Observation of high-temperature macromolecular confinement in lyophilised protein formulations using terahertz spectroscopy.

Characterising the structural dynamics of proteins and the effects of excipients are critical for optimising the design of formulations. In this work we investigated four lyophilised formulations containing bovine serum albumin (BSA) and three formulations containing a monoclonal antibody (mAb, here mAb1), and explored the role of the excipients polysorbate 80, sucrose, trehalose, and arginine on stabilising proteins. By performing temperature variable terahertz time-domain spectroscopy (THz-TDS) experiments it is possible to study the vibrational dynamics of these formulations. The THz-TDS measurements reveal two distinct glass transition processes in all tested formulations. The lower temperature transition, T g , ß , is associated with the onset of local motion due to the secondary relaxation whilst the higher temperature transition, T g , α , marks the onset of the α -relaxation. For some of the formulations, containing globular BSA as well as mAb1, the absorption at terahertz frequencies does not increase further at temperatures above T g , α . Such behaviour is in contrast to our previous observations for small organic molecules as well as linear polymers where absorption is always observed to steadily increase with temperature due to the stronger absorption of terahertz radiation by more mobile dipoles. The absence of such further increase in absorption with higher temperatures therefore suggests a localised confinement of the protein/excipient matrix at high temperatures that hinders any further increase in mobility. We found that subtle changes in excipient composition had an effect on the transition temperatures T g , α and T g , ß as well as the vibrational confinement in the solid state. Further work is required to establish the potential significance of the vibrational confinement in the solid state on formulation stability and chemical degradation as well as what role the excipients play in achieving such confinement.

Cellular changes in motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis as revealed by monoclonal antibody Py.

Transgenic mice (G93A) carrying the human amyotrophic lateral sclerosis (ALS) linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. The affected motoneurons are characterized by the presence of cellular alterations. The antigen recognized by the monoclonal antibody Py is suggested to be associated with the neurofilamentous and microtubular elements of the cytoskeleton of specific neuron populations including the spinal motoneurons. The aim of the present study was to measure changes in the relative Py-immunoreactivity per identified Choline-Acetyl-Transferase (ChAT)-immunoreactive motoneuron during the disease progression. The relative Py-immunoreactivity of identified spinal motoneurons was measured on double stained (Py and ChAT) motoneurons using a digital imaging system coupled to an inverse microscope. A significant decrease of Py-immunoreactivity was already noted in the pre-symptomatic stages of the disease even before the onset of massive motoneuron degeneration. It is concluded that the Py-antibody detects early intracellular abnormalities related to neurodegenerative changes in spinal motoneurons of transgenic SOD1-(G93A) mice.

Branchiogenic motoneurons innervating facial, masticatory, and esophageal muscles show aberrant distribution in the reeler-phenotype mutant rat, Shaking Rat Kawasaki.

Shaking Rat Kawasaki (SRK) is an autosomal recessive mutant rat that is characterized by cerebellar ataxia. Although previous studies indicated many points of similarity between this mutant rat and the reeler mouse, nonlaminated structures such as the facial nucleus have not been studied in this mutant rat. Nissl-stained sections through the brainstem showed that the cytoarchitecture of the facial, motor trigeminal, and ambiguus nuclei was abnormal in SRK, especially in the lateral cell group of the facial nucleus and the compact formation of the ambiguus nucleus. To examine whether orofacial motoneurons are also malpositioned in the SRK rat, horseradish peroxidase (HRP) was injected into the facial, masticatory, and abdominal esophageal muscles of the SRK rats and normal controls to label facial, trigeminal, and ambiguus motoneurons, respectively. HRP-labeled facial, trigeminal, and ambiguus motoneurons of the SRK rat were distributed more widely than those of their normal counterparts, as in the case of the reeler mouse, with the one exception that labeled facial motoneurons innervating the nasolabial muscle were distributed more widely in the ventrolateral-to-dorsomedial direction in comparison with those of the reeler mutant. These data demonstrate that nonlaminated structures in the brainstem of the SRK rat are affected severely, as is the case in the reeler mutant mouse.

Aberrant trajectory of entorhino-dentate axons in the mutant Shaking Rat Kawasaki: a Dil-labelling study.

The Shaking Rat Kawasaki (SRK) is a neurological mutant that exhibits abnormalities of cell migration and lamination, with many similarities to the mouse reeler mutant. We recently used lamina-specific antibody staining to show that despite severe aberrations in the laminar organization of the SRK dentate gyrus, the entorhinal terminal field in the outer dentate molecular layer appeared relatively normal (Woodhams & Terashima, 1999, J. Comp. Neurol. 409 p57). However, neurofilament immunostaining suggested that entorhino-dentate afferents take an abnormal trajectory in reaching their appropriate targets, the granule cells dendrites. In the present study, anterograde tracing with the carbocyanine dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) has been used to delineate directly the path that entorhinal axons take to the dentate gyrus, confirming that in SRK entorhinal axons do indeed reach their appropriate terminal fields in the molecular layer, with laminar segregation between projections from the lateral and medial entorhinal cortices. However, these fibres fail to cross the hippocampal fissure between the subiculum and the dentate gyrus, coursing instead parallel to it until they curve round the deepest point of the fissure in field CA3. Similar findings were seen in the murine reeler mutant. Insertion of DiI crystals into the entorhinal cortex of neonatal rats also retrogradely labelled the developmentally transient Cajal-Retzius cells at the hippocampal fissure; these survive for longer in SRK than in normal littermates. The presence of a marked astrogliosis at the SRK hippocampal fissure may play a part in determining the abnormal trajectory taken by entorhino-dentate afferents in this mutant.

Differential distribution of immunoreactivity in the developing rat spinal cord revealed by the monoclonal antibody Py.

Monoclonal antibody Py was developed as a useful tool for the identification of large diameter neurons of the adult rat central nervous system [Woodhams et al., J. Neurosci., 9 (1989) 2170-2181]. Here, we present a detailed light-microscopic study of the distribution of Py-immunoreactivity in the developing rat spinal cord. The first cells which demonstrated Py-immunoreactivity were the motoneurons in layer IX of the gray matter at embryonic day 15. These cells, including their axons and dendrites, remained Py-immunoreactive throughout subsequent developmental stages into adulthood and were the most intensely stained cells in the adult rat spinal cord. Other cell populations which became Py-immunoreactive during development were neurons in layers III-VIII, and large-to-medium diameter neurons of the dorsal root ganglion (DRG). Transient Py-immunoreactivity was observed in the distal portions of DRG axons as well as in the ascending fibers in the dorsal funiculus. Py-immunoreactive fibers could be detected in the ventral most part of the dorsal funiculus (corticospinal tract area), even at embryonic ages prior to the arrival of corticospinal fibers. The localization and transient expression of the antigen recognized by the Py-antibody in developing rat spinal cord strongly suggests an important role of this molecule in stabilization and/or plasticity of the neuronal cytoskeleton. The results presented here form the foundation for the use of Py-immunocytochemistry to study well-defined cell populations under a range of experimental and pathological conditions.

Laminar boundaries persist in the hippocampal dentate molecular layer of the mutant Shaking Rat Kawasaki despite aberrant granule cell migration.

The present report provides the first detailed description of the hippocampus in the Shaking Rat Kawasaki (SRK) mutant by using a panel of antibody markers to delineate its laminar organization. The mutant was characterised at postnatal day 21 by severe malformations of both neuronal position and orientation, the most striking of which was the presence of a rounded central granule cell mass in the dentate gyrus rather than the normal V-shaped granule cell layer. Despite this finding, the SRK dentate gyrus not only retained a cell-sparse molecular layer (thinner but similar in gross appearance to that of control littermates), but the sharp laminar boundary between its inner and outer parts was as clearly marked by IM1 and OM4 antibody staining as it was in the normal dentate gyrus. These immunocytochemical data suggest that the entorhinal terminal field of the dentate gyrus may be relatively normal in the mutant, despite entorhinal afferents appearing to take an abnormal trajectory after they fail to cross the hippocampal fissure. Laminar malformations included disruption of the SRK pyramidal cell layer, with spreading of the CA3 mossy fibre projection to an ectopic infrapyramidal position, radial displacement of CA1 pyramids, and transposition of a hitherto unremarked longitudinal fibre bundle immunoreactive for calretinin from its normal position in the stratum lacunosum-moleculare of field CA2 to an alvear position in SRK. The SRK malformations were very like but not identical to those seen in the mouse reeler mutant, suggesting similar underlying developmental mechanisms.

A novel early component of the cell body response in axotomized Clarke's nucleus neurons revealed by monoclonal antibody Py.

The monoclonal antibody Py was initially developed as a tool for the identification of subpopulations of hippocampal neurons. Recently it has also been demonstrated to be a useful marker for other populations of midbrain and spinal cord neurons in which the antigen showed a strong colocalization with cytoskeletal elements. To assess the possible usefulness of Py as a tool for studying lesion-induced cell body changes, densitometric analysis of altered Py-immunoreactivity (Py-IR) has been compared with that of microtubule-associated protein 2 (MAP2) in Clarke's nucleus following axotomy. One week after a unilateral transection of the dorsal spinocerebellar tract at Th9-10, Py-IR in the Clarke's nucleus ipsilateral and caudal to the lesion was reduced by approximately 40%. By 21 days, Py-IR was reduced by approximately 50% (a near maximal reduction) and remained constant up to 5 months after the lesion (the longest survival time studied). Alterations of MAP2-IR in Clarke's nucleus were later in onset, slower to develop, and less marked. The differential distribution of the Py antigen in the CNS and its rapid and long lasting loss indicate that the Py antibody is a sensitive tool for studying novel early alterations of the cytoskeleton which may be important molecular events in axotomy-induced pathological processes.

Structural alterations and changes in cytoskeletal proteins and proteoglycans after focal cortical ischemia.

In order to study structural alterations which occur after a defined unilateral cortical infarct, the hindlimb region of the rat cortex was photochemically lesioned. The infarcts caused edema restricted to the perilesional cortex which affected allocortical and isocortical areas differently. Postlesional changes in cytoskeletal marker proteins such as microtubule-associated protein 2, non-phosphorylated (SMI32) and phosphorylated (SMI35, SMI31 and 200,000 mol. wt) neurofilaments and 146,000 mol. wt glycoprotein Py as well as changes in proteoglycans visualized with Wisteria floribunda lectin binding (WFA) were studied at various time points and related to glial scar formation. The results obtained by the combination of these markers revealed six distinct regions in which transient, epitope-specific changes occurred: the core, demarcation zone, rim, perilesional cortex, ipsilateral thalamus and contralateral homotopic cortical area. Within the core immunoreactivity for microtubule-associated protein 2 and SMI32 decreased and the cellular components showed structural disintegration 4 h post lesion, but partial recovery of somatodendritic staining was seen after 24 h. Microtubule-associated protein 2 and SMI32 persisted up to days 7 and 5 respectively in the core, whereas the number of glial fibrillary acidic protein- and WFA-positive cells decreased between days 7 and 14. The demarcation zone showed a dramatic loss of immunoreactivity for all epitopes 4 h post lesion which was not followed by a phase of recovery. In the inner region of the demarcation zone there was an invasion and accumulation of non-neuronal WFA-positive cells which formed a tight capsule around the core. Neuronal immunoreactivities for microtubule-associated protein 2, SMI31 and Py as well as astrocytic glial fibrillary acidic protein increased strongly within an approximately 0.4-1.0 mm-wide rim region directly bordering the demarcation zone. Py immunoreactivity increased significantly in the perilesional cortex, whereas glial fibrillary acidic protein-positive astrocytes became transiently more numerous in the entire lesioned hemisphere including strongly enhanced immunoreactivity in the thalamus by days 5-7 post lesion. Glial fibrillary acidic protein immunoreactivity increased in the corpus callosum and the homotopic cortical area of the unlesioned hemisphere by days 5-7. In this homotopic area additional changes in SMI31 immunoreactivity occurred. Our results showed that a cortical infarct is not only a locally restricted lesion, but leads to a variety of cytoskeletal and other structural changes in widely-distributed functionally-related areas of the brain.

Axotomy-induced alterations in the red nucleus revealed by monoclonal antibody, Py, following a low thoracic spinal cord lesion in the adult rat.

The monoclonal antibody Py was previously developed as a tool for the identification of subpopulations of hippocampal neurons. Here, the differential distribution of Py immunoreactivity in the mid-brain is described showing that Py also serves as a useful marker for other populations of neurons. Medium to strong immunoreactivity was observed in the cell body and dendrites of neurons of the oculomotor nucleus, superior colliculus and substantia gelatinosa reticulata. However, particularly intense Py-immunoreactivity was identified in the magnocellular neurons in the caudal pole of the red nucleus. Unilateral transection of the rubrospinal tract at Th9-10 induced a marked reduction of Py immunoreactivity in the ventrolateral territory of the caudal pole of the axotomised red nucleus. A small but statistically significant reduction of Py-immunoreactivity was first seen at 7 days after surgery and a maximal loss of immunoreactivity (reduced to 66% of control levels) was observed by 21 days after surgery. Immunoreactivity in the axotomised red nucleus was reduced for the duration of the experiment but at the longer survival times studied (3 and 6 months) a small degrees of recovery of staining was observed in small-medium diameter atrophic neurons. These results indicate that monoclonal antibody Py, may be a useful novel and sensitive tool for investigating the cell body reaction of particular populations of axotomised CNS neurons following spinal cord injury.

Differential distribution of immunoreactivity in the adult rat spinal cord revealed by the monoclonal antibody, Py: a light and electron microscopic study.

The monoclonal antibody Py has previously been shown to be a useful marker for subpopulations of neurons in the rat brain. However, the distribution of Py immunoreactivity in other regions of the CNS and PNS is not known. Here, we present a light and electron microscopic investigation into the distribution of Py immunoreactivity in the adult rat spinal cord, dorsal root ganglia, and peripheral nerves. Py immunoreactivity was associated with cytoskeletal elements in the cell body and dendrites of large-diameter neurons (particularly motoneurons, Clarke's nucleus neurons, and some dorsal root ganglion cells). Small-diameter neurons of lamina II (substantia gelatinosa) were Py negative. Py immunoreactivity was also detected in some populations of nerve fibers, notably axons located in the corticospinal tract, axons in the region of the white matter bordering the gray matter (presumably propriospinal axons), and also motor axons of the ventral root, but not in peripheral nerve. Dorsal roots were largely unstained. The present observations suggest a possible involvement of the Py antigen in the function or maintenance of the cytoskeleton of some populations of neurons and that the antibody may be a potentially useful tool for studying lesion-induced cytoskeletal alterations, particularly in alpha-motoneurons and Clarke's nucleus neurons.

Entorhinal axons perforate hippocampal field CA3 in organotypic slice culture.

In growing towards their hippocampal targets, incoming afferent axons from the entorhinal cortex arrive at the subicular pole of the hippocampus and normally turn pialwards from the alvear path, crossing (perforating) the subiculum and field CA1, but never the more distally situated field CA3. To address the question of whether a specific repulsive characteristic of field CA3 might explain this behaviour, artificial confrontation were set up in vitro. Embryonic entorhinal explants were placed in restricted contact with 8-day-old rat hippocampal slices, orientated so that outgrowing axons could only grow into either the dentate gyrus, the subiculum/field CA1, or field CA3. Anterograde biotin-dextran labelling of projections after 2 weeks in culture showed that entorhinal axons perforated the stratum oriens, pyramidal cell layer, and stratum radiatum of CA3 just as readily as they did along their normal trajectory across CA1/subiculum. It is therefore concluded that spatiotemporal cues are more likely than specific chemorepulsive molecules to be involved in setting up this part of the entorhinal pathway.

Regeneration of entorhino-dentate projections in organotypic slice cultures: mode of axonal regrowth and effects of growth factors.

Explants of Embryonic Day 18 (E18) rat entorhinal cortex were cocultured with Postnatal Day 6 mouse hippocampal slices to study CNS regeneration in vitro. The present report describes a double-labeling paradigm for quantitative analysis of the type of new growth seen in immature cultures. Entorhinal projection neurons in living static cocultures were retrogradely labeled with DiI or Texas red-dextran at 6 days in vitro and with dextran-FITC at 13 days. An intervening lesion to the entorhinodentate pathway was made at 8 days by replacing the hippocampal slices with fresh ones. About one-third of the new efferent entorhinal projections labeled with the second tracer could be characterized as true regeneration of axons from previously projecting entorhinal neurons by virtue of their being double labeled. The remaining two-thirds comprised new, late-arriving axons from previously nonprojecting cells. Earlier studies have shown that rat entorhinal axons will reinnervate hippocampal slices only if the lesions are made before 2-3 weeks in culture, equivalent to a postnatal age of 11-18 days. In a second series of experiments we tested whether treatment with trophic factors could overcome this age-related failure of regeneration characteristic of mature preparations. E18 explants were lesioned after 4 weeks in vitro and grown for a further 2 weeks in medium supplemented with either Schwann cell conditioned medium or acidic fibroblast growth factor plus heparin. A significant increase in outgrowth was seen in both cases, although the effects of each factor were not additive when they were applied in combination. These results show that our model of CNS lesions in vitro can be used to assess the effectiveness of growth factors in ameliorating the decline in regenerative ability with increasing developmental age.

Rapid decline in the ability of entorhinal axons to innervate the dentate gyrus with increasing time in organotypic co-culture.

We have used the species-specific monoclonal antibodies OM1 and OM4 to identify the histiotypic pattern of projection from late embryonic rat entorhinal explants to the outer molecular layer of the dentate gyrus in organotypic cultures of 6-day postnatal mouse hippocampal slices. The presence of this entorhinal projection was detectable with the rat-specific OM1 and OM4 markers after 3-7 days in co-culture, and confirmed by use of the later-forming rat neuron-specific marker THy-1.1, which appeared during the second week. Hippocampal slices confronted with control explants of superior colliculus for 4 weeks in culture showed only sparse, non-specific growth of axons with no histiotypic pattern in the dentate gyrus. In order to assess whether the formation of specific entorhino-dentate projections in vitro is age-dependent, embryonic rat entorhinal cortical explants were cultured alone for periods of 1-5 weeks before cutting across the halo of axons radiating into the collagen matrix and presenting each with 6-day-old mouse hippocampal slices as targets to innervate. After allowing a 2 week period for fibre growth to take place, the density of immunostained axonal outgrowth was scored on a five-point scale for each weekly interval. The amount of new axon growth when the cuts were made after 1 week was slightly reduced compared to undamaged control cultures. However, outgrowth was greatly diminished when the cuts were made after 2 or 3 weeks, and essentially abolished if the interval was extended to > or = 4 weeks. Thus we demonstrate that, although hippocampal slices can survive in organotypic co-culture with entorhinal explants and maintain previously formed connections, the explants show an age-related failure in the ability to form new connections. Such a system provides a possible in vitro model for study of the factors influencing the failure of regeneration in the adult central nervous system.

Monoclonal antibody G10 which recognises microtubule-associated protein 1x identifies growing axons of neurons microtransplanted into adult rat hippocampus.

Monoclonal antibody G10 recognises an epitope on microtubule-associated protein 1x, a developmentally regulated cytoskeletal protein expressed in immature axons of the central and peripheral nervous systems. Here we report that G10 can be used to identify the axonal projections of syngeneic embryonic (E14-E15) hippocampal cells microtransplanted by a minimally traumatic technique into intact adult host hippocampus.

Monoclonal antibodies reveal molecular differences between terminal fields in the rat dentate gyrus.

We have derived a number of monoclonal antibodies which detect molecular differences correlating with the afferent inputs to the molecular layer of the adult rat hippocampal dentate gyrus. One group, dubbed OM-1 to OM-4, strongly stain the outer zone of the molecular layer, which receives its major innervation from the ipsilateral entorhinal cortex. A second group, IM-1 and IM-2, show a complementary pattern and preferentially stain the inner molecular layer, which receives inputs from the ipsilateral and contralateral hippocampus. These antigens are not, however, restricted to these layers, being found outside the hippocampus in several other areas of neuropil in the adult brain. In the developing brain the IM-1 antigen appears ubiquitously from the earliest age studied, embryonic day 12. Within the dentate gyrus, its restriction to the inner terminal field of the molecular layer only occurs during the second postnatal week. In contrast, OM staining appears only sparsely and late in the prenatal brain, appearing in developing cortical white matter between embryonic days 18 and 20. The outer dentate molecular layer becomes OM-positive from birth onwards, corresponding to the time of arrival of entorhinal axons during the first postnatal week. These two groups of monoclonal antibodies recognize a number of different glycoproteins. Ultrastructural immunohistochemistry shows they are cell surface molecules, and as such may be involved in the recognition events required for the establishment of specific patterns of neuronal connectivity.

The OM series of terminal field-specific monoclonal antibodies demonstrate reinnervation of the adult rat dentate gyrus by embryonic entorhinal transplants.

Monoclonal antibodies OM-1 to OM-4 and IM-1 [Woodhams et al. (1991) Neuroscience 46, 57-69] have complementary immunostaining patterns in the molecular (dendritic) layer of the adult rat dentate gyrus, with OM-1 to OM-4 selectively recognizing the outer (distal) two-thirds (i.e. the entorhinal afferent zone), and IM-1 the inner (proximal) one-third (i.e. the hippocampal commissural/associational zone). Immunoblotting suggests that OM-1 recognizes a single glycoprotein antigen of mol. wt around 93,000, and OM-2, OM-3, and OM-4 all recognize a second glycoprotein antigen of mol. wt around 36,000. At four weeks after removal of the ipsilateral entorhinal cortex the background OM immunostaining of the entorhinal afferent zone is abolished and replaced by a network of densely stained granules, which we interpret as degenerating entorhinal afferent axons. At the same time, the proximal, IM immunoreactive zone expands by about 10 microns in width (while the distal deafferented zone shrinks by about 80 microns). Attempts were made to restore the OM immunoreactivity of the distal zone by grafting either small pieces or cell suspensions of embryonic day 18 entorhinal cortex directly into the dentate molecular layer of entorhinally deafferented adult hosts. About half (14/26) of the animals with successfully positioned grafts showed restoration of OM-2 to OM-4 immunostaining throughout the entire width of the outer two-thirds (entorhinal afferent zone) of the dentate molecular layer. Strikingly, however, in adjacent serial sections the restoration of OM-1 immunoreactivity was restricted to the "middle" molecular layer, i.e. the most proximal part of the distal (entorhinal) two-thirds of the dentate molecular layer. In no case did the OM-1 immunoreactivity extend to the outer margin of the molecular layer. This did not appear to be associated with incompleteness of the removal of the host entorhinal projection, since it occurred in grafted cases where the hippocampus had been completely isolated from the entorhinal area. The simplest explanation of the observed pattern of OM loss and restitution is that the epitopes are located on the entorhinodentate axons, but it is not clear whether the antigens recognized by OM-1 and OM-2 to OM-4 are expressed in different parts of the same group of axons, or in different subsets of entorhinodentate axons. Nor is it clear why the pattern of OM-1 is only restored to the "middle" molecular layer, while that of OM-2 to OM-4 is restored to the entire outer two-thirds.(ABSTRACT TRUNCATED AT 400 WORDS)

A monoclonal antibody, Py, distinguishes different classes of hippocampal neurons.

A monoclonal antibody, Py, was produced by immunizing mice with a glycoprotein fraction isolated from 3-week-old rat hippocampus. Py antibodies gave strong immunocytochemical staining of the perikarya and dendrites of large neurons in many areas of the rat brain, including the cerebral cortex, hippocampus, cerebellum, brain stem, and olfactory bulb. Immunoelectron microscopy showed the antigen to be predominantly intracellular, although its presence on the neuronal cell surface was not excluded. The antibody gave differential staining of adult hippocampal neurons, large pyramids of field CA3 being strongly immunoreactive, while CA1 pyramids and the dentate granule cells were unstained. Some interneurons were positive in each of the hippocampal fields. In developing hippocampus, the Py antigen appeared by the middle of the first postnatal week, and the adult pattern of staining was achieved by the end of the second week. Immunoblotting showed the antigen to have a relative mobility of 146 kDa with an additional faint band at 166 kDa. Differential Py staining of neurons was seen in dissociated cultures of embryonic hippocampus and in subdissected hippocampal fragments transplanted into adult host brains. This antibody can therefore be used for identification of hippocampal neurons that have been removed from their normal anatomical context.

A developmentally regulated axonal glycoprotein (7-8D2 antigen) with a restricted distribution in mature rat brain.

7-8D2 is a mouse monoclonal IgGl antibody which recognizes a neuronal cell surface antigen in rat brain. Immunohistochemical techniques reveal the antigen to be present most abundantly in the cerebellum of the adult brain, where it is expressed by the cell bodies and fibres of the granule neurons. Lower levels of staining were found in the neuropil of the hippocampus, in some fibres of the corpus callosum and in the most superficial layer of the cerebral cortex. Immunoelectron microscopy confirmed that the antigen was present on the surface of the parallel fibres in the cerebellum and showed that it was absent from glial, Purkinje or stellate cell membranes. The antigen had a more widespread distribution early in development, and the restricted adult distribution was achieved by the end of the second postnatal week. Immunoblotting of samples of adult rat brain shows that the antigen appears as a close doublet of bands at 211,000 mol. wt. This result was confirmed by immunoprecipitating the antigen from metabolically labelled glycoproteins prepared from cultured cerebellar interneurons. Immunoblotting and immunohistochemical experiments were in agreement that the cerebellum contained high levels of the antigen, and that lower but significant amounts could be found in other brain regions, notably the hippocampus and the cerebral cortex. The localization data and the changes in the distribution of the antigen may suggest some role for this molecule during early brain development.