEV), the thalamic distribution and densities of retrogradely labeled neurons were analyzed. Projection CPDs and ratios of projection/total CPDs were determined and compared within the different thalamic components in the kitten and adult cat. A significant decrease in total cell packing density was observed in the various thalamic components of the adult cat, varying between 43% and 65%, and a marked increase in mean cell body diameter in the A, A1 and C laminae and MIN from kitten to adult (8.4+/-1.8 and 11.8+/-2.8 microm respectively) compared to the LP subnuclei (9.0+/-1.3 and 9.1+/-1.5 microm). The ratios of projection/total CPDs decreased significantly for projections upon areas 17/18 stemming from layers A and A1 (20 and 25%, respectively) and from LPi upon both PMLS (34%) and AEV (16%). Thalamocortical projections observed in the kitten from LPi upon areas 17/18 and from the A-laminae upon PMLS were absent in the adult cat. The data indicate that, in comparison to the lateral posterior nucleus, the maturation of neurons within the dLGN and MIN is incomplete with respect to cell body size during the early postnatal period. In addition, the developmental changes observed involve both reductions in the total number of thalamic neurons and a differential loss of cortical projections. The selective elimination of early cortical connections stemming from dorsal lateral geniculate laminae A and A1 and from the intermediate division of the lateral posterior nucleus may occur through a process of axon collateral withdrawal from the expanded cortical sites, thereby giving rise to the adult pattern.

Impairment of synaptic transmission by transient hypoxia in hippocampal slices: improved recovery in glutathione peroxidase transgenic mice.

There is increasing evidence that oxygen free radicals contribute to ischemic brain injury. It is unclear, however, to what extent specific antioxidant enzymes can prevent or reverse the impairment of synaptic function caused by transient hypoxia. In this study, we investigated in transgenic (Tg) mice whether a moderate increase in glutathione peroxidase-1 (GPx1) may improve the capacity of CA1 pyramidal cells to recover synaptic transmission after a short period of hypoxia in vitro. In control hippocampal slices, transient hypoxia (7-9 min) produced irreversible loss of excitatory postsynaptic potentials. Complete recovery of synaptic transmission was observed with homozygous Tg-MT-GPx-6 mice after reoxygenation, and, after repeated episodes of hypoxia, synaptic transmission was still viable in most Tg slices, in contrast to non-Tg slices. Moreover, hypoxic episodes abolished the capacity of hippocampal slices to generate long-term potentiation in area CA1 of control mice, whereas a significant extent of long-term potentiation expression was still preserved in Tg tissues. We also demonstrated that susceptibility to N-methyl-d-aspartate-mediated oxidative injury was reduced in Tg hippocampal slices. In conclusion, our results suggest that a moderate GPx increase can be sufficient to prevent irreversible functional damage produced by transient hypoxia in the hippocampus and to help maintain basic electrophysiological mechanisms involved in memory formation.

AMPA receptor modulation in previously frozen mouse brain sections: opposite effects of calcium in the cortex and hippocampus.

Various forms of synaptic plasticity in the brain have been proposed to result from modifications in the properties of glutamate receptors by calcium-dependent mechanisms. In the present study, changes in glutamate receptors elicited by calcium treatment of previously frozen mouse brain sections were evaluated by qualitative as well as quantitative analysis of tritiated ligand binding to both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptor subtypes. Quantitative analysis revealed that 3H-AMPA binding was reduced in a dose-dependent manner by calcium in the cerebral cortex and striatum formations. However, an opposite change in AMPA receptor properties was observed in the hippocampus, as calcium generated an increase of AMPA binding in all hippocampal fields. Analysis of the saturation kinetics of 3H-AMPA binding showed that the calcium-induced augmentation of AMPA binding in the stratum radiatum of the CA1 region was due to an alteration in the maximal number of sites, while the reduction of binding elicited by calcium in the cortex appeared to be due to modified AMPA receptor affinity. Calcium-induced downregulation of AMPA receptor affinity in the cortex and striatum was affected by baicalein, a selective inhibitor of the lipoxygenase pathways of arachidonic acid metabolism, whereas the same inhibitor did not modify calcium-mediated upregulation of receptor number in the CA1 region of the hippocampus. On the other hand, the effect of calcium appeared to be specific for the AMPA receptor, as the same treatment did not affect glutamate binding to the NMDA glutamate receptor subtype. Our results suggest the possibility that, depending on the brain regions, calcium ions may generate opposite modulation of AMPA receptor properties. Because the regulation of AMPA receptors by calcium-dependent enzymes has been implicated in synaptic plasticity, our results suggest that regional variations in the effect of calcium on AMPA binding account for differential plasticity at glutamatergic synapses.

Cholinergic systems and long-term potentiation in memory-impaired apolipoprotein E-deficient mice.

Impairments in cholinergic neurotransmitter systems of the basal forebrain are a hallmark of Alzheimer's disease pathophysiology. The presence of the epsilon4 allele of apolipoprotein E was recently implicated as a major risk factor in both familial and sporadic Alzheimer's disease. The present study examined the integrity of cholinergic and non-cholinergic systems in apolipoprotein E-deficient, memory-impaired mice. Choline acetyltransferase activity, hippocampal acetylcholine release, nicotinic and muscarinic (M1 and M2) receptor binding sites and acetylcholinesterase cell or terminal density showed no signs of alteration in either three-month or 9.5-month-old apolipoprotein E-deficient mice compared to controls. In contrast, long-term potentiation was found to be markedly reduced in these mice, but increases in the strength of stimulation induced the same level of long-term potentiation as that observed in controls. These alterations did not appear to be the consequence of modifications in the binding properties of glutamatergic receptors (N-methyl-D-aspartate and [RS]-alpha-amino-3-hydroxy-5-methylisoxazole propionic acid) but from defective regulation of the (RS)-alpha-amino-3-hydroxy-5-methylisoxazole propionic acid receptor by phospholipase A2 activity. These results support the notion that apolipoprotein E plays a fundamental role in neuronal plasticity, which could in turn affect cognitive performance through imbalances in extra- and intracellular lipid homeostasis.

Hypoxia-induced loss of synaptic transmission is exacerbated in hippocampal slices of transgenic mice expressing C-terminal fragments of Alzheimer amyloid precursor protein.

To investigate the possible involvement of beta-amyloid (A beta) in disrupting neuronal function during ischemia, we examined whether overexpression of C-terminal fragments of beta-amyloid precursor protein (beta-APP) in transgenic (Tg) mice is capable of altering the capacity of hippocampus slices to recover synaptic transmission after transient hypoxic episodes. Recovery of synaptic transmission was monitored in area CA1 of perfused hippocampal slices prepared from both control and Tg mice. The results obtained indicate that hippocampal slices prepared from Tg mice exhibited a much lower level of recovery in synaptic transmission following reoxygenation. This reduction in the capacity of Tg slices to recover from hypoxia-induced impairment of synaptic transmission in the hippocampus does not appear to be related to pre-existing alterations in either functional or biochemical properties of glutamate receptors in Tg mice. The present results provide the first experimental evidence that overexpression of the C-terminal fragment of APP exacerbates functional damage of hippocampal neurons after hypoxic episodes.

Bidirectional modulation of AMPA receptor properties by exogenous phospholipase A2 in the hippocampus.

The synaptic modifications underlying long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission in various brain structures may result from changes in the properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors. In the present study, we report that treatment of rat synaptoneurosomes with increasing concentrations of phospholipase A2 (PLA2) produces a biphasic effect on AMPA receptor binding, with low concentrations causing a decrease and high concentrations an increase in agonist binding. Analysis of the saturation kinetics of 3H-AMPA binding revealed that the biphasic effect of PLA2 was due to modifications in receptor affinity and not to changes in the maximum number of binding sites for AMPA receptors. The 12-lipoxygenase inhibitors preferentially reduced PLA2-induced decrease in AMPA binding and treatment of hippocampal synaptoneurosomes with arachidonic acid (AA) or 12-HPETE, the first metabolite generated from the hydrolysis of AA by 12-lipoxygenases, decreased 3H-AMPA binding. Moreover, electrophysiological experiments indicated that the 12-lipoxygenase inhibitor baicalein totally blocked LTD formation in area CA1 of hippocampal slices. The decrease in 3H-AMPA binding elicited by low concentrations of PLA2, as well as the level of LTD, were partially reduced by AA-861, a 5-lipoxygenase inhibitor, while the cyclooxygenase inhibitor indomethacin did not prevent LTD formation or the effects of PLA2 on 3H-AMPA binding. Our results provide evidence for a possible involvement of lipoxygenase metabolites in the regulation of AMPA receptor during synaptic depression. In addition, they strongly support the idea that the same biochemical pathway, i.e., NMDA receptor activation and endogenous PLA2 stimulation, may represent a common mechanism resulting in AMPA receptor alterations for both LTP and LTD formation.

AMPA receptor properties in adult rat hippocampus following environmental enrichment.

In adult rats, environmental enrichment has been shown to selectively increase -AMPA binding in the hippocampus but the molecular mechanisms underlying this effect remain unknown. We used in situ hybridization with antisense oligonucleotides to determine possible changes in the hippocampal expression of messenger RNAs for different subunits of AMPA receptors in adult rats following exposure to an enriched environment. Quantitative analysis revealed that mRNA levels for three subtypes of AMPA glutamate receptors (GluR1-3; Flip and Flop variants) were not modified in any hippocampal region after environmental enrichment. In addition, no differences were detected in the levels of GluR1 and GluR2/3 proteins in Western blots of hippocampal membranes from enriched rats. Nevertheless, quantitative ligand binding autoradiography indicated that environmental enrichment evoked a significant and uniform decrease in the capacity of calcium or phosphatidylserine (PS) to up-regulate -AMPA binding in various hippocampal regions but not in the cerebral cortex. These findings support previous observations suggesting that post-translational changes in AMPA receptor properties, as a result of the activation of calcium-dependent processes, may represent an important mechanism underlying long-term modifications of synaptic efficacy in the rat hippocampus.

Impaired modulation of AMPA receptors by calcium-dependent processes in streptozotocin-induced diabetic rats.

The mechanisms by which diabetes impairs cognitive function are not well-established. In the present study, we determined the electrophysiological and biochemical nature of disturbances in the mechanism of long-term potentiation (LTP) in diabetic rats. As previously reported, the administration of streptozotocin (STZ) was found to reduce the magnitude of LTP in the CA1 region of the hippocampus, while the same treatment did not interact with the capacity of the hippocampus to generate long-term depression induced by low-frequency stimulation. In addition, STZ treatment did not modify the component of excitatory postsynaptic potentials mediated by activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, suggesting that NMDA receptor function remained intact in STZ-treated slices. At the biochemical level, the capacity of calcium to increase [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (3H-AMPA) binding to glutamate/AMPA receptors in rat brain tissue sections was markedly affected in most regions of the hippocampus of STZ-treated rats. Moreover, changes in 3H-AMPA binding properties elicited by both exogenous phospholipase A2 and melittin, a potent activator of endogenous phospholipases, were also altered in synaptoneurosomes from diabetic rats. Taken together, the present data suggest that the loss of LTP maintenance in STZ-treated rats is more likely the result of disruption of calcium-dependent processes that are suspected to modulate postsynaptic AMPA receptors during synaptic potentiation. Understanding the biochemical factors participating in the impairment of AMPA receptor modulation might provide important clues revealing the very basis of memory deficits in diabetes.

Impaired learning and LTP in mice expressing the carboxy terminus of the Alzheimer amyloid precursor protein.

Proteolytic processing of amyloid precursor protein (APP) through an endosomal/lysosomal pathway generates carboxy-terminal polypeptides that contain an intact beta-amyloid domain. Cleavage by as-yet unidentified proteases releases the beta-amyloid peptide in soluble form. In Alzheimer's disease, aggregated beta-amyloid is deposited in extracellular neuritic plaques. Although most of the molecular mechanisms involving beta-amyloid and APP in the aetiology of Alzheimer's disease are still unclear, changes in APP metabolism may be important in the pathogenesis of the disease. Here we show that transgenic mice expressing the amyloidogenic carboxy-terminal 104 amino acids of APP develop, with ageing, extracellular beta-amyloid immunoreactivity, increased gliosis and microglial reactivity, as well as cell loss in the CA1 region of the hippocampus. Adult transgenic mice demonstrate spatial-learning deficits in the Morris water maze and in maintenance of long-term potentiation (LTP). Our results indicate that alterations in the processing of APP may have considerable physiological effects on synaptic plasticity.

Binding properties of glutamate receptors in streptozotocin-induced diabetes in rats.

The biochemical mechanisms by which diabetes modulates cognitive function are not well established. Here, we determined the effects of streptozotocin (STZ) administration on the binding properties of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors in rats, using quantitative autoradiographic analysis of (3)H-AMPA and [(3)H]glutamate binding on brain tissue sections. The STZ injection (70 mg/kg intraperitoneally) produced a reduction of (3)H-AMPA binding in various brain regions, an effect that is due to a decrease in receptor affinity. The STZ-induced reduction of (3)H-AMPA binding varied in different brain structures, being more pronounced in the striatum, cerebral cortex, and hippocampus and almost absent in the cerebellum. Western blots performed on hippocampal membranes revealed that the decrease in (3)H-AMPA binding is possibly associated with changes in immunologic properties for one glutamate receptor subunit (GluR1). Finally, the effect of STZ-induced diabetes appeared to be specific to the AMPA subtype of glutamate receptors, as the same treatment did not modify [(3)H]glutamate binding to NMDA receptors. These changes in AMPA receptor properties may have important implications for understanding the biochemical mechanisms underlying cognitive impairment in diabetes.

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats.

The effects of phosphatidylserine (PS) on the binding properties of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [3H]AMPA, [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]glutamate binding on rat brain tissue sections. Preincubation of brain sections with PS produced an increase in [3H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [3H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [3H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [3H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties, which could be involved in producing long-lasting changes in synaptic operation.

Mechanism of altered synaptic strength due to experience: relation to long-term potentiation.

An increase in medial perforant synaptic strength can be observed for hippocampal slices from rats exposed to environmental enrichment. The expression of enhanced synaptic strength exhibits properties similar to long-term potentiation (LTP), a physiological model of memory storage. Similarities include an increase in strength of the synaptic response in the absence of an altered paired-pulse ratio and an increase in the binding of the glutamate agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. Furthermore, environmental enrichment interacts with the mechanisms responsible for the induction of LTP by inhibiting further increases in synaptic strength following LTP-inducing stimulation. The results provide evidence for experience-mediated influences on postsynaptic mechanisms regulating medial perforant path synaptic strength.

Kainic acid increases the expression of the prohormone convertases furin and PC1 in the mouse hippocampus.

Prohormone convertases (PCs) belong to the mammalian family of subtilisin/kexin-like enzymes which have been implicated in the posttranslational processing of precursor proteins. Several PCs are produced in the central and peripheral nervous system, and only a few specific precursor-substrates have been identified in vivo. In the nervous system, PCs may be involved in intracellular processing of precursors for neuropeptides, hormones and neurotrophic factors, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). To study the interrelationships between the convertases furin, PC1 and PC2, and the neurotrophins NGF, BDNF and NT-3, we compared their mRNA distribution in different tissues. We also examined their expression in the hippocampus of mice undergoing kainic acid-induced seizures. In this experiment, in situ hybridization (ISH) demonstrated that the levels of mRNA for furin, PC1 and BDNF increased maximally at 3 h after kainic acid administration, followed by a decline to normal levels by 96 h. NGF showed small changes, while NT-3 was downregulated with minimal expression levels between 3 to 12 h. Double ISH with radioactively-labeled riboprobes and digoxigenin-labeled riboprobes demonstrated colocalization of furin with NGF and BDNF in the mouse submaxillary gland, and of furin and PC1 with BDNF in the trigeminal ganglion. Based on colocalization studies and evidence of coordinate expression with NGF and BDNF, we suggest the involvement of furin in processing of proNGF, and of both furin and PC1 in processing of proBDNF.

Involvement of the 12-lipoxygenase pathway of arachidonic acid metabolism in homosynaptic long-term depression of the rat hippocampus.

Low-frequency stimulation is associated with long-term depression (LTD) of synaptic efficacy in various brain structures. Like long-term potentiation (LTP), homosynaptic LTD in area CA1 of the hippocampus appears to require NMDA receptor activation, changes in postsynaptic calcium concentration and phospholipase A2 (PLA2) activation. Arachidonic acid (AA) is released after the activation of calcium-dependent phospholipases and free AA is rapidly metabolized to a family of bioactive products (the eicosanoids) which are thought to be both intracellular and extracellular messengers. In the present study, we investigated the involvement of the cyclooxygenase and lipoxygenase pathways of AA metabolism in the formation of homosynaptic LTD in the rat hippocampus. Stimulation at 1 Hz for 15 min was used to produce homosynaptic depression in area CA1 of hippocampal slices. LTD induction was partially blocked by bromophenacyl bromide (50-100 microM), a selective PLA2 inhibitor, and by the a nonselective lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 100 microM). In contrast, the specific cyclooxygenase blocker indomethacin (100 microM) did not significantly reduce hippocampal LTD. Since NDGA interferes with LTD formation, we examined whether specific inhibitors of 5- and 12-lipoxygenases were capable of blocking LTD expression. The 12-lipoxygenase inhibitor baicalein at a concentration of 50 microM reduced LTP formation when given in the bath, an effect that was less pronounced with the 5-lipoxygenase inhibitor AA-861. These data suggest that the activation of endogenous PLA2 and the formation of 12-lipoxygenase metabolites of AA may be important factors controlling the expression of hippocampal LTD.

Developmental changes in depolarization-mediated AMPA receptor modifications and potassium-induced long-term potentiation.

In the present study, we examined the KCl-induced increase in [3H] amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding in telencephalic synaptoneurosomes and potentiation of synaptic transmission (KLTP) in hippocampal slices during development in rats. As previously reported, KCI-induced depolarization of telencephalic synaptoneurosomes resulted in a 40 +/- 5% increase in [3H]AMPA binding to membrane fractions in adult rats (3 months old). KCI-induced increase in [3H]AMPA binding was reduced to 24 +/- 5% and 15 +/- 5% at postnatal days (PND) 25-30 and PND 15-20 respectively, and was only 6 +/- 5% at PND 5-10. KLTP in area CA1 of hippocampus was most pronounced in adult slices (40 +/- 5%), and was reduced to 30 +/- 5% in slices prepared from PND 25-30 animals; KCI-induced LTP was absent in CA1 hippocampal slices prepared from PND 5-10 animals (3 +/- 5%). The decrease in KCI-induced changes in AMPA receptor binding in young animals was also associated with an altered capacity of the bee venom peptide, mellitin (a phospholipase A2 (PLA2) activator), to increase [3H]AMPA binding in synaptoneurosomes. The smaller effect of mellitin on [3H]AMPA binding in young animals was not due to a decreased ability of this peptide to release [3H]arachidonate from synaptoneuro-somes. The parallel modifications in the extent of depolarization-induced change in AMPA receptor binding and excitatory synaptic transmission during development further support the hypothesis that alterations in AMPA receptor properties may play a critical role in synaptic plasticity.