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

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 phosphorylation 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-Dependent Increase in Release of Arachidonic Acid Following Passive Avoidance Training in the Day-Old Chick

Abstract: We have investigated the role of arachidonic acid, a putative retrograde messenger, in a one-trial aversive learning task in the day-old chick. The left and right intermediate medial hyperstriatum ventrale (IMHV) in the chick forebrain have previously been implicated in the formation of memory for this task. Using an ex vivo technique we have determined the concentrations of various fatty acids liberated from prisms prepared from these brain regions at different time points up to 24 h following passive avoidance training. At 30, 60, and 75 min post-training the concentration of arachidonic acid, but not of other fatty acids, in prisms prepared from the left IMHV, but not the right IMHV, was enhanced compared with that in chicks trained on a nonaversive water-coated bead. To test whether arachidonic acid liberation from the left IMHV was receptor-stimulated we showed that (a) liberation of endogenous arachidonic acid from homogenate prepared from the left and right IMHV of untrained chicks was stimulated by depolarization with KCl (50 m M ) and that (b) glutamate agonists of the NMDA and metabotropic subtypes of glutamate receptor stimulated release of preloaded [¹⁴C]arachidonic acid from prisms prepared from the left IMHV but not the right IMHV. These results indicate that arachidonic acid is liberated from the left IMHV following passive avoidance training in the day-old chick and may play a role as a retrograde messenger in this memory task.     

Synaptic Vesicle Proteins and Acetylcholine Levels in Chick Forebrain Nuclei Are Altered by Passive Avoidance Training

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), paleostriatum 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 titre of anti-p65 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive avoidance training increases fucose incorporation into glycoproteins in chick forebrain slices in vitro

When day old chicks are trained to avoid pecking at a bright bead coated with methyl anthranilate, many neurochemical changes, both transient and longer lasting, have been found. These include an increased fucose incorporation in vivo into particulate glycoproteins, which persists for at least 24 hrs after training. We have now developed an in vitro method for studying fucose incorporation and have been able to replicate effects of training found in vivo. Chick forebrain slices incubated at 42° in a glucose containing-medium incorporatel-[U¹⁴C]fucose linearly for up to 3 hrs at rates of 30–35 nmol/g prot/hr. Incorporation was only 60% inhibited by cyclohexmide indicating that some fucosylation is occuring on preexisting proteins. Fucose incorporation was compared in slices from trained and control chicks and, as in vivo, a 16% increase in incorporation into the right forebrain base of trained birds was found. This increase was confined to the microsomal fraction. When cycloheximide was added to the incubation medium, the enhanced fucose incorporation in slices from trained birds was still observed.Dedicated to Professor Yasuzo Tsukada.     

Phosphorylation of the Postsynaptic Density Glycoprotein gp 180 by Ca2+/Calmodulin-Dependent Protein Kinase

Postsynaptic densities (PSDs) were prepared by the aqueous two-phase extraction of synaptic membranes in the presence of n-octyl glucoside. Incubation of postsynaptic densities with [gamma-32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P-labelled PSDs by affinity chromatography on concanavalin A-agarose identified the postsynaptic glycoprotein of apparent Mr 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13-fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin-dependent increases in phosphorylation of 68-fold. The phosphorylation of gp180 was increased 7.5-fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 X 10(-6) M. The amounts 12.6 micrograms/ml and 9.1 micrograms/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180.     

Passive Avoidance Training Increases Fucokinase Activity in Right Forebrain Base of Day-Old Chicks

Fucokinase (EC 2.7.1.52) activity was estimated in supernatants of homogenate from day-old chick forebrain. Enzyme kinetic studies gave a Km of 4.5 X 10(-6) M and Vmax of 3.72 nmol fucose converted into fucose-1-phosphate/mg prot/h. The pH optimum was 7.5. The enzyme is thus considerably more active than was reported for other species and tissues. There were no differences in enzyme activity between the four forebrain regions studied. One hour after chicks were trained on a one-trial passive avoidance learning paradigm, enzyme activity in the right forebrain base increased 14% over control values (p less than 0.02). The 11.3% increase in activity in the left forebrain base and 10.3% increase in the left roof were not statistically significant. The relationship of this change to the increased fucose incorporation into glycoproteins known to occur over a similar time period and the significance of the lateralization of the increase 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.     

Potentiation of synaptic responses in slices from the chick forebrain.

Coronal slices, containing part of the medial hyperstriatum ventrale (MHV), were cut from the left forebrains of domestic chicks and maintained in vitro. Records were made of the field responses evoked in the MHV by local electrical stimuli provided at 0.1 Hz. Two 1 min periods of stimulation at 5 Hz, separated by 10 min, were used in attempts to induce a persistent increase in the size of the postsynaptic response to test stimulation at 0.1 Hz. This procedure produced a potentiation which usually lasted longer than 2 h. The probability of inducing this persistent potentiation of the response (PPR) is not distributed evenly over the whole anteroposterior length of the MHV but is higher in slices that also contain the septo-mesencephalic tract ventrally. These are the slices that contain the intermediate part of the medial hyperstriatum ventrale (IMHV); an area that is essential for early behavioural learning. At this level PPR is not confined to the IMHV. It can also be produced in the lateral neostriatum in response to similar local stimulation at 5 Hz. No PPR was observed in either the caudal ectostriatum, or the paleostriatum.     

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

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]deoxygalactose 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 methylanthranilate-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.     

Predispositions and preferences. Effects on imprinting of lesions to the chick brain

Domestic chicks were exposed to a moving, stuffed jungle fowl or a rotating red box. The effects on the imprinting process of lesions to a restricted part of the hyperstriatum ventrale (IMHV) were studied in a series of experiments. Sham-operated control chicks developed a strong preference for the training object. Damage to IMHV impaired chicks' preferences for the training stimulus. However, the effect on chicks exposed to the red box was profound, whereas the effect on chicks exposed to the jungle fowl was relatively weak. The results suggest that information about a complex object, which in the experiments described resembled the chicks' own species, is stored in a different way from information derived from a relatively simple artificial object.     

Increased Fucosylation of Chick Brain Proteins Following Training: Effects of Cycloheximide

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.     

Genotype differences in catecholamine concentrations in hypothalamus,intramedial hyperstriatum ventrale,and optic tectum of newly hatched chicks

This study was designed to compare catecholamine concentrations among three brain areas of four pureline populations of visually isolated chicks. The purelines used were a commercial male line, a fertility selected line, an unselected fertility control line, and unselected White Jersey Giants. In general, male chicks had significantly larger brain weights than females. Six catecholamine-related compounds (norepinephrine, epinephrine,l-DOPA, dopamine, DOPAC, and MHPG) were measured via HPLC-ECD. No significant differences in neurochemical concentration were observed for any line or brain area due to sex of the chick. The hypothalamus (HT) contained the greatest concentration of catecholamines in all lines, followed by the intramedial hyperstriatum ventrale (IMHV) and optic tectum (OT). The HT exhibited consistent lateralization in all lines with the right HT containing ca. 30% more catecholamines than the left HT. While no consistent lateralization was observed among the other brain areas, the IMHV exhibited significantly different degrees of lateralization among the populations. Neuronal activity, as measured by MHPG:NE and DOPAC:DA ratio varied by line within each brain area. There were line differences for MHPG:NE in the HT, IMHV, and OT, while line differences for DOPAC:DA were observed in the HT. Since differences among purelines have been demonstrated in this study, care must be given to precisely define the genotype of chicks used in behavioral and neurochemical research.     

Passive avoidance learning in the young chick results in time- and locus-specific elevations of alpha-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.     

In Vivo Phosphorylation of the Postsynaptic Density Glycoprotein gp180

Rats received intraventricular injections of [32P]PO4 and were killed after 30 min for the preparation of postsynaptic densities (PSDs). Gel electrophoretic analysis identified a number of PSD proteins that incorporated 32P under these conditions. Major proteins that were labelled with 32P had Mr of 185,000, 165,000, 140,000, 92,000, and 51,000. Of these p185, p165, and p140 were also labelled when PSDs were incubated with [gamma-32P]ATP in vitro. In contrast p92 and p51 were relatively poorly labelled under in vitro conditions. Analysis of glycoproteins isolated by chromatography on concanavalin A (Con A)-agarose demonstrated that greater than 70-80% of the 32P present in the glycoproteins eluted from Con A-agarose with alpha-methyl-D-mannopyranoside (Con A+ glycoproteins) was associated with the PSD specific glycoprotein gp180 following both in vivo and in vitro labelling. Phosphopeptide maps and phosphoamino acid analysis of gp180 indicated that similar sites were labelled in vitro and in vivo. Analysis of the subcellular distribution of glycoproteins that incorporated 32P during in vivo labelling demonstrated that gp180 was highly concentrated in PSDs, in accord with the previously suggested exclusive association of this glycoprotein with postsynaptic structures.     

日龄雏鸡的学习记忆模型及其分子机制和药理学研究进展

日龄雏鸡一次性被动回避学习和厌恶性条件化学习模型被广泛用于学习记忆机制的研究,并取得了很大的进展. 上纹体和旁嗅核是参与雏鸡学习记忆的主要脑区. 结合相关的分子机制研究,药理学实验发现了多种能影响不同记忆阶段的药物,如去甲肾上腺素对长时记忆有增强和调控作用. 由于鸟类和哺乳动物与记忆相关的脑结构和功能具有一定可比性,上述工作可为了解大脑的学习记忆功能提供重要参考.     

The effects of age and visual experience on potentiation of responses in slices from the chick forebrain.

A single coronal slice, containing the intermediate part of the medial hyperstriatum ventrale (IMHV) was cut from the left forebrain of a series of domestic chicks and maintained in vitro. Records were made of field responses evoked in the IMHV by local electrical stimuli. Two 1-min periods of 5 Hz stimulation, separated by 10 min, were used in attempts to induce persistent potentiation of the responses (PPR) to test stimulation at 0.1 Hz. In dark-hatched chicks the probability of producing PPR is much higher in slices from chicks aged 2-5 days post-hatch than in those from either younger or older birds. As an independent measure of plasticity in dark-hatched chicks, the probability of eliciting unit responses to repeated stimulation of remote sites in the slice at 3.3 Hz was analysed. This probability was greater in slices from chicks aged 2-5 days than in those from either younger or older birds. In light-hatched chicks the probability of inducing PPR is significantly higher during the first day post-hatch, than in dark-hatched chicks of this age. The probability of producing PPR in slices from light-hatched chicks aged 2-3 days is less than that in slices from either younger or older birds. It is clear that both the age and past experience of the domestic chick affect the neurophysiological properties of slices of brain, tested in vitro.     

The mechanisms of memory reorganization during the retrieval of acquired behavioral experience in chicks: the effects of protein synthesis blockade in the brain]

It is currently assumed that disruption of memory formation by inhibitors of protein synthesis can occur in a relatively short time interval before and after training. However, there is some evidence that memory may be disrupted by delayed injections of protein synthesis inhibitors during "reminder" treatment, i.e., environmental cue that was presented earlier during the training procedure. Our experiments were conducted to test the late effects of protein synthesis inhibitor cycloheximide on memory in chicks using a reminder treatment. A standard passive avoidance task was presented to day-old chicks. A reminder (a dry bead of the same color as during training) was delivered within 2, 24, or 48 hours after the training. Chicks were bilaterally intracranially injected with cycloheximide (20 micrograms) into the IMHV area 5 min prior to reminder administration. Testing was conducted 0.5, 1, 3, 24, and 48 hours after the reminder. Administration of cycloheximide before the reminder resulted in transient amnesia. Duration of amnesia decreased with increasing interval between the training and reminder procedures. These results suggest that memory reactivated by the reminder treatment is subjected to reorganization and reconsolidation depending on protein synthesis. The gradual decrease in vulnerability of memory to protein synthesis inhibitor points to development of memory consolidation process in the interval between 2 and 48 h after training.     

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

Isolation of Postsynaptic Densities from Day-Old Chicken Brain

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 glycoprotein 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 alpha- and beta-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 (greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)