Decreased skin sensory innervation in transgenic mice overexpressing insulin-like growth factor-II.

Cutaneous sensory innervation was studied in transgenic mice overexpressing insulin-like growth factor II using a keratin promoter. The skin area of these animals is enlarged providing increased target for sensory neurons. L4 dorsal root ganglion cell counts revealed that the total number of sensory neurons was the same in transgenics as control animals. Levels of nerve growth factor per unit weight of skin were also unchanged. The cutaneous nerves of the hindlimb were immunostained with the pan-neuronal marker PGP 9.5 in transgenic and control mice at postnatal day 0 and 21. The innervation in transgenic mice was markedly reduced, particularly in superficial dermis and epidermis and in some areas innervation was completely absent. The effect was greatest in distal skin regions and increased with age. Since insulin-like growth factor II has been reported to be a sensory neurotrophic factor, its effect on neurite outgrowth was tested on embryonic day 14 and 18 mouse lumbar dorsal root ganglion explants in culture. Under these conditions insulin-like growth factor II (5-100 ng/ml) did not have strong growth promoting activity and at embryonic day 18, in the presence of 5-10 ng/ml nerve growth factor, neurite outgrowth was suppressed by insulin-like growth factor II. The results show that increased skin target and availability of nerve growth factor per se do not alter the number of innervating sensory neurons. However, reduced sensory terminal arborization and skin hypoinnervation does occur in the presence of excess insulin-like growth factor-II. It is possible that insulin-like growth factor-II inhibits terminal axon growth directly via receptors on sensory neurons or peripheral glia.

Voluntary transformation from an institutionally based to a community-based service system.

The transformation of a large, private, not-for-profit, church-affiliated provider of residential services from an institutionally based to community-based service system was described. Closure of a 200-person ICF/MR facility was discussed. Factors influencing the decision to close the institution as well as the guidelines used in effecting the transformation were described. Finally, data were presented indicating that consumer and staff satisfaction and judgments of program quality remained high during the period of transformation.

Long-term sensory hyperinnervation following neonatal skin wounds.

Skin innervation during wound healing was investigated using immunocytochemical staining with the panneuronal marker antiprotein gene product (PGP) 9.5, which labels the entire innervation of the skin throughout development and in the adult. Full-thickness skin wounds in the hairy skin of the foot in neonatal rats result in pronounced hyperinnervation of the tissue that persists long after the wound has healed (at least 12 weeks). Quantification of this hyperinnervation by image analysis indicates that skin innervation density in the wounded area can increase up to 300%. The effect is greatest when wounds are performed at postnatal day (P) 0 or 7, declining when performed at P14 and P21 to resemble the weaker and transient effect in the adult. Staining with selective markers for different neuronal populations innervating skin (monoclonal anti-RT97 staining the myelinated axons of large light sensory ganglion cells; anticalcitonin gene-related peptide staining unmyelinated C axons, thinly myelinated A delta axons, and a subpopulation of large A fibres) reveal that both A- and C-fibre sensory axons contribute to this response. Destruction of the majority of the C-fibre population with neonatal capsaicin pretreatment, which leaves large A fibres intact, significantly reduces the hyperinnervation response at 14 days, confirming a major contribution from both A and C fibres. Sympathetic axons, stained with anti-tyrosine hydroxylase, do not sprout into the wounded area. Furthermore, pretreatment of neonates with 6-hydroxydopamine, which destroys the sympathetic innervation, does not significantly reduce the overall sprouting response, as identified by anti-PGP9.5 staining. Behavioural sensory testing revealed a 50% drop in the mechanical threshold in the wounded area after 3 weeks. These remarkably long-term and specific effects on sensory terminal axons following neonatal skin wounding indicate the plasticity of cutaneous innervation density following alterations in the target tissue at a critical stage of development.

Nerve growth factor levels in developing rat skin: upregulation following skin wounding.

Levels of nerve growth factor (NGF) in rat hindpaw skin, measured with a sensitive two-site enzyme-linked immunosorbent assay, show two peaks during normal development. The first (57 +/- 5 pg mg-1) occurs at embryonic days (E) 18-20 and coincides with the arrival of axon terminals into the hindpaw skin. The second, larger peak (132 +/- 10 pg mg-1), occurs later, around postnatal day (P) 21 and may be involved in maintenance of neuronal phenotype. Levels outside the two peaks stay relatively constant throughout development (30 pg mg-1). Skin wounding at birth produces a marked increase in NGF levels (149 +/- 25 pg mg-1) which declines after 4 days. This large increase is not observed if wounding is performed at older ages and may underlie the sensory hyperinnervation that accompanies neonatal wounds.

GAP-43 expression in primary sensory neurons following central axotomy.

Primary sensory neurons are capable of successful regenerative growth in response to peripheral nerve but not dorsal root injury. The present study is concerned with the differential expression of the mRNA for GAP-43, a growth-associated protein, in these sensory neurons, in response to injury of their central or peripheral axonal branches. Peripheral axotomy resulted in an elevation in message detectable within 24 hr, using Northern blot and in situ hybridization, which was maintained for 30 d, whereas dorsal root section produced no change except a transient and small increase if the axotomy was immediately adjacent to the dorsal root ganglia (DRG). Dorsal root section had no effect on GAP-43 mRNA levels in the dorsal horn or in neighboring intact DRG. It also failed to alter the laminar boundaries of the GAP-43 central terminal labeling produced by peripheral nerve section, even though vacant synaptic sites were produced in unstained laminae by this procedure. This indicates that the location of GAP-43 immunolabeling in the central terminals of primed sensory cells may not depend only on the location of vacant synaptic sites. We conclude that distinct control mechanisms regulate the response of DRG neurons to peripheral nerve and dorsal root injury, and these may be related both to the glial environment and the particular target influences exerted on the central and peripheral branches of the primary sensory neuron. Central denervation alone is insufficient to upregulate GAP-43 levels, and this may explain the relative absence of collateral sprouting after the production of central vacant synaptic sites. The failure of dorsal root section to increase GAP-43 expression may contribute to the poor regenerative response initiated by such lesions.

Partial denervation of the medial gastrocnemius muscle results in growth-associated protein-43 immunoreactivity in sprouting axons and Schwann cells.

Regeneration in the mammalian peripheral nervous system following nerve injury is associated with the upregulation of a developmentally regulated phosphoprotein, growth-associated protein-43 (GAP-43), in the injured neurons. We have examined whether uninjured adult neurons also express GAP-43 when they sprout. The model system investigated has been the sprouting induced in the terminal axons of intact motor neurons by a partial muscle denervation. Partial denervation of the medial gastrocnemius muscle in adult rats was produced by resecting the terminal nerve supply to the anterolateral quadrant of the muscle. Three zones could be identified in the motor endplate region of the muscle after such a denervation using protein gene product (PGP) 9.5, calcitonin gene-related peptide and silver staining as axonal markers and S-100 to identify Schwann cells: a normally innervated zone, a totally denervated zone and a border or intermediate zone between the two which contained axons at the endplates with nodal and terminal sprouts. The endplates in the normally innervated zone were GAP-43 negative. In the denervated zone, Schwann cells were GAP-43 positive and had a distinctive appearance with a lack of any normal endplate organization. Endplates in the intermediate zone were GAP-43 immunoreactive. In approximately half, the GAP-43 immunoreactivity was axonal-like, identical to PGP 9.5 in an adjacent section; in the remainder it was Schwann cell-like, identical to S-100 staining. Partial muscle denervation results, therefore, in the appearance of GAP-43 both in axons and Schwann cells in the endplates bordering the denervated zone. The presence of GAP-43 in these cells may contribute to their capacity to sprout.

Reciprocal Schwann cell-axon interactions.

This article describes the reciprocal interactions between neurones and Schwann cells with particular reference to the role of growth factors and neurokines as signalling molecules between these cells and of the extracellular matrix as a conduit for such signalling. Major recent advances have identified molecules produced by neurones that are responsible for Schwann cell proliferation, as well as some of the Schwann cell factors regulating the expression of molecules shown to play an important role in neuronal survival and differentiation.

Neonatal sciatic nerve section results in thiamine monophosphate but not substance P or calcitonin gene-related peptide depletion from the terminal field in the dorsal horn of the rat: the role of collateral sprouting.

The expression of substance P, calcitonin gene-related peptide (CGRP) and thiamine monophosphatase in the sciatic nerve terminal field of the lumbar dorsal horn of the rat was examined following neonatal sciatic nerve section and ligation. The total terminal field from L3 to L5 was mapped from semi-serial sections on the treated side and compared to equivalent maps on the contralateral intact side. To obtain a detailed time course of events, data were obtained 4, 7, 10, 15-20 and 40-60 days after sciatic nerve section. At 4-7 days thiamine monophosphate was depleted from the cut nerve terminals resulting in a gap in dorsal horn thiamine monophosphate stain similar to that seen after adult nerve section. In contrast, substance P and CGRP-containing terminals showed only a transient fall in expression in the first week following nerve section and then staining was no different from that seen on the control side. The depletion of peptides normally observed after adult nerve section did not occur. This phenomenon was only observed if the sciatic nerve was cut at birth. Nerve section at 10 days of age resulted in the same pattern of peptide depletion as is observed in the adult. A week after neonatal sciatic nerve section, thiamine monophosphate-containing nerve terminals from nearby intact nerves begin to sprout into the sciatic nerve territory in the dorsal horn. This, together with some recovery of thiamine monophosphate from the remaining sciatic terminals themselves, results in a slow filling in of the gap in the thiamine monophosphate stain. Resection of the cut sciatic nerve, together with adjacent intact nerves, re-establishes the depletion. Substance P and CGRP terminals from nearby intact nerves also sprout into the deafferented sciatic field and this can be demonstrated by the larger than normal area of depletion following section of these nerves when adult. Furthermore, resection of the neonatally cut sciatic nerve when adult also causes some depletion of substance P and CGRP within the sciatic field, indicating a degree of recovery or up-regulation of peptides in surviving cut afferents. However, even after resection of the cut sciatic nerve and nearby intact nerves, substance P and CGRP staining remained in the terminal region. We conclude that while central collateral sprouting does take place in both substance P and CGRP-containing afferents following peripheral nerve section, it cannot account for the lack of depletion of peptides observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Denervation of the motor endplate results in the rapid expression by terminal Schwann cells of the growth-associated protein GAP-43.

Developing and regenerating neurons express high levels of the growth-associated phosphoprotein GAP-43. This membrane protein is not confined to neurons, however, as a number of studies have demonstrated GAP-43 immunoreactivity in central and peripheral glia in vitro and in vivo. We have found that the Schwann cells overlying the terminal motor axon at adult rat skeletal muscle endplates, and the motor axons themselves, are normally not GAP-43 immunoreactive. Within 24 hr of denervation, however, the terminal Schwann cells are positive for a GAP-43 mRNA in situ hybridization signal and are GAP-43 immunoreactive. The immunoreactive GAP-43 cells possess elaborate processes that branch from the endplate region into the perisynaptic zone and stain with defined Schwann cell markers: the calcium binding protein S100 and the low-affinity NGF receptor (NGFr), but not with a fibroblast marker, Thy-1. Reinnervating motor axons are GAP-43 positive, with an appearance quite different from the GAP-43-positive Schwann cells. The reappearance of nerve endings at the motor endplate is followed by the disappearance of GAP-43 labeling in the Schwann cells and of a retraction of their processes. GAP-43 expression in Schwann cells is therefore state dependent, apparently regulated by neural contact. This protein, which is associated in neurons with neurite formation, may participate in the elaboration of processes by Schwann cells when their contact with axons is disrupted.

Origin and fate of fetuin-containing neurons in the developing neocortex of the fetal sheep.

The development of the neocortex has previously been extensively studied in carnivores (cat and ferret), rodents (rat and mouse) and primates (monkey and human). In these species, it has been shown that the initial population of cells migrating from the ventricular zone forms the primordial plexiform layer. This is subsequently split into marginal zone and subplate zone by the insertion of later-migrating cells into the primordial plexiform layer, to form the cortical plate proper. Many of the cells derived from the split primordial plexiform layer are transient. The neurons of the subplate zone are found in the deeper part of layer VI, and white matter deep to layer VI in the more mature cortex; most of these neurons disappear by adulthood. [3H]-thymidine labelling in the present study has shown a similar pattern of neocortical development in Artiodactyla (sheep). In addition it has been shown that the previously described staining of subplate and cortical plate cells for the fetal protein fetuin indicates that fetuin is a useful marker for a proportion of this transient population of neurons and defines its extent in neocortical development more clearly. Dividing cells were labelled by a single intra-amniotic injection of [3H]-thymidine at E26 to E35 (birth is at E150). The brains were subsequently examined at E40 or E80 for [3H]-thymidine labelling and fetuin staining by a combination of autoradiography and immunocytochemistry. The earliest generated neocortical cells detected in this study (E26) were found in two layers by E40, the outer marginal zone and inner subplate zone. Neurons of the marginal zone were generated up to E28; those of the early subplate zone were generated up to E31. The cortical plate proper was generated by cells "born" on E32 and later. This sequence is similar to that described in other species, especially the cat. A proportion of the early-generated neurons in the marginal zone, subplate zone and early cortical plate stained for fetuin. By E80 these earliest-generated, fetuin-positive cells were found in the white matter deep to the forming neocortical layers and in layer VI. In adult brains no fetuin-positive neurons could be identified in the neocortex, and neurons had almost entirely disappeared from the white matter. The fetal glycoprotein fetuin seems to be specifically associated with a population of cells that has the same developmental history as the transient marginal zone and subplate neurons described in other species.(ABSTRACT TRUNCATED AT 400 WORDS)

Terminal Schwann cells elaborate extensive processes following denervation of the motor endplate.

Terminal Schwann cells, when stained for S100 (a calcium binding protein), can be seen to cap motor axons at the neuromuscular junction. Within days of denervation the Schwann cells begin to stain for the low affinity nerve growth factor receptor, but remain Thy-1 negative, and elaborate fine processes. These processes become longer and more disorganized over weeks, and cells positive for S100 and nerve growth factor receptor migrate into the perisynaptic area. Reinnervation results in a withdrawal of the processes. The morphology and location of terminal Schwann cells seems to depend on axonal contact. The spread of Schwann cells and their processes away from the synaptic zone following denervation, implies that these cells do not target axons directly to the endplate.

Distribution of the growth associated protein GAP-43 in the central processes of axotomized primary afferents in the adult rat spinal cord; presence of growth cone-like structures.

GAP-43-immunolabelled structures were visualized by electron microscopy in the adult rat L4-L5 superficial dorsal horn 2 weeks after sciatic nerve transection. The majority of immunolabelled elements were unmyelinated axons, but some synaptic terminals and myelinated axons also labelled. The labelled unmyelinated axons were commonly located in prominent bundles which on serial section analysis could be followed into larger single trunks. These enlargements contain many organelles and give rise to smaller processes, which is compatible with their being growth cones. Sciatic nerve transection may result, therefore, in central regenerative processes which reorganize the neuropil and contribute to the decreased sensibility and pain that follows peripheral nerve section.

GAP-43 expression in the developing rat lumbar spinal cord.

The expression of the growth-associated protein GAP-43, detected by immunocytochemistry, has been studied in the developing rat lumbar spinal cord over the period E11 (embryonic day 11), when GAP-43 first appears in the spinal cord, to P29 (postnatal day 29) by which time very little remains. Early GAP-43 expression in the fetal cord (E11-14) is restricted to dorsal root ganglia, motoneurons, dorsal and ventral roots and laterally positioned and contralateral projection neurons and axons. Most of the gray matter is free of stain. The intensity of GAP-43 staining increases markedly as axonal growth increases, allowing clear visualization of the developmental pathways taken by different groups of axons. Later in fetal life (E14-19), as these axons find their targets and new pathways begin to grow, the pattern of GAP-43 expression changes. During the period, GAP-43 staining in dorsal root ganglia, motoneurons, and dorsal and ventral roots decreases, whereas axons within the gray matter begin to express the protein and staining in white matter tracts increases. At E17-P2 there is intense GAP-43 labelling of dorsal horn neurons with axons projecting into the dorsolateral funiculus and GAP-43 is also expressed in axon collaterals growing into the gray matter from lateral and ventral white matter tracts. At E19-P2, GAP-43 is concentrated in axons of substantia gelatinosa. Overall levels decline in the postnatal period, except for late GAP-43 expression in the corticospinal tract, and by P29 only this tract remains stained.

GAP-43 expression in developing cutaneous and muscle nerves in the rat hindlimb.

The expression of the growth associated protein, GAP-43, in developing rat hindlimb peripheral nerves has been studied using immunocytochemistry. GAP-43, is first detected in lumbar spinal nerves at embryonic day (E)12 as the axons grow to the base of the hindlimb. It is expressed along the whole length of the nerves as well as in the growth cones. GAP-43 staining becomes very intense over the next 36 h while the axons remain in the plexus region at the base of the limb bud before forming peripheral nerves at E14. It remains intense along the length of the growing peripheral nerves, the first of which are cutaneous, branching away from the plexus and growing specifically to the skin, their axon tips penetrating the epidermis of the proximal skin at E15 and the toes at E19. GAP-43-containing terminals form a dense plexus throughout the epidermis which subsequently withdraws subepidermally in the postnatal period. GAP-43 staining is also evident along the growing muscle nerves during muscle innervation, which follows behind that of skin. Axons branch over the surface of proximal muscles at E15 but do not form terminals until E17. As target innervation proceeds, GAP-43 staining declines in the proximal part of the nerve but remains intense in the distal portions. Overall GAP-43 expression in the hindlimb decreases in the second postnatal week as axon growth and peripheral terminal formation decline.

Time-dependent differences in the increase in GAP-43 expression in dorsal root ganglion cells after peripheral axotomy.

Peripheral axotomy of primary afferent neurons results in the up-regulation of the growth-associated phosphoprotein GAP-43, by dorsal root ganglion cells. We have studied the temporal sequence of GAP-43 expression in those dorsal root ganglion neurons with unmyelinated axons (the small dark cells) and in those with myelinated axons (the large light cells) after sciatic nerve section in the adult rat. Immunoreactivity for the RT 97 neurofilament epitope, which is detectable only in large light dorsal root ganglion cells, was used to differentiate the two types of dorsal root ganglion cell. Within two days of a sciatic nerve section the number of GAP-43-immunoreactive profiles in the ipsilateral ganglion had increased five-fold and this increase persisted for 80 days post-section. While 50% of the small numbers of GAP-43-positive cells in control ganglia were RT 97 positive, only 8% of the large number of GAP-43-immunoreactive cells four days post-section, were RT 97 positive. By 14 days the number of RT 97-positive/GAP-43-positive cells had increased to 29%. This was paralleled by an increase in GAP-43 immunoreactivity in large diameter profiles at 14 days. The signals that alter GAP-43 expression in unmyelinated (small, RT 97 -ve) and myelinated (large, RT 97 +ve) afferents after peripheral nerve injury appear to operate with different time-courses.

The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury.

When adult dorsal root ganglion cells are dissociated and maintained in vitro, both the small dark and the large light neurons show increases in the growth-associated protein GAP-43, a membrane phosphoprotein associated with neuronal development and plasticity. Immunoreactivity for GAP-43 appears in the cytoplasm of the cell bodies as early as 3.5 h post axotomy and is present in neurites and growth cones as soon as they develop. At early stages of culture (4 h to eight days) satellite/Schwann cells are also immunoreactive for GAP-43. Neurons in isolated whole dorsal root ganglion maintained in vitro become GAP-43-immunoreactive between 2 and 3 h after axotomy. It takes three days however, after cutting or crushing the sciatic nerve in adult rats in vivo, for GAP-43 immunoreactivity to appear in the axotomized dorsal root ganglion cells. GAP-43 immunoreactivity can be detected in the central terminals of primary afferent neurons in the superficial laminae of the dorsal horn of the lumbar enlargement four days after sciatic cut or crush. The intensity of the GAP-43 staining reaches a peak at 21 days and becomes undetectable nine weeks following crush injury and 36 weeks following sciatic nerve cut. The pattern of GAP-43 staining is identical to the distribution of sciatic small-calibre afferent terminals. Little or no staining is present in the deep dorsal horn, but GAP-43 does appear in the ipsilateral gracile nucleus 22 days after sciatic injury. In investigating the mechanism of GAP-43 regulation, blockade of axon transport in the sciatic nerve with vinblastine (10(-5) M-10(-4) M) or capsaicin (1.5%) was found to produce a pattern of GAP-43 immunoreactivity in the dorsal horn identical to that found with crush, while electrical stimulation of the sciatic nerve had no effect. Axotomy of primary sensory neurons or the interruption of axon transport in the periphery therefore acts to trigger GAP-43 production in the cell body. The GAP-43 is transported to both the peripheral and the central terminals of the afferents. In the CNS the elevated GAP-43 levels may contribute to an inappropriate synaptic reorganization of afferent terminals that could play a role in the sensory disorders that follow nerve injury.

Blood-brain, blood-cerebrospinal fluid and cerebrospinal fluid-brain barriers in a marsupial (Macropus eugenii) during development.

1. The blood-brain, blood-CSF and CSF-brain barriers to protein have been studied in post-natal tammar wallabies (newborn to 70 days) using morphological and physiological techniques. 2. Endogenous and exogenous plasma proteins, and in some experiments horseradish peroxidase, were used as indicators of barrier integrity or permeability. 3. Immunocytochemical studies of endogenous tammar proteins showed that these (e.g. albumin) were present in the lumen of vessels in the brain, in cerebrospinal fluid (CSF) and within some cells in the choroid plexus and brain. No staining of the brain extracellular space was obtained; in particular there was no perivascular staining. Possible artifacts that could account for this lack of staining are discussed. 4. Ultrastructural studies showed the presence of well-formed tight junctions between cerebral endothelial cells and between choroid plexus epithelial cells, even as early as the day of birth. A membrane specialization between adjacent neuropendymal cells that had the same ultrastructural appearance as the 'strap junction' previously described in human and sheep fetuses was observed. These junctions may act as a barrier (CSF-brain barrier) to the passage of protein from CSF into brain in these immature animals, as has previously been described in eutherian fetuses. 5. In experiments in which exogenous plasma proteins or horseradish peroxidase were injected intravenously, care was taken to limit both the volume and protein load injected. These proteins penetrated into CSF. The naturally occurring steady-state CSF/plasma ratio for several proteins was approached by several of the injected (human) proteins within a few hours of I.V. injection, suggesting that much of the protein in CSF, at least when sampled from the hindbrain, originates from plasma in this species. No penetration across cerebral vessels was observed. Uptake of some proteins (e.g. albumin), occurred into neuroependymal cells at some ages. 6. These results suggest that the very immature brain of the newborn tammar is protected from protein present in the circulating plasma even at an embryonic stage of development by a combination of a well-formed blood-brain barrier to protein in the cerebral vessels and a CSF-brain barrier to protein at the level of the neuroependyma. The adult-type blood-CSF barrier to protein (tight junctions between adjacent choroid plexus epithelial cells) is present but appears to be bypassed in the immature brain, probably by a transcellular route across the choroid plexus.

A fetuin-related glycoprotein (alpha 2HS) in human embryonic and fetal development.

The human plasma protein, alpha 2HS glycoprotein, has an amino acid composition very similar to that of fetuin, the major protein in fetal calf and lamb serum. Immunohistochemical studies of human fetuses (6-33 weeks gestation) showed that alpha 2HS glycoprotein and fetuin have similar distributions in developing brain and several other tissues, e.g., bone, kidney, gonads, gastrointestinal tract, respiratory and cardiovascular systems. There were notable differences in the liver and thymus in the distribution of the two proteins. Fetuin and alpha 2HS glycoprotein are present in plasma and cerebrospinal fluid of both human and sheep fetuses; their concentrations are reciprocally related: in human plasma and cerebrospinal fluid alpha 2HS glycoprotein concentration is high and fetuin low; the reverse is the case in sheep fetuses. Estimates of the concentration of alpha 2HS glycoprotein in human fetal cerebrospinal fluid and plasma were obtained. It is suggested that alpha 2HS glycoprotein may play a role in developing tissues, especially in the human fetus, similar to that of fetuin in other species.

Fetuin as a marker of cortical plate cells in the fetal cow neocortex: a comparison of the distribution of fetuin, alpha 2HS-glycoprotein, alpha-fetoprotein and albumin during early development.

Fetuin, alpha 2HS-glycoprotein (alpha 2HS), alpha-fetoprotein (AFP) and albumin have been shown to be present in some regions of the neocortex in two early stages of development of the cow brain using PAP immunocytochemistry. In the pre-cortical plate stage fibres of the primordial plexiform layer stained positively for fetuin. No staining was seen for albumin but plasma and cerebrospinal fluid (CSF) were positive for alpha 2HS and AFP. In the early cortical plate stage the strongest fetuin positive staining was seen in the earliest formed cells of the plate. alpha 2HS staining was much less intense but similar in distribution. The possible role of fetuin, or related glycoproteins, in cortical plate differentiation is discussed. Staining for AFP and for albumin was seen mainly in the ventricular zone and marginal zone fibres, and had a similar distribution and intensity for both proteins. Plasma and CSF stained for all four proteins. Tests showed some cross-reactivity between fetuin and anti-alpha 2HS and, to a much lesser extent, between antisera to AFP and albumin and antigens denatured by fixation.