Biochemical studies of trophic dependencies in crayfish giant axons

Medial giant (MGA) and lateral giant (LGA) axons of crayfish were doubly cut in order to selectively isolate axonal segments from perikaryal and transsynaptic sources of trophic input. Isolated MGA segments remained morphologically intact for over 43 days, whereas isolated LGA segments usually degenerated within one week. The glial sheaths around isolated MGA segments had significantly increased in thickness within one week, but severed LGA segments showed no increase in sheath thickness at any time after lesioning. These data suggest that cells of the surrounding glial sheath can provide trophic support to isolated MGA segments but not to isolated LGA segments. Extent of glial hypertrophy seems dependent upon specific spatiotemporal parameters.The diameters of isolated MGA segments decreased more rapidly than the diameters of singly cut MGA segments. These data suggest that the MGA also receives some trophic support from pre- or postsynaptic sources. Conversely, some singly cut LGA segments completely degenerated within one week, whereas other singly cut LGA segments remained intact for at least 43 days after lesioning. Such results suggest that the LGA receives a significant trophic input from pre- or postsynaptic structures.     

Histological studies of trophic dependencies in crayfish giant axons

Medial giant (MGA) and lateral giant (LGA) axons of crayfish were doubly cut in order to selectively isolate axonal segments from perikaryal and transsynaptic sources of trophic input. Isolated MGA segments remained morphologically intact for over 43 days, whereas isolated LGA segments usually degenerated within one week. The glial sheaths around isolated MGA segments had significantly increased in thickness within one week, but severed LGA segments showed no increase in sheath thickness at any time after lesioning. These data suggest that cells of the surrounding glial sheath can provide trophic support to isolated MGA segments but not to isolated LGA segments. Extent of glial hypertrophy seems dependent upon specific spatiotemporal parameters.The diameters of isolated MGA segments decreased more rapidly than the diameters of singly cut MGA segments. These data suggest that the MGA also receives some trophic support from pre- or postsynaptic sources. Conversely, some singly cut LGA segments completely degenerated within one week, whereas other singly cut LGA segments remained intact for at least 43 days after lesioning. Such results suggest that the LGA receives a significant trophic input from pre- or postsynaptic structures.     

Heat-shock proteins in axoplasm: High constitutive levels and transfer of inducible isoforms from glia

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1–3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock. J. Comp. Neurol. 396:1–11, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural basis of impulse propagation failure in a nonbranching axon

The morphological basis of intermittent conduction failure in the excitor axon innervating the crayfish opener and stretcher muscles was investigated using the electron microscope. The connective tissue component of the sheath surrounding the axon was found consistently to be thinner in the region at which blocking occurs than in control regions located one cm either proximally or distally, at which blocking does not occur. Otherwise, in these regions differences in the width of the periaxonal spaces, the length or width of the mesaxons, the density of mitochondria, the width of the adaxonal glial cell layer, or the structure of the lamination of the sheath are not observed. Because of the thinner connective tissue component of the sheath in the joint region, neighboring axons are distributed more densely around the excitor, and the volume of the extracellular space is reduced. The possibility that the reduced extracellular space might allow excessive accumulation of potassium during repetitive discharge, causing conduction block, is discussed. Alternative mechanisms consistent with this morphology are also considered.     

Glutamine cycle enzymes in the crayfish giant nerve fiber: Implications for axon-to-glia signaling

Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis. © 1995 Wiley-Liss, Inc.     

Biochemical studies of trophic dependences in crayfish giant axons.

Data from previous histological studies indicate that long-term survival of crayfish medial giant axons might be due in part to trophic support from cells of the surrounding glial sheath which often hypertrophy in response to transection of the medial giants. The biochemical studies reported herein show that segments from transected ventral nerve cords (VNC) always incorporate more [3H]leucine into protein than do corresponding segments from intact VNCs. Furthermore, the relative amount of [3H]leucine incorporation in severed segments seems to be influenced by distance and direction from the lesion site as well as time after lesioning. Similar spatiotemporal parameters were previously shown to be correlated with extent of glial hypertrophy around severed medial giant axons. Quantitative autoradiography of medial giant axons after incubation in [3H]leucine revealed that the grain density of label in glial sheaths surrounding severed medial giants was over two-fold greater than in sheaths around corresponding control axons. Moreover, the grain density in the axoplasm of severed medial giants was nearly four-fold greater than the grain density in the axoplasm of control axons. Data from experiments using short or long labeling intervals suggests that labeling in the medial giant axoplasm may be due more to transfer from glial sheath cells than from inherent axonal synthetic mechanisms. In light of this and other data, we concluded that long-term survival of severed medial giant axons is probably due to the direct transfer of trophic substances from cells of the glial sheath into the axon.     

Preferential transport and metabolism of glucose in Bergmann glia over Purkinje cells: A multiphoton study of cerebellar slices

Knowing how different cell types handle glucose should help to decipher how energy supply is adjusted to energy demand in the brain. Previously, the uptake of glucose by cultured brain cells was studied in real‐time using fluorescent tracers and confocal microscopy. Here, we have adapted this technique to acute slices prepared from the rat cerebellum by means of multiphoton microscopy. The transport of the fluorescent glucose analogs 2NBDG and 6NBDG was several‐fold faster in the molecular layer of the cerebellar cortex than in Purkinje cell somata and granule cells. After washout of free tracer, it became apparent that most phosphorylated tracer was located in Bergmann glia, which was confirmed by counterstaining with the glial marker sulforhodamine 101. The effective recovery of fluorescence after photobleaching showed that 2NBDG‐P can diffuse horizontally across the molecular layer, presumably through gap junctions between Bergmann glial cells. Our main conclusion is that in acute cerebellar slices, the glucose transport capacity and glycolytic rate of Bergmann glia are several‐fold higher than those of Purkinje cells. Given that the cerebellum is largely fueled by glucose and Purkinje neurons are estimated to spend more energy than Bergmann glial cells, these results suggest substantial shuttling of an energy‐rich metabolite like lactate between glial cells and neurons. © 2008 Wiley‐Liss, Inc.     

Cytophotometric quantification of retrograde axonal transport of a fluorescent tracer (primuline) in mouse facial neurons

A method for cytophotometric quantifications of retrograde axonal transport of a fluorescent tracer in tissue sections is described. As a fluorescent tracer the anionic vital stain primuline proved to be suitable since it resulted in a strong yellow-green fluorescence, which faded very slowly permitting localization of cells during illumination with UV-light. Primuline injected into the muscles of the vibrissae in mice was transported to the corresponding nerve cell bodies in the facial nucleus, where it appeared as fluorescent granules 9 h after the injection. The fluorescence intensity increased with increasing exposure times and concentrations of the injected tracers. The motor endplates showed no ultrastructural changes after the tracer injections.With this method a substantial increase in tracer accumulation in facial neurons could be revealed during nerve regeneration 11 days after crushing the facial nerve. Small alterations in neuronal tracer accumulation could be measured after intoxication of the mice with botulinum and tetanus toxins. Since these toxins should cause a decrease or increase in the degree of synaptic activity the amount of retrograde axonal transport may to a certain extent be dependent on the synaptic function. The findings with this new technique therefore indicate that quantitative changes occur in axonal transport in materials from the periphery during different pathological and physiological conditions, which may be important for an understanding of how a nerve cell body is dependent on its peripheral field of innervation.     

Axonal transport and transcellular transfer of nucleosides and polyamines in intact and regenerating optic nerves of goldfish: speculation on the axonal regulation of periaxonal cell metabolism

The axonal transport, metabolism, and transcellular transfer of uridine, adenosine, putrescine, and spermidine have been examined in intact and regenerating optic nerves of goldfish. Following intraocular injection of labeled nucleosides, axonal transport was determined by comparing left-right differences in tectal radioactivity, and transcellular transfer was indicated by light autoradiographic analysis. The results demonstrated axonal transport, transcellular transfer, and periaxonal cell utilization of both nucleosides in intact axons and severalfold increases of all of these processes in regenerating axons. Experiments in which the metabolism of the nucleosides was studied resulted in data which suggested that uridine and adenosine, when delivered to the tectum by axonal transport, are protected from degradation and thus are relatively more available for periaxonal cell utilization than nucleosides reaching these cells via the blood. In intact axons, the majority of the nonmetabolized radioactivity was present as UMP, UDP, and UTP following [3H]uridine injections, whereas the majority of the radioactivity following [3H]adenosine injections was present as adenosine, with the phosphorylated derivatives constituting a smaller proportion. During nerve regeneration, the relative proportion of nucleosides to nucleotides was reversed, with uridine being the principal labeled compound in the first case, and AMP, ADP, and ATP being the major labeled compounds in the latter case. The nucleosides also were found to be different from each other in that adenosine, but not uridine, can be taken up by optic axons and transported retrogradely from the tectum to retinal ganglion cell bodies in the eye. Following intraocular injection of [3H]spermidine, radioactivity was transported to the optic tectum and transferred to tectal cells in the vicinity of the regenerating axons. Following [3H]putrescine injections, silver grains were found over periaxonal glia, but preliminary findings suggest that they are not present over tectal neurons nor over radial glial cells in the periependymal layers. Analysis of tectal radioactivity showed in each case that it was composed primarily of the injected compounds. These studies indicate that, following axonal transport, the polyamines do not remain within regenerating axons but are transferred to cells surrounding the axon. On the basis of these and previous findings, we speculate that the axonal transport and transcellular transfer of uridine, adenosine, polyamines, and perhaps other small molecules are means of communication between axons and periaxonal cells; that the axon can affect RNA and protein synthesis in periaxonal cells by regulating the availability of these small molecules; and that, during nerve regeneration, the increased metabolic needs of periaxonal cells are met by an increased axonal supply of precursors (adenosine and uridine) and other molecules (polyamines) critical for protein synthesis.     

A sequential double labeling technique for studying changes in motoneuronal projections to muscle following nerve injury and reinnervation

The purpose of this study was to develop an anatomical technique that could directly demonstrate the motoneuron projections to the muscle both before injury and again following reinnervation. Investigation focused on the identification of a long-term retrograde fluorescent tracer that would label original motoneurons and persist long enough for reinnervating motoneurons to become labeled by a second fluorescent tracer. True Blue (TB) was evaluated as a potential long-term tracer, Fluoro-ruby (FR) and Fluoro-emerald (FE) were tested as potential short-term tracers in 45 adult Sprague-Dawley rats. In the initial phase of the study, TB was injected into the tibialis anterior (TA) muscle in 16 rats and sacrificed 1 week to 6 months later, to study its persistence. During the second stage, a short-term tracer was injected into the TA muscles bilaterally in 15 rats with survival time ranging from 4 to 28 days. Sequential double labeling was subsequently performed using the combination of TB and FR in 14 rats. The number and brightness of TB cells did not change over 6 months time, a period sufficient for complete reinnervation. FR and FE showed maximum labeling of motoneurons at 1 week after tracer application. In the double labeling study, we could easily distinguish double-labeled cells from those labeled only by TB or FR. These results suggest that sequential double labeling of TB and FR is a valuable method for long-term muscle reinnervation studies.     

The penetration of fluorescein-conjugated and electrondense tracer proteins into Xenopus tadpole optic nerves following perineural injection.

The permeability of Xenopus tadpole optic nerves to macromolecules was studied in order to evaluate the usefulness of this system for studying mechanisms of serum-induced CNS demyelination in vivo. Single injections of either horseradish peroxidase (HRP), ferritin or fluorescein-conjugated human IgG were injected around the right optic nerve and tadpoles were then sacrificed between 15 min and 48 h. Each of the tracers had penetrated the nerve parenchyma by 30 min. Entry of HRP and ferritin occurred mainly via extracellular clefts between adjacent astrocytic endfeet in the glia limitans region. A similar mode of passage was suggested for IgG. Once within the nerve, the tracers became rapidly associated with myelinated axons. HRP was also seen in the periaxonal space but did not directly penetrate the myelin sheath. By 24 h, extracellular localization of tracer was virtually absent with nearly all of the tracer now being concentrated in vesicles within astrocytic processes and perikarya. The distribution of the tracers was not confined to the optic nerve on the injected side; some was seen in adjacent cranial peripheral nerves and surrounding extraocular musculature. Also, tracers eventually penetrated the pial sheath of the contralateral optic nerve. The results of this study indicate that tadpole optic nerves are permeable to a wide range of macromolecules. Furthermore, the distribution of these tracers to nearby cranial peripheral nerves may provide an important opportunity for testing the differential effect of various substances on central and peripheral myelin sheaths.     

Dye coupling and gap junctions between crustacean neurosecretory cells

Neurosecretory cells in the X organ-sinus gland system of the crayfish were impaled and Lucifer Yellow was intracellularly iontophoresed. In some neurons the injected dye was transferred to neighboring neurons. The interneuronal dye transfer was between adjacent somata. Coupling was also observed between neurons and smaller cells, possibly glia. Gap junctions were identified by freeze-fracture in neuron somata and glial cells in the X organ and also in neurosecretory axons in the sinus gland.     

A glial blood-brain barrier in elasmobranchs

The blood-brain interface in elasmobranchs was examined using physiological and anatomical techniques. Electrolyte analyses of plasma and cerebrospinal fluid (CSF) in control fish and in fish with elevated plasma cation concentration (Ca, Mg or K) demonstrated homeostasis of CSF calcium and magnesium concentrations in dogfish and skates and of CSF potassium concentration in dogfish. In contrast, CSF potassium concentration was not regulated independent of plasma potassium concentration in skates. Ultrastructural examination of the blood-brain barrier in skates showed that brain capillaries are fenestrated and intravenously injected horseradish peroxidase penetrates readily across this endothelium into the basement membrane. Further penetration of tracer into the neuropil is prevented by tight junctions between perivascular glial cells. Results indicate that two hallmarks of the blood-brain barrier —impermeability and homeostatic transport systems — can be localized to the perivascular glial sheath in elasmobranchs. The functional and structural similarity of this glial membrane to a transporting epithelium is discussed.     

Quadruple labeling of brain-stem neurons: a multiple retrograde fluorescent tracer study of axonal collateralization.

Four different fluorochromes were injected into adjacent cervical spinal cord segments, 1 unique tracer per segment. Each tracer, Fluoro-Gold, Fast Blue, Diamidino Yellow dihydrochloride and Propidium Iodide, was taken up by axonal terminals and transported intra-axonally in a retrograde direction to the cell bodies. Some, though by no means all, of these axons were stem axons with terminals in 2, 3 or 4 of the injected spinal segments. Hence as many as 4 different fluorescent tracers could be discerned simultaneously within individual neuronal somata of origin using fluorescent microscopy. These results extend the possibilities for multiple interconnection determinations within the central nervous system. Specifically, the potential for individual neurons of a nucleus to project collateral branches of a stem axon to as many as 4 different central nervous system nuclei now can be studied simultaneously using these 4 fluorescent tracers.     

Maintenance and synthesis of proteins for an anucleate axon

The anucleate (distal) segment of a crayfish medial giant axon (MGA) remains intact for months in vivo after severing the axon from its cell body, a phenomenon referred to as long-term survival (LTS). We collected axoplasm from chronic anucleate MGAs by perfusing 2-cm lengths of axons with an intracellular saline. This axoperfusate was analyzed by SDS-PAGE and silver stained. Axoperfusate proteins from intact MGAs and from chronic anucleate MGAs exhibiting LTS for up to 6 months were the same. Furthermore, immunoreactive levels of actin and β-tubulin were similar in axoperfusates from intact and chronic anucleate MGAs. This maintenance of proteins in chronic anucleate MGAs must be due to a lack of protein degradation and/or to local protein synthesis by a source other than the cell body. To investigate local protein synthesis in vitro, we added [³⁵S]-methionine to the extracellular saline surrounding intact and chronic anucleate MGAs. After 4- to 6-h incubations, radiolabelled proteins were detected in axoperfusates analyzed by SDS-PAGE and fluorography. The similarity between radiolabelled proteins in axoperfusates and MGA glial sheaths indicated a glial origin for the radiolabelled axoperfusate proteins. Various observations and control experiments suggested that glial-axonal protein transfer occurred by a physiological process. Glial-axonal protein transfer may contribute to the maintenance of proteins during LTS of chronic anucleate MGAs.     

Distribution of exudated FITC-dextrans in experimental vasogenic brain edema produced by a focal cryogenic injury

Summary Mice were subjected to cortical cryogenic brain injury, and FITC-dextrans (mol. wt. 20,000 or 150,000) were injected intravenously (i.v.). After a survival period of 4 h the distribution of the FITC-dextrans was determined by a histotechnical procedure described recently (Hultström et al. 1982a). This technique is based on freeze-drying and vapor fixation to immobilize the tracer and to provide tissue fixation.In and around the cryogenic injury both tracers leaked out of the cortical and the leptomeningeal vessels and spread into the brain parenchyma. They were seen as multiple, closely apposed droplets of fluorescent material best recognized by fluorescence microscopy under high magnification. The tracers were also taken up by neuronal perikarya and in glial cell nuclei of, presumably, astrocytic origin.Our study shows that the FITC-dextran technique can be used for experimental studies on the vasogenic form of brain edema. The patterns formed by the extravasated tracers have qualitative similarities to those produced by other more commonly used tracers, such as fluorochrome-labeled serum proteins and peroxidase.Supported by grants from the Swedish Medical Research Council, project no. 12X-03030, Trygg Hansa, Svenska läkarsällskapet, and Söderbergs stiftelser     

Genesis of resting microglia in the gray matter of mouse hippocampus

The genesis of resting microglia in the gray matter of mouse hippocampus was studied by ³H-thymidine autoradiography in combination with electron microscopy. Newborn mice were injected with ³H-thymidine singly or repeatedly at different postnatal stages, and killed shortly after the injection or after various intervals. Tissue specimens of the hippocampus at CA1 and CA2 were processed for light and electron microscopic autoradiography. The results showed that at least 91% of glial cells in the stratum radiatum of the hippocampus are produced after birth. About three-fourths of astroglia in this area are produced before the sixth postnatal day, and a larger part of resting microglia are formed after the ninth postnatal day. Morphological transition can be traced from either proliferating cells in the stratum radiatum at late postnatal days to resting microglia or from those in early postnatal days to astroglia. A continuous morphological transition was observed between the proliferating cells at the late postnatal days (microglial production period) and those at the early postnatal days (astroglial production period). The latter retain some fine structural characteristics similar to small glioblasts in the subependymal layer. These findings strongly suggest that resting microglia, as well as astroglia, are derived from glioblasts, and are of neurectodermal origin.     

Trans-glial channel-facilitated translocation of tracer protein across ventral nerve root sheaths of crayfish.

Trans-glial channels, which traverse the multilamellate glial sheath of crayfish nerves, are easily recognized in freeze-fracture preparations. Their structure and position in the glial layers of the sheath strongly supports the suggestion that they serve to facilitate rapid movement of molecules and fluids from outside the sheath to the surface of axons contained within. Segments of ventral ganglion nerve roots, which were ligated at their free ends, were immersed in crayfish Ringer solution containing 10 mg/ml horseradish peroxidase (HRP). Electron microscopic examination of the nerve sheath 30 sec after exposure to peroxidase showed that the protein had passed across the sheath and was present near the axon surface. Reaction product was present in trans-glial channels as well as in extracellular clefts and adaxonal tubular lattices thereby supporting the notion that these structures constitute a specialized conduit traversing the sheath. Often, 'fronts' of reaction product were observed across the sheath from its exterior to the interior reflecting a gradual accumulation of protein in extracellular clefts toward the axon. After 5 min in HRP-Ringer, protein appeared in all channels, extracellular clefts, and tubular lattices. With increased length of exposure of ligated nerve segments to HRP-Ringer, reaction product was found in vesicles in glial cytoplasm adjacent to axons. Results from this study suggest that trans-glial channels constitute an efficient system for rapid solute movement across nerve sheaths and may represent a mechanism whereby ions and nutrients are made available to nerves isolated in an avascular sheath.     

Intracellular ionic concentration by calibration from fluorescence indicator emission spectra,its relationship to the K(d), F(min), F(max) formula,and use with Na-Green for presynaptic sodium

The emission spectra calibration curves for a fluorescence indicator and the F(min), F(max), and K(d) formula were shown to be related. Using the known calibrated fluorescence emitted by Sodium Green (Na-Green) and photo-multiplier-tube quantum efficiency, we calculated the detection signal over a range of sodium concentrations. The calculated calibration curves were compared for optical filters passing a narrow band, medium band or full spectrum. We found that a method based on the full emission spectrum was the most appropriate. Given a known resting concentration of intracellular sodium, calibrated readings can be converted to concentration values. This method is applicable to any fluorescence indicator when curves for emission spectra over a range of concentrations are available. We measured sodium concentration changes during trains of action potentials (APs) at a crayfish motor axon's presynaptic terminals injected with Na-Green. During low frequency AP trains, net sodium increases asymptotically with frequency. Average net Na-flux per AP decreases for increasing terminal size. The terminals of crayfish motor axon have surface area to volume ratio which is 7700 times larger than for squid. Thus, in comparison to squid, crayfish terminals exhibit a larger change in [Na(+)](i) during equivalent AP activity.     

Extravasation,spread and cellular uptake of Evans blue-labelled albumin around a reproducible small stab-wound in the rat brain

Summary A small stab wound was made in the frontal lobe of the rat brain in order to study both the acute damage and the restitution of the blood-brain barrier to macromolecules under well-defined experimental conditions. Intravenously administered Evans blue-labelled albumin (EBA) was used as a tracer and the brain sections were observed in a fluorescence microscope.EBA leaked into the neuropil only during the first 3 days after the trauma. The maximal leakage occurred during the first day after the injury. The tracer spread from the area surrounding the stab wound in a reproducible way, initially roughly centrifugally. Nerve and glial cells close to the wound displayed a diffuse fluorescence of their cell bodies 1 to 6 h after the injury, i.e. at times with maximal extravasation of the tracer. A more granular distribution of the tracer was seen in neurons and glial cells at both very short and long times after EBA injection.Thus, the blood-brain barrier lesion induced was reproducible and reversible as judged by the pattern of EBA leakage. Some cells immediately adjacent to the injury had a diffuse cytoplasmic distribution of the tracer complex, in contrast to cells more distant from the injury, having a more granular distribution of the tracer in their cytoplasm. However, the appearance of the fluorescence in neurons and glia was to a large extent dependent upon the time after the injury, at which the cells were exposed to the tracer complex and on the time that had elapsed from the time of injection of the tracer complex to the sacrifice of the animal. Thus, it seems likely that two factors contribute to the appearance of the neuronal EBA distribution: on one hand the location and possibly extent of cellular damage of the cell, and on the other hand, the time and amount of EBA to which the cells were exposed.