Reelin function in neural stem cell biology.

In the adult brain, neural stem cells (NSC) must migrate to express their neuroplastic potential. The addition of recombinant reelin to human NSC (HNSC) cultures facilitates neuronal retraction in the neurospheroid. Because we detected reelin, alpha3-integrin receptor subunits, and disabled-1 immunoreactivity in HNSC cultures, it is possible that integrin-mediated reelin signal transduction is operative in these cultures. To investigate whether reelin is important in the regulation of NSC migration, we injected HNSCs into the lateral ventricle of null reeler and wild-type mice. Four weeks after transplantation, we detected symmetrical migration and extensive neuronal and glial differentiation of transplanted HNSCs in wild-type, but not in reeler mice. In reeler mice, most of the injected HNSCs failed to migrate or to display the typical differentiation pattern. However, a subpopulation of transplanted HNSCs expressing reelin did show a pattern of chain migration in the reeler mouse cortex. We also analyzed the endogenous NSC population in the reeler mouse using bromodeoxyuridine injections. In reeler mice, the endogenous NSC population in the hippocampus and olfactory bulb was significantly reduced compared with wild-type mice; in contrast, endogenous NSCs expressed in the subventricular zonewere preserved. Hence, it seems likely that the lack of endogenous reelin may have disrupted the migration of the NSCs that had proliferated in the SVZ. We suggest that a possible inhibition of NSC migration in psychiatric patients with a reelin deficit may be a potential problem in successful NSC transplantation in these patients.

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy.

Reelin is a glycoprotein ( approximately 400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.

Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex.

The expression of telencephalic reelin (Reln) and glutamic acid decarboxylase mRNAs and their respective cognate proteins is down-regulated in postmortem brains of schizophrenia and bipolar disorder patients. To interpret the pathophysiological significance of this finding, immunoelectron microscopic experiments are required, but these cannot be carried out in postmortem human brains. As an alternative, we carried out such experiments in the cortex of rats and nonhuman primates. We found that Reln is expressed predominantly in layer I of both cortices and is localized to bitufted (double-bouquet), horizontal, and multipolar gamma-aminobutyric acid-ergic interneurons, which secrete Reln into extracellular matrix. Reln secretion is mediated by a constitutive mechanism that depends on the expression of a specific signal peptide present in the Reln carboxy-terminal domain. Extracellular matrix Reln is found to aggregate in proximity of postsynaptic densities expressed in apical dendrite spines, which include also the alpha(3) subunit of integrin receptors. Most pyramidal neurons of various cortical layers express the mouse-disabled 1 (Dab1) protein, which, after phosphorylation by a soluble tyrosine kinase, functions as an adapter protein, probably mediating a modulation of cytoskeleton protein expression. We hypothesize that the decrease of neuropil and dendritic spine density reported to exist in the neocortex of psychiatric patients may be related to a down-regulation of Reln-integrin interactions and the consequent decrease of cytoskeleton protein turnover.

Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells.

Reelin regulates telencephalic and cerebellar lamination during mammalian development and is expressed in several structures of the adult brain; however, only traces of reelin were believed to be in peripheral tissues. Because reelin structurally resembles extracellular matrix proteins, and because many of these proteins are expressed in blood, we hypothesized that reelin also might be detectable in the circulation. Reelin (420 kDa) and two reelin-like immunoreactive bands (310 and 160 kDa) are expressed in serum and platelet-poor plasma of rats, mice, and humans, but these three bands were not detectable in serum of homozygous reeler (rl/rl) mice. Reelin plasma levels in heterozygous (rl/+) mice were half of those in wild-type littermates. Western blotting and immunocytochemistry using antireelin mAbs indicated that reelin-like immunoreactivity was expressed in a subset of chromaffin cells within the rat adrenal medulla and in a subset of cells coexpressing alpha-melanocyte-stimulating hormone within the pituitary pars intermedia. However, surgical removal of adrenal or pituitary failed to decrease the amount of reelin (420-kDa band) expressed in serum. Adult liver expressed one-third of the reelin mRNA concentration expressed in adult mouse cerebral cortex. Full-length reelin protein was detectable in liver extracts in situ; acutely isolated liver cells also secreted full-length reelin in vitro. Liver appears to be a prime candidate to produce and maintain the circulating reelin pool. It now becomes relevant to ask whether circulating reelin has a physiologic role on one or more peripheral target tissues.

Keratin expression in astrocytomas: an immunofluorescent and biochemical reassessment.

Several studies have shown that immunoenzymatic staining of formalin-fixed, paraffin-embedded astrocytomas with keratin antibodies frequently yields positive labelling, but no biochemical evidence of keratin expression in astrocytomas has been reported. We have investigated the presence of keratin in astrocytoma and normal brain tissues both by immunofluorescence on frozen sections and by 1D and 2D immunoblotting using seven monoclonal antibodies that, collectively, recognize most keratin polypeptides. Four of these antibodies did not stain neural tissues by immunofluorescence and were also negative by immunoblotting. The remaining three keratin antibodies stained normal brain and/or a high proportion of astrocytomas. Two of these three antibodies only stained glial fibrillary acidic protein (GFAP)-positive cells, while the third only stained GFAP-negative cells. 1D and 2D immunoblotting analysis showed that positive immunofluorescence staining of normal brain and/or astrocytomas seen with these three keratin antibodies was due to cross-reactivity with non-keratin proteins, such as GFAP. These results demonstrate that, contrary to earlier suggestions, keratin polypeptides are not frequently expressed in astrocytomas. Our studies also emphasize that keratin antibodies should be used cautiously for the differential diagnosis of undifferentiated gliomas from tumours of non-glial origin.

A subpopulation of reactive astrocytes at the immediate site of cerebral cortical injury.

We have identified an early-appearing intermediate filament-associated protein (IFAP-70/280 kDa) in radial glia and their immediate derivatives. This IFAP is absent in the adult CNS. In this study, we examined the reexpression of this early glial differentiation trait in rat reactive astrocytes induced by stab injury of the cerebrum. Double-label immunofluorescence microscopy demonstrated that by 36 h postlesion, IFAP-70/280 kDa was present in a few GFAP-positive astrocytes in the area adjacent to the wound. As the gliotic reaction progressed, the number of IFAP-positive reactive astrocytes increased and by 5-6 days postlesion, IFAP-70/280 kDa was present in most of the hypertrophied astrocytes in tissue immediately adjacent to the wound. By 8 days postlesion, while the number of IFAP-negative reactive astrocytes away from the wound diminished, the IFAP-containing reactive astrocytes close to the wound persisted. Concurrently, they began to change from a stellate form to an elongated shape, with their longitudinal axes radiating from the wound. The immunoreactivity of this IFAP started to diminish at 20 days postlesion, and by 30 days postlesion, it was not observed in the remaining gliotic cells. These results demonstrate that reactive astrocytes induced by stab-wound injury can be divided into two subtypes: persistent IFAP-70/280 kDa-containing cells which are close to the wound in the area of the glial scar and transient IFAP-70/280 kDa-negative cells which are farther from the wound. The reappearance of IFAP-70/280 kDa also suggests that some reactive astrocytes have the capacity to recapitulate early developmental stages.

Presence of a 300-kDa intermediate-filament-associated protein (IFAP-300kDa) in bovine chromaffin cells.

Intermediate filaments (IFs) are cell-type-specific filaments that constitute a major part of the cellular cytoskeleton. Neurofilaments (NFs) are representative of a class of IFs which are excellent markers for neurons. NFs are also present in some cells of neural crest origin. A number of proteins have now been identified as being associated with IFs. Previously, a 300-kDa intermediate-filament-associated protein (IFAP-300kDa) was identified in baby hamster kidney cells (BHK-21). This IFAP is developmentally regulated and is not found in the adult CNS. To learn more about the expression of IFAP-300kDa, this study investigated the expression of IFAP-300kDa in neural-crest-derived chromaffin cells, both in situ and in vitro. Immunofluorescence localization of IFAP-300kDa in cryosections of bovine adrenal gland demonstrated IFAP-300kDa immunoreactivity in the dopamine-beta-hydroxylase-positive chromaffin cells of the adrenal medulla. When rounded chromaffin cells in culture were examined, double-label immunofluorescence microscopy revealed an IFAP-300kDa/NF-L-positive juxtanuclear aggregate. The plasma membrane was also IFAP-300kDa positive, but NF-L immunoreactivity was lacking. In cells which have spread under the influence of NGF, slender IFAP-300kDa-positive immunofluorescent strands were frequently seen radiating from a juxtanuclear area of immunoreactivity. Double-labeling revealed these filaments and juxtanuclear area to also be positive for NF-L immunoreactivity. The presence of IFAP-300kDa in situ and in vitro was further confirmed by immunoblot analysis. This study is the first demonstration of an IFAP in neuron-like cells located outside the central nervous system. In addition, these data indicate that the developmentally regulated IFAP-300kDa may persist in some cells of neural crest origin.

Immunomicroscopy of neurofilaments in chromaffin cells of the adult bovine adrenal gland.

Neurofilaments (NFs) represent a class of intermediate filaments which are highly specific for neurons. The most abundant of the native NFs is the 68 kD subunit (NF-L). Chromaffin cells of the adrenal medulla express NF subunits under culture conditions. However, NF expression in situ is questionable. It has been reported that as chromaffin cell precursors mature and begin to express catecholamine-synthesizing enzymes, their neuronal traits are extinguished and they become endocrine-like cells. This study reports that while NF-L may be lacking in the adrenal medulla of some species, NF-L immunoreactivity is clearly present in the adult bovine adrenal medulla. Immunofluorescence microscopy of bovine chromaffin cells in culture demonstrated NF immunoreactivity localized to both thin, highly ramified filaments present throughout the cell and frequently to an intensely immunofluorescent spot located near the nucleus. Double-label immunofluorescence microscopy and immunoblot analysis also demonstrated NF-L immunoreactivity in mature chromaffin cells of the bovine adrenal gland. In vitro and in situ immunofluorescence results were confirmed by immunogold and immunoperoxidase labelling, respectively. In both cases, NF-L immunoreactivity was associated with filaments in close proximity to the nucleus. Additionally, a spheroidal aggregate of immunogold-labelled NFs was seen adjacent to the nucleus in cultured cells. In conclusion, NF-L in bovine chromaffin cells demonstrates that at least one neuronal trait persists in these catecholamine-producing cells of the mature adrenal gland. In addition, this study emphasizes the fact that interspecies comparisons must be interpreted with caution, especially when attempting to formulate a generalized hypothesis.

The fine structure of large dense-core organelles in human locus coeruleus neurons.

Protein bodies, the characteristic spherical organelles present in human monoamine neurons, have been shown in previous electron microscope studies to originate as dense bodies in mitochondria. This study was designed to investigate the presence of catecholamine reaction products in the dense bodies of locus coeruleus neurons, in frozen fresh post-mortem brain tissue with the use of potassium permanganate (KMnO4) fixation. This fixation procedure forms a dense KMnO4/catecholamine reaction product, visible in the electron microscope, in the large dense-core vesicles of experimental animals. Our results demonstrate the localization of KMnO4 dense product in the cores of double membrane-bound spherical organelles, as well as in spherical structures in the matrix of typical mitochondria. No typical large dense-core vesicles were observed in these catecholamine neurons of the tissues studied. Our findings are consistent with the notion that altered mitochondria may contribute to the formation of a new type of large dense-core vesicle in the locus coeruleus neurons of man, which is probably an evolutionary adaptation of amine-storing organelles.

Proteins of the intermediate filament cytoskeleton as markers for astrocytes and human astrocytomas.

There is a pressing need for a more accurate system of classifying human astrocytomas, one that is based on morphologic characteristics and that could also make use of distinctive biochemical markers. However, little is known about the phenotypic characteristics of astrocytomas. Recent studies have shown that the expression of proteins comprising the intermediate filament (IF) cytoskeleton of astrocytic cells is developmentally regulated. It is our hypothesis that this changing protein profile can be used as the basis of a system for clearly and objectively classifying astrocytomas. A spectrum of human astrocytomas has been examined by immunofluorescence microscopy employing antibodies to several IF structural subunit proteins (GFAP, vimentin, and keratins) and an IF-associated protein, IFAP-300kDa. These proteins occupy unique temporal niches in the cytogenesis of the astrocytic cells: keratins in cells of the neuroectoderm; vimentin and IFAP-300kDa in radial glia and immature glia; GFAP in mature astrocytes; and vimentin in some mature astrocytes. In agreement with previous reports, our immunofluorescence studies have revealed both GFAP and vimentin in all astrocytoma specimens. Two new observations, however, are of particular interest: IFAP-300kDa is detectable in all astrocytic tumors, and the proportion of keratin-containing cells present in the astrocytomas is in direct relationship to the degree of the malignancy. Because IFAP-300kDa is not present in either normal mature or reactive astrocytes, this protein appears to represent a specific marker of transformed (malignant) astrocytes. If it is presumed that higher malignancy grades represent the most dedifferentiated cellular state of the astrocytes, the presence of keratin-containing cells is not totally unexpected, given the ectodermal (epithelial) origin of the CNS. Specific developmentally regulated proteins of the IF cytoskeleton thus appear to hold great potential as diagnostic markers of astrocytomas and as tools for investigating the biology of these tumors.

Immunotyping of radial glia and their glial derivatives during development of the rat spinal cord.

The differentiation of glia in the central nervous system is not well understood. A major problem is the absence of an objective identification system for involved cells, particularly the early-appearing radial glia. The intermediate filament structural proteins vimentin and glial fibrillary acidic protein have been used to define the early and late stages, respectively, of astrocyte development. However, because of the non-specificity of vimentin and the temporal overlap in expression patterns of both proteins, it is difficult to refine our view of the process. This is especially true of the early differentiation events involving radial glia. Using the developmentally-expressed intermediate filament-associated protein IFAP-70/280 kD in conjunction with vimentin and glial fibrillary acidic protein markers, a comprehensive investigation of this problem was undertaken using immunofluorescence microscopy of developing rat spinal cord (E13-P28 plus adult). The phenotypes of the cells were defined on the basis of their immunologic composition with respect to IFAP-70/280 kD (I), vimentin (V) and GFAP (G). A definitive immunotype for radial glia was established, viz, I+/V+/G-; thus reliance upon strictly morphological criteria for this early developmental cell was no longer necessary. Based upon the immunotypes of the cells involved, four major stages of macroglial development were delineated: (1) radial glia (I+/V+/G-); (2) macroglial progenitors (I+/V+/G+); (3) immature macroglia (I-/V+/G+); and (4) mature astrocytes (I-/V+/G+ primarily in white matter and I-/V-/G+, the predominant type in gray matter). It is of interest to note that the cells of the floor plate were distinguished from radial glia by their lack of IFAP-70/280 kD immunoreactivity. Introduction of the IFAP-70/280 kD marker has therefore provided a more refined interpretation of the various differentiation stages from radial glia to mature astrocytes.

Expression and distribution of cytoskeletal IFAP-300kD as an index of lens cell differentiation.

By their implication in the organization of the intermediate filament (IF) cytoskeleton, IF-associated proteins (IFAPs) can delineate subsets of the same IF type within a cell; moreover, they are proving useful as markers of the differentiation states of certain cells. For these reasons the expression of the vimentin-associated IFAP-300kD was investigated in the constantly differentiating cell lineage of the adult bovine lens. Immunofluorescence microscopy and immunoblot analysis were employed using a monoclonal anti-IFAP-300kD and a rabbit anti-lens vimentin. Cultures of adult lens epithelial cells were immunopositive for the IFAP. By double-label studies the IFAP-300kD pattern co-localized with that of the vimentin-type IF; moreover, the IFAP pattern co-distributed with that of both colchicine-sensitive and -insensitive IF systems. IFAP-300kD was also present in a co-distributing pattern with vimentin IF in fresh lens epithelial cells on whole mounts. There was a differential expression of the IFAP in the lens fiber cells in that those of the cortex exhibited the IFAP and vimentin IF, while both proteins were absent from the nuclear fiber cells. Furthermore, there was a differential distribution of the IFAP within the cortical fiber cells in that the IFAP localized only with a paramembranal subset of IF. Immunoblot analysis supported the presence of IFAP-300kD in the lens cytoskeletal fraction. IFAP-300kD thus identified a subset of vimentin IF whose location may have functional significance for the cortical fiber cell. The changes in the IFAP's expression and distribution pattern throughout lens cell differentiation in the adult organ suggest the usefulness of IFAP-300kD as a potential marker in studying lens cell differentiation in vitro.

Localization of ATPase activity in dendritic spines of the cerebral cortex.

An ATPase activity has been demonstrated in dendritic spines of the adult rat cerebral cortex using cerium to capture inorganic phosphate that is liberated during the enzymatic hydrolysis of ATP. Small pieces of cerebral cortex were fixed and incubated in a standard incubation medium containing both Ca2+ and Mg2+ at pH 7.2; other modifications of the incubation medium are described below. Electron microscopic examination of the cerium phosphate reaction product showed an electron dense precipitate localized in the cytoplasm of the spine behind the postsynaptic density. Whereas the postsynaptic density, itself, is not reactive, dense reaction product is seen immediately underneath the postsynaptic density and extending into the subsynaptic web. Reaction product is also associated with membranous cisternae within the dendritic spine. The reaction occurred in the presence of Ca2+ and Mg2+ and either of these two ions alone. However, virtually no reaction product is seen when the tissue was incubated in a medium devoid of Ca2+ and Mg2+, or in a medium containing Mg2+ and EGTA, suggesting that trace Ca2+ is necessary, but not sufficient for the reaction. Addition of p-chloromercurobenzoate, which selectively blocks SH groups, inhibited the reaction in the presence of Ca2+ and Mg2+, or both of these ions. The effect of pH on the reaction was determined using a lead precipitation method. The reaction occurred at pH 9.2 in the presence of Ca2+ alone. In the presence of Mg2+ alone, the reaction product appeared somewhat reduced at this pH. The presence of an ATPase activity, which is dependent upon Ca2+ in dendritic spines where actin and actin-binding proteins have also been localized, suggests that this activity may be involved in the dynamics of cytoskeletal function leading to shape changes in dendritic spines and synapses, as seen with various physiological and behavioral paradigms.

Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor.

Carboxypeptidase M, a plasma membrane-bound enzyme, is present in many human organs and differs from other carboxypeptidase that cleave basic COOH-terminal amino acids. Cultured Madin-Darby canine kidney (MDCK) distal tubular cells contain a kininase I-type enzyme that inactivates bradykinin by releasing Arg9. We found the properties of this kininase to be identical with carboxypeptidase M. In fractionated cells, carboxypeptidase activity sediments with membranes; and detergents, trypsin, and phosphatidylinositol-specific phospholipase C solubilize it, similar to results with human placental carboxypeptidase M. Ten microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and 1 mM o-phenanthroline inhibit, whereas 1.0 mM CoCl2 activates the enzyme. It has a neutral pH optimum and cleaves COOH-terminal Arg or Lys in bradykinin and in shorter peptides. The relative hydrolysis rates of peptides in the presence or absence of 1 mM CoCl2 were similar to those obtained with human carboxypeptidase M. The carboxypeptidase in MDCK cells (54 kDa) cross-reacts with antibodies to human carboxypeptidase M in Western blotting, but not with antibodies to plasma carboxypeptidase N. The enzyme is a glycoprotein; chemical deglycosylation reduced the size to 48 kDa. The presence of the enzyme on the cell membrane of MDCK cells was also shown with transmission electron microscopy using immunogold, which indicated that the enzyme is on the apical side. In addition, MDCK cells contain neutral endopeptidase 24.11 (enkephalinase) and prolylcarboxypeptidase (angiotensinase C) activities. Partitioning of solubilized carboxypeptidase M into Triton X-114 and water indicates that trypsin and phospholipase C remove a hydrophobic tail, while detergent solubilization leaves the hydrophobic moiety intact. Labeling of MDCK cells with [3H]ethanolamine resulted in the synthesis of radiolabeled carboxypeptidase M as determined by immunoprecipitation and fluorography. Thus, MDCK cells contain membrane-bound carboxypeptidase M, which is anchored to the plasma membrane via phosphatidylinositol-glycan. As a major kininase of the distal tubules, it may regulate salt and water excretion.

Calcium channel blocker influences the density of alpha-actinin labeling at the rat neuromuscular junction.

Alpha-actinin is a muscle protein located along the Z-disc. Incubation of frog muscle with the calcium ionophore, A23187, can decrease the immunogold labelling of alpha-actinin. Pyridostigmine (PYR) is an inhibitor of acetylcholinesterase, which causes disruption of Z-discs only in the region of the motor endplate. This is probably due to excess influx of calcium ions, leading to activation of proteases. Pretreating animals with the calcium channel blocker diltiazem can significantly reduce damage to the Z-discs at the motor endplate caused by PYR. It was of interest to determine whether the distribution of alpha-actinin had been altered following PYR administration and whether diltiazem could prevent those changes. There was less alpha-actinin labelling at the motor endplate compared to away from this region for all treatment groups. Animals administered diltiazem showed less labelling compared to PYR, but with no disruption of Z-discs at the motor endplate following diltiazem. Pretreatment with diltiazem reduced the incidence of Z-disc damage, but the degree of alpha-actinin labeling at the endplate was less than that seen with diltiazem alone. The greater effect seen at the endplate implies that neuromuscular activity is an important factor. The drugs may be causing a reduction in alpha-actinin labelling by different mechanisms.

Immunocytochemical localization of alpha-actinin in frog muscle treated with the ionophore A23187.

Incubation of frog skeletal muscle with the ionophore A23187 induces severe damage in the muscle. At the level of the Z line, the ionophore induces redistribution and release of the protein alpha-actinin, as shown by immunocytochemical techniques. The ionophore does not induce damage in denervated preparations or in preparations pretreated with d-tubocurarine, which indicates that the effect is indirect and neurally mediated. It is concluded that the increase in spontaneous transmitter release produced by the ionophore induces Ca2+ influx through the acetylcholine receptor. Calcium ions activate neutral proteases which release alpha-actinin from the Z line.

Fine structural localization of Ca2+-ATPase activity at the frog neuromuscular junction.

Ca2+-ATPase activity has been shown to be associated with the nerve terminal plasma membrane at the frog neuromuscular junction. Using a modification of the Wachstein-Meisel procedure for localization of phosphatases, a dense reaction product forms at the neuronal plasma membrane/Schwann cell interface. It has been determined that this reaction product is associated with the plasma membrane of the nerve terminal and not the plasma membrane of the Schwann cell. No ATPase activity is demonstrated at the presynaptic portion of the plasma membrane facing the synaptic gap. When a preparation is denervated, a Schwann cell process moves into the space previously occupied by the nerve. There is no ATPase activity associated with the Schwann cell plasma membrane. Conversely, when the Schwann cell is selectively injured, dense reaction product continues to be associated with the nerve terminal plasma membrane. There is some indication that this ATPase activity is dependent on the presence of Ca2+ and Mg2+. Incubation in the calmodulin inhibitor, R24571, shows little inhibition of labelling.

The effect of 2450 MHz microwave radiation on the ultrastructure of snail neurons.

An electron microscopical study of snail neurons was undertaken to verify whether any ultrastructural alterations accompany microwave-induced electrophysiological changes observed in these neurons. Subesophageal ganglia from Helix aspersa snails were exposed to 2450 MHz microwave radiation in vitro at SAR 12.9 mW/g for 60 minutes. It was found that exposure at 21 degrees C causes minor changes in Golgi complexes and slight swelling of the endoplasmic reticulum.