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Notes: Abstract: In a search for biochemical markers of modified synaptic function following training of day-old chicks on a passive avoidance task, we have assayed two monoclonal antibodies to synaptic vesicle proteins (anti-p65 and anti-SV2) and one raised to postsynaptic densities (411B). We have also measured total acetylcholine (ACh) content. Measurements were made on three forebrain regions known to show metabolic and morphological change consequent on training—the lobus parolfactorius (LPO), paleostriatuni augmentatum (PA), and medial hyperstriatum ventrale (MHV)—in the right and left hemispheres 2 and 24 h after training chicks on a passive avoidance task, in which they learn to avoid pecking a bead coated with methylanthranilate [methylanthranilate-trained (M-trained)]. Control chicks were trained on a water-coated bead [water-trained (W-trained)]. Twenty-four hours after training, 411B levels showed no differences between W-trained and M-trained chicks in any region. M-training reduced the litre of antip65 by 16% in the left PA and 15% in the left MHV and that of anti-SV2 by 19% in the left PA. M-trained chicks showed reduced total ACh content in the LPO by up to 40% and in the PA by up to 48% but had no change in ACh level in the MHV. The decreases in antibody titre were not seen in forebrains analysed 2 h after training, but tendencies toward increases in levels in the right PA and MHV were observed with all three antibodies. Significant differences between right and left hemispheric regions, independent of training, were observed for all the antibodies and for ACh content. In the LPO, antibody litres were some 10% lower in the left compared with the right hemisphere and 34% lower for ACh content, but in the PA, the left hemisphere titre was higher by 14–86%. We discuss these results in the context of morphometric and electrophysiological data oblained from W-trained and M-trained chicks.

Notes: Abstract: Synaptic plasma membranes from chicken brain were used to isolate a postsynaptic density (PSD) fraction using an aqueous two-phase polymer system and the detergent n-octyl glucoside. The protein and glyco-protein composition and the morphology of the day-old chicken brain PSD fraction were compared with a PSD fraction isolated from 12-week-old chicken brain. The PSD fraction from day-old chicken brain contained predominantly PSDs although, like the fraction from 12-week-old chicken, there was some membrane contamination. The major polypeptides in the day-old chicken fraction resolved by polyacrylamide gel electrophoresis comigrated with α- and β-tubulin (Mr 57,000 and 55,000) and actin (Mr 45,000). The major PSD polypeptide (mPSDp) of 12-week-old chicken forebrain, which has a molecular weight of 52,000 was not a major component in day-old chicken. A polypeptide of molecular weight 63,000 was also far more prominent in the 12-week-old chicken PSD fraction whereas the reverse was true for a polypeptide of 31,000. Day-old chicken brain PSDs contained at least 14 concanavalin A-binding glycoproteins of high (〉85,000) molecular weight, the two most prominent having molecular weights of 170,000 and 180,000. In contrast to the polypeptide composition, the glycoprotein pattern of day-old chicken PSDs was very similar to that of the 12-week-old bird. Intraperitoneally injected [3H]fucose was incorporated into the glycoproteins of synaptic plasma membranes and PSDs from day-old chickens. In the synaptic plasma membranes, maximum specific radioactivity relative to homogenate (2.4) was reached after a 24-h pulse, whereas in the PSDs, relative specific radioactivity continued to rise up to 72 hr after injection to reach a value of 3 times that of the homogenate.

Notes: Abstract: Using 411B, a monoclonal marker raised to chick forebrain postsynaptic densities (PSDs), we have been able to demonstrate by enzyme-linked immunosorbent assay that the antigen recognised by this monoclonal exists in brain tissue from adult Wistar rats but not in liver, heart, or lung. Moreover, 411B immunoreactivity estimated in various cortical and subcortical brain structures exhibited remarkable differences. The pattern of subcellular distribution of 411B antiserum titre in rats was found to be qualita-tively similar to that in day-old chicks, indicating an enrichment of the antigen concentration in the PSD fraction by about 60 times over that observed in the lysed homogenates. One aim of this study was to investigate whether 411B is a useful biochemical marker for plastic changes of postsynaptic structures in the rat brain. Antigen was assayed in lysed homogenates from various brain regions dissected from dopaminergically supersensitive rats. Dopaminergic supersensitivity induced by treating animals with haloperidol (1 mg/kg i.p.) for 21 consecutive days resulted in a significant increase in the titre of 411B in corpus striatum (+21%) and hippocampus (+45%) whereas the titre of Q155, a monoclonal marker for an integral synaptic vesicle protein, was unchanged. Our results support the hypothesis that drug-induced dopaminergic supersensitivity is based on plastic changes at the postsynaptic site. In addition, monoclonal antibody 411B does appear to be a useful tool for further investigation of plastic changes occurring in postsynaptic brain components.

Notes: Abstract: Slices from the forebrains of day-old chicks represent a highly active in vitro protein-synthesising system. The in vitro incorporation of l[14C]leucine into protein of slices was estimated to be 2.5 mmol/mg protein/h. Incorporation was linear over 90 min of incubation and was suppressed by 92% by 1 mM cycloheximide. The highest incorporation was into microsomal and cell-soluble fractions. Under the electron microscope, slices appeared vacuolated near the cut surfaces, but well preserved internally (〉40 μm from the edge). Autoradiography showed that radioactivity was incorporated evenly across the slice with no decrease in label in the central part of the tissue. The rate of incorporation was only weakly dependent on leucine concentration in the medium (0.04–1 mM). Addition of a mixture of unlabelled amino acids (1 mM) produced a 20–50% inhibition of incorporation of radioactive l-leucine depending on the amino acids involved. In slices prepared from chicks 1 h after training on a one-trial passive avoidance paradigm, l-[14C]leucine incorporation was 23% higher (p 〈 0.01) in the forebrain roof than in slices from control chicks. This figure is comparable to the one previously reported in vivo. Subcellular fractionation of incubated slices from the forebrain roof of trained and control birds revealed that the increased protein synthesis was due mainly to an elevated leucine incorporation into the soluble fraction.

Notes: 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.

Notes: Abstract: The interaction of the amnesic agent 2-deoxygalactose with fucose incorporation into glycoproteins in day-old chick forebrain has been studied with the aim of identifying glycoproteins whose synthesis is modified during memory formation. 2-Deoxygalactose inhibited total exogenous [14C]fucose incorporation into the forebrain glycoproteins by 26%. Sodium dodecyl sulphate-polyacrylamide gradient gel analysis revealed that intracerebrally injected 2-[3H]deoxygalatose labelled the same eight major glycoprotein bands as were identified using [14C]fucose labelling. Subsequent investigations focussed on these selected components. Subcellular fractionation showed that between 4 and 24 h after administration of the deoxy-sugar, the incorporated radioactivity was found predominantly at the synaptic sites, some glycoproteins being more abundant in synaptic plasma membranes and others in postsynaptic densities. This distribution pattern varied according to the time after injection. The effect of passive avoidance training, using a methylan-thranilate-coated bead, on [14C]fucose incorporation into forebrain was to decrease fucose uptake into components of molecular mass 150–180 kilodaltons but to increase significantly labelling of glycoproteins of molecular mass 33 and 28 kilodaltons. The possible implications of these training-induced changes are discussed.

Notes: 411B is a monoclonal antibody raised to chick forebrain postsynaptic densities (PSDs) which also recognises an antigen in brain tissue from adult Wistar rats but not liver, heart, or lung. This antigen is enriched in the PSD fraction and appears to be a useful biochemical marker for plastic changes of postsynaptic structures in the rat brain. The aim of this study was to investigate whether 411B immunoreactivity is changed in various hippocampal subregions by post-tetanic long-term potentiation (LTP). LTP was elicited in freely moving rats by applying four trains of 300 square-wave pulses (frequency 200 Hz, pulse duration 0.2 ms, and intensity 300 mA) into the right perforant path; this included an increase in transmission efficacy at the ipsilateral perforant path-granular cell synapse of the dentate gyrus lasting several days. Eight hours after tetanisation, antigens recognised by monoclonal 411B and a polyclonal anti-actin antiserum were assayed in lysed homogenates of ipsi- and contralateral CA1, CA3, and CA4/dentate area hippocampal subfields as well as in visual cortex, cerebellum, and olfactory bulb dissected from LTP rats, and compared to passive controls. Under these experimental conditions, tetanisation of the perforant path resulted in a significant increase in the titre of 411B in the ipsilateral CA4/dentate area subfield (+34.0%; p 〈 0.001) compared with passive controls, whereas in all other brain regions studied no differences between experimental and control rats were observed. In no region were anti-actin titres significantly different from controls. Our results support the hypothesis that the “late phase” of posttetanic LTP is accompanied by, or even based on, macromolecular changes elicited at the postsynaptic site of perforant path-granular cell synapses.

Notes: Abstract: We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphor ylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks. After 30 min, this decrease was greater (-34%, p 〈 0.05) and was significant. There was a concomitant but nonsignificant increase (+22%) in phosphorylation of a 45 kDa component in the SPM fraction. No significant changes in phosphorylation patterns were seen in PSD fractions or in SPMs and PSDs isolated from cerebella of chicks with passive avoidance training at any time.

Notes: Abstract: To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyper-striatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150–180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass ∼50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150–180, 100–120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxyga-lactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.

Notes: Abstract: When chicks are trained to avoid pecking a bead coated with methylanthranilate in a one-trial passive avoidance task there is an increase in fucose incorporation in vivo and in vitro in the right forebrain base of methylanthranilate (M)-trained compared to water (W)-trained chicks. The relation of this increase to de novo protein synthesis in vivo and in vitro has been examined. Cycloheximide (Cx), 1 mM, inhibited in vitro fucosylation of chick brain slices by 60% after 3 h. However, the training-related increase in in vitro fucosylation still persisted. When Cx was injected intraventricularly 10 min before training, the subsequent increase in in vitro fucosylation due to training was still apparent. When Cx was injected and [14C]leucine and [3H]fucose incorporation studied in vivo in M-trained and W-trained chicks, there was no increase in fucosylation due to training in the Cx-treated M-trained over the W-trained chicks. These results are taken to indicate that in vitro fucosylation and its increase subsequent to training is not protein synthesis-dependent, but that both in vivo and in vitro there are interactions between Cx and fucosylation steps that are independent of Cx's effects on protein synthesis.