The potent non-competitive mGlu1 receptor antagonist BAY 36-7620 differentially affects synaptic plasticity in area cornu ammonis 1 of rat hippocampal slices and impairs acquisition in the water maze task in mice.

In this study we evaluated the effects of the novel, potent non-competitive metabotropic glutamate receptor (mGluR) 1 antagonist (3aS,6aS)-6a-naphthalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on (BAY 36-7620) on different types of synaptic plasticity in the hippocampal cornu ammonis (CA) 1-region and on hippocampus-dependent spatial learning. After having confirmed the presence of mGluR1 in the hippocampal CA1 region of our rat strain by confocal microscopy, we tested the effects of BAY 36-7620 on: 1) long-term potentiation (LTP) induced by weak and strong stimulation; 2) 3,5-dihydroxyphenylglycine (DHPG, 30 microM)-induced depression of synaptic transmission; and 3) learning of the hidden platform version of the water maze by mice. BAY 36-7620 (10 microM) amplified LTP but, like the mGluR1 antagonists 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt, 10 microM) and 4-carboxyphenylglycine (4-CPG, 50 microM), diminished LTP at 1 microM. The mGluR5 antagonist 6-methyl-2-(phenylethynyl)-pyridine (MPEP, 10 microM) had no effect. BAY 36-7620 (10 microM) did not affect strong LTP. Thus, mGlu 1, but not mGlu 5, receptors modulate LTP elicited by weak stimulation in vitro. DHPG-induced depression of synaptic transmission was only marginally affected by BAY 36-7620 (1 microM) or 4-CPG (100 microM). In a mouse water maze study, BAY 36-7620 (10 mg/kg, i.v.) increased the escape latency and impaired water escape task acquisition during the first 4 days. Drug- and vehicle-treated groups showed comparable performance at day 5. Our data support a role for mGluR1 in LTP and in the acquisition of spatial memory.

Upregulation of ICAM-1 and MCP-1 but not of MIP-2 and sensorimotor deficit in response to traumatic axonal injury in rats.

The pathophysiology of traumatic axonal injury (TAI) is only partially understood. In this study, we investigated the inflammatory response as well as the extent of neurological deficit in a rat model of traumatic brain injury (TBI). Forty-two adult rats were subjected to moderate impact-acceleration brain injury and their brains were analyzed immunohistochemically for ICAM-1 expression and neutrophil infiltration from 1 hr up to 14 days after trauma. In addition, the chemotactic factors MIP-2 and MCP-1 were measured in brain homogenates by ELISA. For evaluating the neurological deficit, three sensorimotor tests were applied for the first time in this model. In the first 24 hr after trauma, the number of ICAM-1 positive vessels increased up to 4-fold in cortical and subcortical regions compared with sham operated controls (P < 0.05). Maximal ICAM-1 expression (up to 8-fold increase) was detected after 4 days (P < 0.001 vs. 24 hr), returning to control levels in all brain regions by 7 days after trauma. MCP-1 was elevated between 4 hr and 16 hr post-injury as compared with controls. In contrast, neither neutrophil infiltration nor elevation of MIP-2, both events relevant in focal brain injury, could be detected. In all neurological tests, a significant deficit was observed in traumatized rats as compared with sham operated animals from Day 1 post-injury (grasping reflex of the hindpaws: P < 0.001, vibrissae-evoked forelimb placing: P = 0.002, lateral stepping: P = 0.037). In conclusion, after moderate impact acceleration brain injury ICAM-1 upregulation has been demonstrated in the absence of neutrophil infiltration and is paralleled by a selective induction of chemokines, pointing out that individual and distinct inflammatory events occur after diffuse vs. focal TBI.

Inhibition of evoked glutamate release by the neuroprotective 5-HT(1A) receptor agonist BAY x 3702 in vitro and in vivo.

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT(1A) receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC(50)=1 microM). This effect was blocked by the selective 5-HT(1A) receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT(1A) receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 microg/kg or 10 microg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.

Effects of subdural haematoma on sensorimotor functioning and spatial learning in rats.

Twenty per cent of all strokes are haemorrhagic in character and are associated with severe disturbances in sensorimotor behaviour and cognition. Although spontaneous recovery of pre-stroke functioning occurs in some cases, the process is demanding, slow, and often incomplete. A first step in the preclinical testing of new putative, neuroprotective and recovery-supporting therapeutics is to validate animal models of brain injury. In a series of four experiments we evaluated the behavioural impairments and the time course of recovery of functional deficits in rats with an experimentally induced subdural haematoma. We found that unilateral subdural haematoma resulted in dysfunction in both simple reflexive (experiment 1) and skilled sensorimotor behaviour (experiment 2). Reflexive behaviour did not recover, or recovered only marginally, and neither did the deficits in skilled forepaw use. Bilateral subdural haematoma impaired the learning and memory performance of adult (experiment 3) and old rats (experiment 4) in the Morris water escape task. Considering the diversity of the deficits found in our experiments, we conclude that different models are needed to cover the broad range of deficits seen in stroke patients.

Phorbol ester-induced changes in rat hippocampal glycoprotein fucosylation.

Studies on glycoprotein fucosylation were carried out using hippocampal slices from rat brain. These slices were incubated in the presence of the protein kinase C (PKC) activating phorbol ester, 4 beta-phorbol-12,13-dibutyrate (PDBu), or an inactive isoform, 4 alpha-phorbol-12,13-didecanoate (PDD), respectively, for 7 min followed by a 60 min pulse of [3H]fucose. PDBu caused an increase in [3H]fucose incorporation into glycoproteins by 29% as well as an activation of the fucokinase enzyme reaction by 21%. The PDBu-induced stimulation of [3H]fucose insertion into hippocampal glycoproteins was abolished by the PKC inhibitors, staurosporine and H7. The importance of a PKC-regulated glycoprotein fucosylation in mechanisms underlying changes in neuronal plasticity is discussed.

Distribution of (fucogalactosyl-)epitope expressing glycoconjugates in rat brain.

Glycoconjugates are known to be concentrated in plasma membranes, especially in synaptic junctions, where they subserve various functions in neural connectivity. Here we report the cellular distribution of a new monoclonal antibody recognizing (fucogalactosyl) sequences in carbohydrate structures. The most pronounced immunoreactivity was found in fibrous astrocytes, in many parts of the brain and with lower density in various neuronal elements. This points to the expression of identical carbohydrate sequences on molecules within certain glial and neuronal elements. Previous intracerebral injections of the antibody interfered with long term memory formation. Therefore, functions mediated by corresponding glycoproteins in neurons and glia cells or even neuron-glial interactions, might be relevant for information-processing.

The amnesic substance 2-deoxy-D-galactose suppresses the maintenance of hippocampal LTP.

Male Wistar rats were intraventricularly injected with 2-deoxy-D-galactose (do-gal), a substance interfering with the fucosylation of glycomacromolecules and impairing memory consolidation in various learning tasks. Do-gal was found to have no influence on the monosynaptically evoked field potential (MEFP) recorded in the dentate gyrus upon stimulation of the perforant pathway. However, hippocampal long-term potentiation (LTP) induced in do-gal-pretreated animals by fractionated tetanization of the perforant pathway declined to control levels 2 h after tetanization, whereas it remained constant for 24 h in saline-treated rats. Similar effects were observed in the CA1 region of hippocampal slices. The results indicate a participation of fucosylated macromolecules in the maintenance of LTP. The possible significance of processes involved in LTP for memory formation is discussed.

Enhanced development of morphine tolerance in rats treated with 2-deoxy-D-galactose.

Rats treated subcutaneously for 6 days with morphine developed a weak tolerance which was characterized by a decrease in the analgesic action of the opioid. Under those experimental conditions a simultaneous intracerebroventricular application of 2-deoxy-D-galactose enhanced development of morphine tolerance, while other deoxy-sugars like 2-deoxy-D-glucose and 6-deoxy-D-galactose were ineffective. In contrast, no influence of 2-deoxy-D-galactose on a more enhanced morphine tolerance after a 11-day pretreatment with morphine was found. The results are discussed in the light of a rather specific interference of 2-deoxy-D-galactose with neuronal glycoprotein processing and related cellular mechanism underlying adaptive processes involved in the development of morphine tolerance.

Accumulation of c-fos mRNA in rat hippocampus during acquisition of a brightness discrimination.

Training rats to attain a foot-shock-motivated brightness discrimination in a Y-maze results in an early and transient increase of hippocampal c-fos mRNA levels. Maximal accumulation was observed immediately after training, returning to basal levels during the following 2 h. A similar increase was obtained when rats were subjected to a pseudotraining with an equal number of runs, but with random pairing of the choice of bright and dark alleys with foot shock. It is suggested that induction of hippocampal c-fos mRNA expression is a necessary, but not sufficient, prerequisite for the formation of long-term memory trace. This early gene expression seems rather to correspond to an initial stage induced by complex stimulus presentation of both the training and the pseudotraining procedure. The subsequent late synthesis or processing of target proteins finally contributing to the formation of a permanent trace requires the action of further convergent signals to principal cells, probably mediating reward or emotional influences.

[Determination of fucokinase activity in the blood of schizophrenic patients]. / Bestimmung der Fukokinaseaktivität im Blut schizophrener Patienten.

An investigation was conducted into whether dopamine induces an alteration in the fucolysation of glycoproteins, starting from the dopamine hypothesis of schizophrenia and taking the fucokinase activity determined in erythrocytes of schizophrenic patients as parameter. As with patients with "schizoaffective psychosis" and those with manic-depressive disorders, who were likewise examined, it was found that the enzyme activity of schizophrenic patients was no different than that found in the blood of a control group.

Polymyxin B, an inhibitor of protein kinase C, prevents the maintenance of synaptic long-term potentiation in hippocampal CA1 neurons.

The involvement of protein kinase C (PKC)-mediated processes in mechanisms of long-term potentiation (LTP) was suggested by recent studies which have demonstrated a correlation between PKC activation and LTP. However, it was not possible to tell whether there is a causal relationship between the two events. Therefore, we have examined the induction and maintenance of LTP in rat hippocampal slices in the presence of a relatively selective PKC inhibitor, using extracellular electrophysiological techniques. Bath application of 0.1-100 microM polymyxin B did not influence the occurrence of post-tetanic and long-term potentiation usually seen in test responses 1 and 10 min after a 100-Hz/1 s tetanic stimulation of stratum radiatum fibers. However, 20 microM polymyxin B significantly depressed the increase in population spike amplitude and population excitatory postsynaptic potential (EPSP) slope from 30 to 120 min onwards, following repeated tetanization. Immediately after the drug application only weak and reversible effects were seen by the same parameters in test responses of a non-tetanized control input. A late (greater than 6 h) heterosynaptic potentiation of the population spike in the control input was blocked by polymyxin B treatment. Whereas the EPSP-LTP was fully blocked, some potentiation of the population spike still remained, suggesting the independence of PKC of the additional spike (E/S) potentiation for the first 6 h. These results provide direct evidence that the PKC activation is not essential for the initial phase of LTP, but is a necessary condition for a medium and a late, protein synthesis-dependent phase in this monosynaptic pathway, i.e. for the maintenance of synaptic LTP.

Different effects of 2-deoxy-sugars on memory formation.

When rats were trained on a brightness discrimination task, intrahippocampal injections of 2-deoxy-galactose interfered with long-term memory formation while galactose and 2-deoxy-glucose were ineffective altogether. No differences were found in the morphology of hippocampal CA1 neurons investigated light-microscopically after application of the sugars. The amnestic action of 2-deoxy-galactose is discussed in the light of a rather specific interference with fucosylation of proteoglycans involved in mechanisms underlying the formation of a memory trace.

Amnesic action of 2-deoxy-D-galactose is not related to alterations in the release of dopamine and noradrenaline.

2-deoxy-D-galactose, an inhibitor of glycoprotein fucosylation, did neither affect the K+-stimulated release of noradrenaline from rat striatal slices nor that of dopamine from striatal synaptosomes. The results are discussed in the light of an amnesic action of the deoxy-sugar in animal learning experiments.

Long-term memory formation in chicks is blocked by 2-deoxygalactose, a fucose analog.

When day-old chicks are trained on a passive avoidance task there is enhanced synthesis of glycoproteins. Bilateral intracerebral injections of 20 mumole of 2-deoxygalactose (2-D-gal), administered just before and just after training on the task, produce amnesia for the avoidance. Amnesia develops slowly over the first hour and persists for at least 24 h subsequently. If 2-D-gal injections are administered 4 h prior to the training or delayed for 3 h after training, no amnesia occurs. Apart from a brief initial suppression of pecking following injection there are no effects of 2-D-gal on other observed behaviors of the birds. Within the first hour this dose of 2-D-gal inhibits [3H]fucose incorporation into acid-insoluble material by 26% (or 68%, calculated relative to free pool fucose). The amnestic effect of 2-D-gal is not shown by galactose, glucose, fucose, or 2-D-glucose. Injecting 40 mumole of galactose simultaneously with the 2-D-gal abolishes the 2-D-gal-induced amnesia; 40 mumole of fucose, however, does not abolish the amnesia. The utility of 2-D-gal as an agent for analyzing the role of glycoproteins in memory formation is discussed.

4-Aminopyridine affects synaptosomal protein phosphorylation in rat hippocampal slices.

Rat brain hippocampal slices were incubated with or without the convulsant 4-aminopyridine (4-AP). From these slices a crude mitochondrial/synaptosomal membrane fraction was prepared and analyzed for endogenous protein phosphorylation. 4-AP (10(-5) M) stimulated the phosphorylation of a 50 kDa protein by 86%. The phosphorylation of this 50 kDa protein is Ca2+/calmodulin-dependent and we suggest that this protein is the lower molecular weight subunit of Ca2+/calmodulin-dependent protein kinase II (CaMK II).

Impairment of glycoprotein fucosylation in rat hippocampus and the consequences on memory formation.

The intraventricular injection of 2-deoxy-D-galactose led to a dose- and time-dependent decrease in the fucosylation of hippocampal glycoproteins in rats whereas the incorporation of 3H-N-acetyl-glucosamine was not influenced. This effect is not related to an interference with fucose activating or transferring enzymes but can be abolished by an application of D-galactose. Thus, it seems likely that also in brain tissue a deoxy-galactose induced decrease in the fucosylation is due to a hindering of a glycosidic linkage of fucose to the deoxy-sugar incorporated into glycoprotein chains. As a consequence of an intrahippocampal injection of the deoxy-sugar the retention performance of the animals in a foot-shock motivated brightness discrimination task was considerably impaired. But deoxy-galactose is effective only when administered before and immediately after training whereas either a pre- or a post-training injection did not influence the retention performance of the rats. Thus, an effective metabolic inhibition of the glycoprotein completion by the deoxy-sugar starting at the time of training seems to be crucial to interfere with such morphofunctional alterations in the neuronal network underlying the formation of a memory trace.

The influence of experimental conditions on the morphological preservation of hippocampal slices in vitro.

The influence of different brain cutting techniques and various conditions of in vitro incubation on the morphological preservation of hippocampal slices was investigated comparatively. In order to obtain well-preserved slices great attention has to be paid to the immediate cooling of brain tissue to +4 degrees C and of all instruments as well. Under these conditions both the cutting technique and the incubation medium will be of subordinate importance. The incubation of 'interface slices' on a net in a slice chamber where the incubation medium flows constantly through the system and where the slices lay on the border to moistened oxygen is superior to the incubation of slices which are floating submerged in tubes--especially if the incubation time will last longer than 1.5 h. Different reliable incubation media did not show striking differences with regard to their effect on the preservation of hippocampal slices. New possibilities for incubation of slices for biochemical long-term investigations were tested and are discussed.

Dopamine-stimulated fucosylation of brain proteins in vitro is not inhibited by puromycin.

In slices of rat hippocampus, the influence of puromycin (0.5 mM) on dopamine (0.5 mM)-stimulated L-fucose incorporation into glycoproteins was studied. Puromycin (15 min, 2 h and 4 h pretreatment) inhibited protein synthesis by some -70% (estimated by the in vitro incorporation rate of L-leucine), whilst L-fucose incorporation into rat hippocampal glycoproteins was only inhibited about -20% (4 h pretreatment) compared to control values (100%). Under these experimental conditions, the dopamine-induced increase in both the L-fucose incorporation into glycoproteins and the activity of fucokinase was not affected by puromycin. These data suggest that these short-lasting dopamine actions do not depend on protein synthesis.

Inhibition of brain protein fucosylation by sulfated sugar molecules.

Brain fucosyltransferase reaction (GDP-fucose: glycoprotein fucosyltransferase, EC 2.4.1.68) was found to be inhibited by heparin. Also a variety of other sulfated polysaccharides led to a decrease in the fucosyltransferase reaction comparable to that induced by heparin. This action seems not to be related to the structural and functional integrity of the glucosaminoglycan since the same effect of the enzyme reaction was seen using its breakdown products obtained by hydrolysis or ultrasonics. In contrast, unsulfated polysaccharides and constituents of heparin, respectively, did not influence the enzyme reaction at all. The same was true for phosphorylated monosaccharides. Therefore, it seems likely that the inhibitory effect of appropriate polysaccharides or their breakdown products is related to the action of sulfated sugar units. Moreover, investigating the influence of a heparin hydrolysate on the incorporation of [3H]fucose into the glycoproteins of hippocampal slices, also an inhibition of the sugar incorporation was found. Thus, the inhibitory influence of sulfated sugar units on brain fucosyltransferase reaction seems to be effective under both conditions, in the assay system of the enzyme in vitro as well as in intact cells of the hippocampal structure.