Milton W. Brightman

Neurone-specific enolase is a molecular marker for peripheral and central neuroendocrine cells

NEURONE-SPECIFIC ENOLASE (NSE) is the most acidic brain isoenzyme of the glycolytic enzyme enolase (EC4.2.1.11) and has been shown to be homologous to the 14-3-2 protein isolated from bovine brain by Moore1–3. Whereas NSE is exclusively localised in neurones in mammalian nervous tissue4,5, another brain enolase isoenzyme termed non-neuronal enolase (NNE) is localised in glial elements5. As NSE and NNE are structurally, functionally and immunologically distinct isoenzymes that represent separate gene products6,7, they are useful markers for cell classes in the nervous system. Although NNE is probably identical to liver enolase, NSE has to date been considered to be localised in neurones. We now report that NSE is also present in peripheral and central neuroendocrine cells, also termed amine precursor uptake and decarboxylation (APUD) cells8–10. Immunocytochemistry using the unlabelled antibody enzyme method of Sternberger11 demonstrates that APUD cells in both laboratory rat and rhesus monkey (Macacca mulatta) stain positively with NSE antiserum. Using a sensitive radioimmunoassay (RIA) for NSE, it is possible to confirm their localisation in adrenal gland and to support the finding in other APUD-cell-containing tissues in rat, monkey and man. The results in human tissues suggest that the immunocytochemical localisation in rat and monkey will be valid for man and prove useful in the study of human diseases involving the APUD cell class.

Dendrodendritic synaptic pathway for inhibition in the olfactory bulb

Abstract Anatomical and physiological evidence based on independent studies of the mammalian olfactory bulb points to synaptic interactions between dendrites. A theoretical analysis of electric potentials in the rabbit olfactory bulb led originally to the conclusion that mitral dendrites synaptically excite granule dendrites and granule dendrites then synaptically inhibit mitral dendrites. In an independent electron micrographic study of the rat olfactory bulb, synaptic contacts were found between granule and mitral dendrites. An unusual feature was the occurrence of more than one synaptic contact per single granule ending on a mitral dendrite; as inferred from the morphology of these synaptic contacts, a single granule ending was often presynaptic at one point and postsynaptic at an adjacent point with respect to the contiguous mitral dendrite. We postulate that these synaptic contacts mediate mitral-to-granule excitation and granule-to-mitral inhibition. These dendrodendritic synapses could provide a pathway for both lateral and self inhibition.

The intracerebral movement of proteins injected into blood and cerebrospinal fluid of mice.

Publisher Summary The entry of radioactively labeled protein from blood into cerebrospinal fluid (CSF) and into cerebral tissue is much slower and less in amount, than that of tritiated water and certain ions. The barrier that impedes the entry of labeled protein into the CSF has been interpreted as being responsible also for the exclusion of intravascularly injected acid dyes which, because of their negative electric charge, bind to serum proteins. The second portion of this paper is concerned with this direction of transfer and offers a brief, preliminary description of the movement of peroxidase from ventricular CSF not only toward the vessels of the choroid plexus but also toward the vessels of the cerebral parenchyma. Some observations on the passage of the protein ferritin in this direction are also included. Movement of peroxidase from choroidal blood to epithelium and also the movement of peroxidase and ferritin from ventricle into choroidal epithelium and cerebral parenchyma are given in detail in the abstract. As a conclusion, the chapter states that the anatomical barriers to the movement of peroxidase from blood to ventricular CSF consist of: (a) the structures (which are probably tight junctions) between the apices of the choroidal epithelial cells and, perhaps, (b) their ventricular surface. This surface has been regarded as the barrier to the movement of fluorescent proflavine dyes out of the choroid plexus, though the only part of the epithelial cells enclosing demonstrable dye was the nuclear membrane rather than any portion of the cell membrane itself.

Neurons switch from non-neuronal enolase to neuron-specific enolase during differentiation

The enolase (EC 4.2.1.11) isoenzymes, neuron-specific enolase (NSE, gamma gamma) and non-neuronal enolase (NNE, alpha alpha), are markers for neurons and glia, respectively, in adult mammalian brain. In developing fetal and early postnatal brain, levels of non-neuronal enolase (NNE) are high. Neuron-specific enolase (NSE) appears only after neurogenesis begins in a given region and only slowly attains adult levels. Immunocytochemistry in developing rat and rhesus monkey brain reveals that proliferative zones that give rise to neurons are NNE(+). Thus, nerve cells must undergo a switch from NNE to NSE. In addition, study of neurons in cerebellum and neocortex reveals that they are NNE(+) during migration and only become NSE(+) in their final location, presumably after making full synaptic connections. Such migrating cells may contain hybrid enolase (alpha gamma) and some (e.g. cerebellar stellate/basket cells) may not completely switch over to NSE even in the adult. Neuron-specific enolase is not only a specific molecular marker for mature nerve cells, but is closely correlated to the differentiated state.

Morphology of blood-brain interfaces

The blood-brain barrier to the protein horseradish peroxidase (HRP, tool. wt. 40 000) is the consequence of both an obstruction to passive extracellular movement and the absence of an active transcellular migration. Although the endothelium of some cerebral arterioles can transfer protein from blood to perivascular basement membrane by means of vesicles, the endothelium of cerebral capillaries cannot. The barrier may be passed when the junctions between endothelial cells become patent as in some brain tumors and in vitally- induced choroid plexus papillomas. The vessels in the latter tumors also have fenestrae and numerous endothelial pits and vesicles, features that may also account for the increased permeability of the tumor vessels. The barrier is also opened by infusing hyperosmotic non-electrolytes into the internal carotid artery. Within 60 sec after the infusion of 1.4 M-arabinose into one carotid artery of adult rats that had received HRP intravenously, the vessels on the infused side of the brain extravasate peroxidase. The escape of HRP is probably through junctions or actively formed channels and not by means of vesicular transport. When hyperosmotic arabinose is injected first, the brain fixed and then, 1 hr after fixation, HRP is infused into the aorta, perivascular exudates appear primarily on the saccharide-infused side of the brain. Since vesicular transfer requires energy and is halted by chemical fixation, the protein must have crossed the endothelium through pre-existing channels. Comparable to the effects of hyperosmotic urea on the vessels of the rabbit brain, only a few endothelial junctions were obviously patent. It is likely, therefore, that most of the passage is by way of channels formed, perhaps, by confluent vesicles. The channels, once formed, do not close im- mediately but remain patent long enough to be fixed in the open position. Neurons deep within the brain stem are inaccessible to protein circulating within capillaries immediately adjacent to them. The same neurons, however, can pinocytose HRP at their axon terminals which innervate peripheral organs such as muscle and mucous membrane. The blood vessels of these tissues are permeable to protein which, upon crossing the vessels, are incorporated by the axon terminals and transported within the axons back to lysosomes within the neuronal soma. In this way, large molecules circulating in the systemic blood may enter certain neurons even though the same molecules are barred from crossing the cerebral capillaries supplying these neurons. In contrast to hydrophilic molecules, there is no appreciable restriction to the passage of lipids from blood to brain. Fatty acids (FA) injected into blood are directly incorporated into cerebral lipids. When large concentrations of the polyunsaturated FA linolenic acid, wbich is strongly osmiophilic, is infused into one carotid artery, the soap droplets formed fuse with endothelial cell membranes. The osmiophilic droplets enter the endothelium and penetrate cytomembranes as well as cell membranes except, perhaps, for the inner nuclear membrane; droplets readily enter the perinuclear cistern but are never found within the nucleus of any cell, be it endothelial, smooth muscle, epithelial or neuronal. Some of the FA enters endothelial pits and vesicles and may represent that fraction of the FA which normally circulates as a complex with serum albumin. Mitochondria are infiltrated by droplets as are synaptic vesicles and axoplasm of perivascular and intracerebral neurons.

Assemblies of particles in the cell membranes of developing, mature and reactive astrocytes

SummaryOrthogonal arrays of small intramembranous particles characterize freeze-fractured astrocytic plasma membranes. The normal variation of assemblies in plasma membranes of subpial astrocytic processes in mature and developing rats was established and compared with assemblies in plasma membranes of reactive astrocytes. In mature rats, subpial astrocytic processes had the greatest number of assemblies. As the parenchyma was approached, this number decreased in each successive layer of astrocytic processes. The advent of assemblies within the plasma membrane is a new criterion of astrocytic differentiation. The foot processes of subpial astrocytes in foetal rats began to acquire assemblies between day 19 and 20 and continued to mature postnatally by a constant addition and rearrangement of assemblies. In contrast to the paucity of assemblies in deeper laminae of the normal brain, reactive astrocytes comprising the lower lamellae in glial scars had an increased number of assemblies while the most striking feature of the superficial, astrocytic processes within the scar was a rearrangement of assemblies. Although the function of these intramembranous particles is still unknown, it appears from our measurements that they are localized primarily in the outermost astrocytic foot process and to a progressively lesser degree in the underlying astrocytic lamellae.

Pathway across blood-brain barrier opened by the bradykinin agonist, RMP-7

The route taken by lanthanum (MW 139) across cerebral endothelium was delineated when the blood-brain barrier was opened by RMP-7, a novel bradykinin agonist. Balb C mice were infused through a jugular vein with LaCl3 with or without RMP-7 (5 micrograms/kg). Ten minutes later, the brains were fixed with aldehydes and processed for electron microscopy. The patency of the junctions between endothelial cells was estimated by counting the number of junctions penetrated by LaCl3. Tracer penetrated the junctions in about 25% of microvessels in vehicle infused, control mice and about 58% in the RMP-7 group, where more junctions per vessel were also penetrated. The LaCl3 then penetrated the basal lamina in about 20% of all microvessels in the RMP-7 group, versus 0.50% in the control group. From the basal lamina, the tracer entered perivascular spaces in about 13% of all microvessels in the RMP-7 group and about 0.07% in the controls. Very few endocytic pits or vesicles in the RMP-7 group were labeled, so LaCl3 did not cross endothelium by transcytosis. The increased number of tight junctions penetrated by tracer and its spread into periendothelial basal lamina and interstitial clefts indicated, therefore, a paracellular route of exudation in the RMP-7 treated animals.

The sarcolemma of Aplysia smooth muscle in freeze-fracture preparations.

Smooth muscle cells in the sheath covering the visceral ganglion of Aplysia californica were examined with the techniques of freeze-fracture and conventional electron microscopy. The sarcolemma of these muscle cell invaginates to form myriad caveolae that have an intrinsic marker within their membrane. This intrinsic structure of the caveolar membrane is revealed by freeze-fracture and consists of rows of large particles in the outer half and matching grooves on the complementary inner half of the membrane. In thin plastic sections, parallel striations or shelves within the caveolar membrane appear to be the equivalent of the particles and grooves of the fractured membrane. Physical fixation of some specimens by rapid freezing in supercooled liquid nitrogen or in liquid helium suggests that in their natural state, the caveolar ostia are not uniform in size and that at any given moment a number of caveolae are flattened. When segments of the connective nerves which link the visceral ganglion to the cephalic ganglia are stretched in vitro two to three times their in situ length, the caveolae lose their invaginated shape and are fully exposed to the extracellular space. The caveolar membrane, so stretched, is pulled into the line of fracture with the result that the large particles rather than the ostia appear on the cleaved surface. This flattening of the caveolae is reversible and suggests that they might serve as miniature stretch-receptors within the membrane of the smooth muscle cells. The caveolae are accessible to extracellular horseradish peroxidase but do not appear to pinocytose the protein.

Development of membrane interactions between brain endothelial cells and astrocytes in vitro.

To ascertain whether there is a mutual influence on the structure of their cell membranes, brain endothelial cells and their closest neighbor, astrocytes, were grown alone or together in vitro and freeze‐fractured. When cultured separately, the brain endothelial cells had a low frequency of short, fragmented tight junctions. Many gap junctions, which are absent from mature brain capillaries in vivo, intercalated among the tight junctional strands, or were separate from them. The separately cultured astrocytes had low concentrations of randomly distributed assemblies (1–30/μm2) in their membranes. When the two cell types were co‐cultured, the endothelial tight junctions were greatly enhanced in frequency, length, width and complexity, and the gap junctional area enclosed by the tight junctional strands were markedly reduced. Thus, the in vitro endothelial junctional complex resembled their in vivo counterpart, the tight junctions of brain capillaries, when co‐cultured with astrocytes. Reciprocally, brain endothelial cells induced the astrocytic membrane assemblies to increase in concentrations by 5̃ fold, and sometimes to form aggregates with very high concentrations (400/μm2) which approached the concentration of the perivascular astrocytic membranes in vivo. Substituting astrocytes with fibroblasts or smooth muscle cells in co‐cultures did not enhance the tight junctions in the brain endothelium. On the other hand, substituting brain endothelium with endothelium from pulmonary artery or aorta in cocultures did not increase the concentration or induce aggregation of the assemblies in the astrocytes. Thus, the two close neighbors in vivo, brain endothelium and astrocytes, interact specifically in vitro to induce development of membrane specializations which resemble those at the site of the blood‐brain barrier.

Localization of neuron-specific enolase in mouse spinal neurons grown in tissue culture

Neuron-specific enolase (NSE) is an isoenzyme of the glycolytic enzyme enolase (EC 4.2.1.11) which also has a muscle and liver isoenzyme. Previous work has shown NSE to be specifically localized to neurons and neuroendocrine cells, but the application of NSE as a marker for cell cultures has not been investigated. Primary culture of central nervous system tissue derived from mice have been used to study optimal fixation procedures. The results show that NSE can serve as a useful alternative to non-specific histochemical strains or strictly morphologic criteria for identifying nerve cells.