Specific regulation of N-CAM/D2-CAM cell adhesion molecule during skeletal muscle development

The expression of the N-CAM/D2-CAM cell adhesion molecule was studied in skeletal muscle. In cell cultures derived from adult human muscle N-CAM/D2-CAM was found at the cell surface of myoblasts and myotubes but not fibroblasts, showing that N-CAM/D2-CAM is a specific gene product of muscle. Western blots showed that the anti N-CAM/D2-CAM antibody reacted with a single protein band of 180 000 daltons in these cultures that differed in mobility from the broad band of 150 000-200 000 daltons found in brain. N-CAM/D2-CAM is also expressed by muscle at certain stages of development. Human foetal muscle of 10 and 20 weeks gestation showed N-CAM/D2-CAM around developing myofibres while both fast and slow adult muscle fibres did not express N-CAM/D2-CAM, suggesting that the protein is down regulated during myofibre maturation. This was studied further in developing rat muscle where N-CAM/D2-CAM was found on myofibres in the day 1 neonate, but had disappeared by day 9. N-CAM/D2-CAM is, however, re-expressed in human muscle disease where there is muscle regeneration such as in polymyositis, and here is associated with classic regenerating myofibres. N-CAM/D2-CAM expression is temporally regulated and is expressed only at times of synapse formation consistent with the idea that it may be involved in early nerve-muscle interactions.     

Postnatal D2-CAM/N-CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [¹⁴C]sialic acid was found to be associated with the high-molecular-weight [>200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.     

The effect of a neuron-specific antiserum,BPM, on thein vitro development of cerebellar granule cells

Anti-BPM is a neuron-specific antiserum which specifically recognizes the D2 cell adhesion molecule in crossed immunoelectrophoresis of Triton X-100-solubilized brain extracts. Here the effect of this antiserum on the in vitro development of cerebellar neuronal cultures is described. The initial adhesion of cells and neurite outgrowth were not influenced by immunoglobulin fractions of anti-BPM. However, after 5 days in vitro the cultures had become completely disorganized, with the majority of cells being dead at immunoglobulin concentrations greater than 0.5 mg/ml culture medium. This effect was seen only with immunoglobulins and their F(ab')2 fragments, the F(ab') fragments being without effect. The addition of anti-BPM to 8-day-old cultures resulted in a more rapid and pronounced rate of cell death. In many instances this was preceded by a rapid "destabilization" of culture organization. The cytotoxic effect of anti-BPM was neuron specific and the small numbers of astrocytes and fibroblasts found in the cultures were unaffected by prolonged exposure to this serum.     

Pre- and post-training infusion of prazosin into the bed nucleus of the stria terminalis impaired acquisition and retention in a Morris water maze task

The bed nucleus of the stria terminalis (BNST) is interconnected with the amygdala that is implicated in memory modulation. In view of the innervation of this structure by the hippocampus and brain stem noradrenergic nuclei, this study examined the role of BNST noradrenergic activity in acquisition, formation and expression of spatial memory. Male Wistar rats with indwelling cannulae in the BNST were trained on a spatial navigation task in the Morris water maze. Groups of rats received intra-BNST infusion of vehicle, norepinephrine, prazosin or both drugs shortly before or after each daily training session, or shortly before retention tests. Results showed that pre- or posttraining infusion of 1.0 microg prazosin impaired acquisition and retention, but the treatment had no effect on a cued response task. Posttraining infusion of 1.0 microg norepinephrine enhanced acquisition and retention, and this enhancing effect was blocked by simultaneous infusion of 0.3 microg prazosin. Pretest intra-BNST of prazosin or norepinephrine at a dose of 1.0 microg did not impair expression of the spatial navigation memory. These findings suggest that the BNST noradrengergic function is involved in modulating acquisition and formation of spatial memory that engage the hippocampus.     

Characterisation of Antibodies Specific for Chick Brain Neural Cell Adhesion Molecules Which Cause Amnesia for a Passive Avoidance Task

Abstract: Antisera were prepared against six postsynaptic density glycoprotein fractions (150–180, 62–80, 50, 41, 33, and 28 kDa) that show enhanced fucosylation during memory formation after training day-old chicks in a one-trial passive avoidance task. Each antiserum was tested for its possible effect on memory retention. Bilateral intracranial injections of two of the antisera, R-1 and R-6, or their IgGs (IgG-1 and IgG-6), resulted in amnesia for the passive avoidance task when chicks were tested 24 h later. IgG-1 and IgG-6 antibodies were amnestic only when injected 5.5 h after training, and had no effect when injections were made 30 min before training, thus resembling an effect previously observed with polyclonal or monoclonal anti-N-CAM antibodies. IgG-1 and IgG-6 antibodies were found to be specific for protein epitopes of glycoproteins that contain a high amount of N-linked mannose and fucose, and a very low amount of polysialic acid and O-linked galactose. Absorption of IgG-6 antibodies with neural cell adhesion molecule (N-CAM) isolated from synaptic plasma membranes derived from day-old chick brain resulted in loss of amnestic effect. As we have previously shown that long-term memory for the passive avoidance task requires two waves of glycoprotein synthesis, the first occurring immediately after training and the second 5–8 h later, the present results suggest strongly that isoforms of N-CAM molecules with a low level of sialic acid are involved specifically in the establishment of an enduring memory for the experience of the passive avoidance task in chicks, possibly by stabilising changes in synaptic connectivity that encode the memory.     

Bryostatin enhancement of memory in Hermissenda

Bryostatin, a potent agonist of protein kinase C (PKC), when administered to Hermissenda was found to affect acquisition of an associative learning paradigm. Low bryostatin concentrations (0.1 to 0.5 ng/ml) enhanced memory acquisition, while concentrations higher than 1.0 ng/ml down-regulated the pathway and no recall of the associative training was exhibited. The extent of enhancement depended upon the conditioning regime used and the memory stage normally fostered by that regime. The effects of two training events (TEs) with paired conditioned and unconditioned stimuli, which standardly evoked only short-term memory (STM) lasting 7 min, were--when bryostatin was added concurrently--enhanced to a long-term memory (LTM) that lasted about 20 h. The effects of both 4- and 6-paired TEs (which by themselves did not generate LTM), were also enhanced by bryostatin to induce a consolidated memory (CM) that lasted at least 5 days. The standard positive 9-TE regime typically produced a CM lasting at least 6 days. Low concentrations of bryostatin (<0.5 ng/ml) elicited no demonstrable enhancement of CM from 9-TEs. However, animals exposed to bryostatin concentrations higher than 1.0 ng/ml exhibited no behavioral learning. Sharp-electrode intracellular recordings of type-B photoreceptors in the eyes from animals conditioned in vivo with bryostatin revealed changes in input resistance and an enhanced long-lasting depolarization (LLD) in response to light. Likewise, quantitative immunocytochemical measurements using an antibody specific for the PKC-activated Ca2+/GTP-binding protein calexcitin showed enhanced antibody labeling with bryostatin. Animals exposed to the PKC inhibitor bisindolylmaleimide-XI (Ro-32-0432) administered by immersion prior to 9-TE conditioning showed no training-induced changes with or without bryostatin exposure. However, if animals received bryostatin before Ro-32, the enhanced acquisition and demonstrated recall still occurred. Therefore, pathways responsible for the enhancement effects induced by bryostatin were putatively mediated by PKC. Overall, the data indicated that PKC activation occurred and calexcitin levels were raised during the acquisition phases of associative conditioning and memory initiation, and subsequently returned to baseline levels within 24 and 48 h, respectively. Therefore, the protracted recall measured by the testing regime used was probably due to bryostatin-induced changes during the acquisition and facilitated storage of memory, and not necessarily to enhanced recall of the stored memory when tested many days after training.     

Extracellular matrix heparin induces alteration of the cell adhesion during brain development

The studies of neuronal cell-glycosaminoglycan interactions indicate an increasing interest in the question of how heparin can mediate adhesion properties of the cell. We have found that high levels of both N-CAM concentration and heparin-binding activity were noticed in the early stages of brain formation. According to electron microscopy data, an elevation of free heparin in the substratum leads to a decrease of the N-CAM content and changing of its distribution on the membrane of cultured hippocampal neurons. Spatial arrangement of immunogold labelled N-CAM molecules in plasma membrane profiles of cultured neurones was quantified with image analysis software using an interlabel distance estimate. To convert these estimates into two dimensional (2D) quantities, namely the 2D pattern and density of labelling, a computer simulation technique was used. Heparin added to the substratum in a concentration of 40 microg/ml decreased the 2D N-CAM labelling density by 50% - 39.8 labels/microm(2) compared with the control values of 88.9 labels/microm(2).     

Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle

The neural cell adhesion molecule (N-CAM) is present in both embryonic and perinatal muscle, but its distribution changes as myoblasts form myotubes and axons establish synapses (Covault, J., and J. R. Sanes, 1986, J. Cell Biol., 102:716-730). Levels of N-CAM decline postnatally but increase when adult muscle is denervated or paralyzed (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA., 82:4544-4548). To determine the molecular forms of N-CAM and N-CAM-related RNA during these different periods we used immunoblotting and nucleic acid hybridization techniques to analyze N-CAM and its RNA in developing, cultured, adult, and denervated adult muscle. As muscles develop, the extent of sialylation of muscle N-CAM decreases, and a 140-kD desialo form of N-CAM (generated by neuraminidase treatment) is replaced by a 125-kD form. This change in the apparent molecular weight of desialo N-CAM is paralleled by a change in N-CAM RNA: early embryonic muscles express a 6.7-kb RNA species which hybridizes with N-CAM cDNA, whereas in neonatal muscle this form is largely replaced by 5.2- and 2.9-kb species. Similar transitions in the desialo form of N-CAM, but not in extent of sialylation, accompany differentiation in primary cultures of embryonic muscle and in cultures of the clonal muscle cell lines C2 and BC3H-1. Both in vivo and in vitro, a 140-kD desialo form of N-CAM and a 6.7-kb N-CAM RNA are apparently associated with myoblasts, whereas a 125-kD desialo form and 5.2- and 2.9-kb RNAs are associated with myotubes and myofibers. After denervation of adult muscle, a approximately 12-15-fold increase in the levels of N-CAM is accompanied by a approximately 30-50-fold increase in N-CAM RNA, suggesting that N-CAM expression is regulated at a pretranslational level. Forms of N-CAM and its RNA in denervated muscle are similar to those seen in perinatal myofibers.     

Cell-Free Synthesis of the D2-Cell Adhesion Molecule: Evidence for Three Primary Translation Products

The D2-cell adhesion molecule (D2-CAM) is a membrane glycoprotein that is involved in cell-cell adhesion in the nervous system. To study the biosynthesis of D2-CAM we have translated free and membrane-bound polysomes from rat brain in vitro in the rabbit reticulocyte lysate system. D2-CAM was exclusively synthesized on membrane-bound polysomes. The primary translation products of D2-CAM were three polypeptides of apparent molecular weights 187,000, 134,000, and 112,000. No interconversion between these polypeptides was detected. In contrast to previous suggestions, we conclude that all three D2-CAM polypeptides are primary translation products. When translating polysomes from embryonic and postnatal rat brain, we found that the relative amounts of the three polypeptides synthesized varied with age. Their molecular weights, however, were not age-dependent.     

Chronic Low-Level Lead Exposure Impairs Embryonic to Adult Conversion of the Neural Cell Adhesion Molecule

The neural cell adhesion molecule (N-CAM) is a complex of surface glycoproteins that are developmentally regulated and believed to be intimately involved in the orderly structuring of the CNS. Here the effect of chronic low-level lead exposure on their expression in the postnatally developing cerebellum is described. Rat pups were chronically exposed to lead via their dam's drinking water which contained either 200 or 400 mg PbCl2/L from time of birth. Pup postnatal blood lead levels ranged between 10 and 20 micrograms/dl until day 16 after which they became elevated to 40 micrograms/dl on day 20. During this period the developmental sialylation state of N-CAM, which is believed to regulate cell-cell interaction, fibre outgrowth, and synapse formation, was monitored by rocket immunoelectrophoresis. In control animals the expected desialylation of N-CAM was found to occur at times coincident with postnatal synaptogenesis. In contrast, desialylation in animals chronically exposed to lead was found to be significantly impaired when blood lead levels exceeded 20 micrograms/dl. This could not be attributed to lead-induced undernutrition or alterations in immunoprecipitate formation. These observations could account for the neurobehavioural deficits that are known to be induced at similar blood lead levels, and the potential contribution of impaired N-CAM desialylation to synaptic elaboration is discussed.     

Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes

Posttranslational modifications and intracellular transport of the D2- cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A) and 131,000-158,000 (B) were synthesized using radiolabeled precursors in cultured neurons. A and B were found to contain only N-linked complex oligosaccharides, and both polypeptides appeared to be polysialated as determined by [14C]mannosamine incorporation and precipitation with anti-polysialic acid antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination by photoactivation employing a hydrophobic labeling reagent. In rat forebrain explant cultures, D2-CAM was synthesized as four polypeptides: A (195,000 Mr), B (137,000 Mr), C (115,000 Mr), and a group of polypeptides in the high molecular weight region (HMr) between 250,000 and 350,000. Peptide maps of the four polypeptides yielded similar patterns. Biosynthesis of C and HMr increased with age, relative to A and B. A and B were sulfated in embryonic brain, however, sulfation was not noticeable at postnatal ages. Phosphorylation, on the other hand, of A and B was observed at all ages examined. We suggest that D2-CAM function may be modified during development by changes in the relative synthesis of the different polypeptides, as well as by changes in their glycosylation and sulfation.     

Paradoxical transitory facilitation of performance in the Hebb-Williams labyrinth after lesion of the cingulate cortex in mice

The effects of lesions of the anterior (Ant) or posterior (Post) regions of the cingulate cortex were tested using the closed-field maze tests of Hebb-Williams. Subjects were male BALB/c mice which had received restricted bilateral electrolytic lesions of the Ant or Post cingulate cortex. Their performances in acquisition or retention were compared to those of sham operated mice at different time intervals after the lesion. In a first series of experiments, naive animals were lesioned. The effects of lesions on acquisition were then tested at different time intervals (up to 45 days) after surgery. In a second series of experiments, the animals were lesioned only after prior complete acquisition of the maze tests and tested in a retention paradigm. The results show a differential effect of Ant versus Post cingulate lesions. Ant lesions had no significant effects whereas Post lesions induced a facilitation of performance at time intervals between 19 and 33 days (acquisition paradigm) or 11 and 25 days (retention paradigm). Conversely a reversal (impairment) of the effect was observed when Post animals were tested 28 days (acquisition) or 48 days (retention) after surgery. Taking into consideration the changing nature of the behavioral paradigm used, we suggest that the paradoxical and transitory facilitatory effects of the Post cingulate lesions may indicate that lesioned mice exhibited a greater degree of adaptability in each new learning situation. However, we can also postulate that these facilitatory effects resulted from a difficulty of lesioned mice in addressing long-term memory stores. This would therefore paradoxically protect them from interference generated by the high degree of familiarity in the behavioural testing apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cold narcosis on memory acquisition,consolidation and retrieval in honeybees(Apis mellifera)

In learning and memory studies on honeybees(Apis mellifera),cold-induced narcosis has been widely used to temporarily immobilize honeybees.In this study,we investigated the effects of cold narcosis on the associative memories in honeybees by using the proboscis extension response(PER)paradigm.Severe impairments in memory acquisition was found when cold narcosis was performed 30 min,instead of 1 h before training.Locomotor activities were reduced when honeybees were tested 15 min,instead of30 min after cold narcosis.These results indicate that cold narcosis impairs locomotor activities,as well as memory acquisition in a time-dependent manner,but by comparison no such effects on memory retrieval have yet been observed.[0]     

Emotional and cognitive reinforcement of rat hippocampal long-term potentiation by different learning paradigms

In earlier studies we have shown that a protein-synthesis-independent, early, long-term potentiaton (early-LTP) that lasts up to 4-5 hours can be transformed (reinforced) into a protein-synthesis-dependent late-LTP that lasts > or = 8 hours by either an emotional challenge (e.g. swim stress) or mastering a cognitive task (e.g. spatial learning). In the present study we show that LTP-reinforcement by spatial training depends on the specific constraints of the learning paradigm. In a holeboard paradigm,LTP-reinforcement is related to the formation of a lasting reference memory whereas water-maze training gives more heterogenous results. Thus, cognitive aspects interfere with emotionally challenging components of the latter paradigm. These data indicate that different spatial-learning tasks are weighted distinctly by the animal. Thus, we show that aspects of specific spatial learning paradigms such as shifts of attention and emotional content directly influence functional plasticity and memory formation.     

Characterisation of a chromatographically produced anti-D immunoglobulin product

A chromatographic fractionation method has been developed for the production of a liquid-stable anti-D immunoglobulin product for intravenous and intramuscular use. An immunoglobulin fraction, highly enriched with anti-D immunoglobulins, was isolated by cation-exchange column chromatography and further polished, first by anion-exchange chromatography, followed by an aluminium hydroxide gel treatment. The process includes two specific steps for virus inactivation and removal, namely S/D treatment and nanofiltration. The overall anti-D process yield is about 56%. The final product is stabilised with human albumin and glycine and placed in ready-to-use syringes. The anti-D product was shown to be stable in liquid state for at least 30 months at 4°C.     

Galnon Facilitates Extinction of Morphine-Conditioned Place Preference but Also Potentiates the Consolidation Process

Learning and memory systems are intimately involved in drug addiction. Previous studies suggest that galanin, a neuropeptide that binds G-protein coupled receptors, plays essential roles in the encoding of memory. In the present study, we tested the function of galnon, a galanin receptor 1 and 2 agonist, in reward-associated memory, using conditioned place preference (CPP), a widely used paradigm in drug-associated memory. Either before or following CPP-inducing morphine administration, galnon was injected at four different time points to test the effects of galanin activation on different reward-associated memory processes: 15 min before CPP training (acquisition), immediately after CPP training (consolidation), 15 min before the post-conditioning test (retrieval), and multiple injection after post-tests (reconsolidation and extinction). Galnon enhanced consolidation and extinction processes of morphine-induced CPP memory, but the compound had no effect on acquisition, retrieval, or reconsolidation processes. Our findings demonstrate that a galanin receptor 1 and 2 agonist, galnon, may be used as a viable compound to treat drug addiction by facilitating memory extinction process.     

Participation of the dopaminergic brain system in effects of glucocorticoid hormones

Following the conditioning with dexamethasone, a dose-dependent place preference in non-preferred compartment was observed on the second test day in male Wistar rats. Amphetamine in subthreshold dose exerted no effect if administered alone and induced a place preference in an unbiased paradigm after pre-treatment with dexamethasone. Administration of D2-dopamine receptors' antagonist sulpiride 30 min prior to dexamethasone conditioning completely blocked the acquisition of the place preference. The D1-dopamine receptors' antagonist SCH23390 exerted no effect on the place conditioning. The findings suggest that the D2-dopamine receptors take part in conditioned place preference with dexamethasone.