Sarah Bullock

Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training

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.

Effects of the Amnesic Agent 2‐Deoxygalactose on Incorporation of Fucose into Chick Brain Glycoproteins

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.

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.

Characterisation and Regional Localisation of Pre‐ and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

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.

Synaptic vesicle proteins and acetylcholine levels in chick forebrain nuclei are altered by passive avoidance training.

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.

Training Chicks on a Passive Avoidance Task Modulates Glutamate‐stimulated Inositol Phosphate Accumulation

The effects of glutamate, N‐methyl‐d‐aspartate (NMDA), (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo‐(a,d)‐cyclohepten 5,10‐imine maleate (MK801), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) and quisqualate on the accumulation of inositol phosphates (IP) from the breakdown of phosphoinositides in vitro have been studied in tissue prisms derived from a region of the chick forebrain, the intermediate medial hyperstriatum ventrale (IMHV). In prisms from the left IMHV, glutamate stimulated IP accumulation by 10–20%, AMPA by 55% and quisqualate by 650%. These effects were more marked in the right IMHV, where AMPA stimulated IP accumulation by 157% and quisqualate by 920%. MK801 and NMDA had no significant effect on IP accumulation in either hemisphere. The left IMHV is known to be the site of a biochemical cascade resulting in synaptic remodelling following training day‐old chicks on a one‐trial passive avoidance task. The effect of such training was to reduce glutamate‐stimulated accumulation of IP by 27% (P < 0.05) in prisms taken 30 min after training. There was no effect on prisms taken at 5 or 180 min after training, and no effect at any time in the right IMHV. MK801, injected intraperitoneally before training at a concentration known to produce amnesia for the passive avoidance task, abolished the training‐induced decrease without itself affecting IP accumulation. Taken in conjunction with pharmacological and autoradiographic evidence, these results indicate that memory formation for the passive avoidance task involves the activation of NMDA receptor channels, but not quisqualate or AMPA receptors, in the left IMHV of the chick 30 min after training.

Passive avoidance learning in the young chick results in time- and locus-specific elevations of α-tubulin immunoreactivity

A monoclonal antibody was used to examine changes in immunoreactivity of the cytoskeletal protein, alpha-tubulin, following passive avoidance learning in day-old chicks. Postmitochondrial fractions (16,000 g supernatants) were prepared from specific forebrain loci taken at several time points after training and assayed with the anti-alpha-tubulin antibody, YL1/2. Of the regions examined, elevations in the titre of YL1/2 were found in the left intermediate hyperstriatum ventrale 1 h, 6 h and 24 h following training, in the left lobus parolfactorius 1 h following training and in the right lobus parolfactorius 6 h and 24 h following training. No training-related changes were detected in a third forebrain region, the paleostriatum augmentatum. These results regarding the cellular dynamics of memory formation in the chick confirm and expand on earlier findings from our laboratory.

Posttetanic long-term potentiation in rat dentate area increases postsynaptic 411B immunoreactivity

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.

411B: A Monoclonal Postsynaptic Marker for Modulations of Synaptic Connectivity in the Rat Brain

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.

Preparation and Characterisation of a Monoclonal Antibody to an Antigen Enriched in Chick Brain Postsynaptic Densities

Abstract: The preparation and characterisation of a monoclonal antibody to an antigen enriched in day‐old chick brain postsynaptic densities (PSDs), with respect to other subcellular loci, are described. Immunolabelling with this antibody produced a dendritic immunoprecipitate that was markedly stronger in PSDs than in other subcellular loci. Thus, the antiserum could be used as a marker for PSDs during their purification by subcellular fractionation, as well as in the study of PSD assembly. Monoclonal antibody 411B has already been shown to be a useful tool in the chemical determination of changes in synapse density after various experimental manipulations in both the chick and rat. In the present study, we have used the antiserum to monitor the appearance and maintenance or redundancy of synaptic components in the developing chick forebrain.