Applications of Monoclonal Antibodies to Neuroscience Research

Ann. Rev. Neurosci. 1985. 8:199-232Copyright © 1985 by Annual Reviews Inc. All rights reserved

APPLICATIONS OFMONOCLONAL ANTIBODIES TONEUROSCIENCE RESEARCHKaren L. Valentino, Janet Winter, and Louis EReichardt

Department of Physiology, University of California, School of Medicine, SanFrancisco, California 94143

INTRODUCTION

The introduction of immunological methods has revolutionized neurobiology.Anatomical studies have profited from immunocytochemical localization ofneurotransmitters and peptides for a better understanding of the organizationof the nervous system. Antibodies have also bccn useful in identifying dif-ferent cell classes in the nervous system, and antibodies to subcellular organ-elles have contributed to our knowledge of neuron function. Monoclonal anti-bodie~are increasingly used by neurobiologists to build on this knowledge.

This review consists of four major sections. In the first three, applicationsof monoclonal antibodies are reviewed with regard to the cell biology of theneuron and the anatomy and development of the nervous system. The finalsection is an overview of the strategies involved in isolating and applyingmonoclonal antibodies to studies of the nervous system. This is not intendedto be a comprehensive review of the literature of these subjects, and, wherepossible, reviews of individual subjects are indicated in the text.

In some areas, such as peptide and transmitter research, the main contri-butions of monoclonal antibodies have been to make scarce antibodies morewidely available and to make antibodies to antigens that have been difficult topurify. In developmental neurobiology, monoclonal antibodies have made apromising beginning in identifying molecules that might be involved in cell-cell recognition and cell adhesion. Some of the most interesting contributionsof monoclonal antibodies have been in determining protein structure (e.g.acetylcholine receptor and sodium channel), and we discuss these areas ingreater detail.

199

0147- 006X/85/0301 - 0199502.00

www.annualreviews.org/aronlineAnnual Reviews

Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.

http://www.annualreviews.org/aronline

200 VALENTINO, WINTER & REICHARDT

NEURONAL CELL STRUCTURE AND FUNCTION

Channels, Pumps, and Receptors

SODIUM CHANNEL Voltage sensitive Na ÷ channels play a key role in con-ducting the action potential and are the one channel type that has been ~ignif-icantly purified and studied with traditional biochemical methods (reviewedin Reichardt & Kelly 1983). The Na÷ channel has been purified substantiallyfrom Electrophorus electroplax, rat brain, and rat muscle. Purified channelsfrom the latter two preparations have been successfully reconstituted in lipidbilayers. All channel preparations contain a very large (ca 270 kDa) glyco-protein. Smaller subunits have been seen in the purified channel preparationsfrom rat brain and rat muscle. During the last two years, conventional antiseraand monoclonal antibodies have been raised that bind the large glycoproteinsubunit of the channel, both fro:m Electrophorus electroplax and rat muscle(Moore et al 1982, Ellisman & Levinson 1982, Casadei et al 1983). A mon-oclonal antibody that binds the Electrophorus channel subunit has been usedto complete the purification of that subunit by affinity chromatography (Nakay-ama et al 1982). Another monoclonal antibody has shown that Na+ channelsare concentrated on the electrically excitable caudal face of the electrocytesand not on the inexcitable rostral face (Fritz & Brockes 1983). There was detectable binding with this antibody to sections of Electrophorus spinal cord,muscle, or nerve, or to electrically excitable tissues of other species. In con-trast, a polyclonal antiserum to a highly but not completely purified Electro-phorus Na+ channel preparation binds to both the innervated surfaces ofelectrocytes and to nodal regions in myelinated axons (Ellisman & Levinson1982). The monoclonal antibody appears to recognize an antigenic determi-nant found only on the Na+ channel in the electroplax. The polyclonal serumappears to recognize a more widely shared determinant. The specificity of thepolyclonal antiserum for the Na + channel, however, has not been assessedusing total membrane proteins. The distribution of Na + channels in dendrites,axons, and nerve terminals is functionally important in modulating integrationof different synaptic inputs, initiation and conduction of the action potential,and neurotransmitter release. Detailed studies using these antibodies and anti-bodies to other channels will be very important in understanding the electricalproperties of excitable cells. Monoclonal antibodies to the small Na+ channelsubunits should be useful in understanding the role of these subunits in Na +

channel function.

SODIUM PUMPS The major mechanism for removing internal Na+ and re-storing internal K ÷ after action potentials is the ATP driven Na +-K + exchangepump. It is detected in biochemical assays as a Na+, K+-ATPase, whichcontains two polypeptide subunits of approximate Mr 120,000 and 50,000.


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


MONOCLONAL ANTIBODIES IN NEUROBIOLOGY 201

Distribution of the Na+, K+-ATPase on neurons in fish brain has been exam-ined with immunocytochemical methods using a conventional antiserum (Woodet al 1977). On neurons, it is restricted to the plasmalemma, where it isdistributed over the surface of cell somata and dendrites. It appears to beconcentrated in nodes of Ranvier in myelinated axons. Two classes of synapticterminals were observed, one with high and one with lower concentrations ofthe ATPase. Differences in concentration between different nerve terminalsare likely to be important in regulating the response properties of these ter-minals to stimulation.

There is biochemical evidence for multiple forms of the Na÷, K÷-ATPasein neurons (Sweadner 1979). One form is the standard form 0 that is seenin kidney, muscle, and astrocytes in addition to cultured sympathetic neurons.A specific neuronal form (o~+) is found in both vertebrate and invertebratebrain and is the only form found in myelinated axons. Recently, a monoclonalantibody to the Na +, K +-ATPase has been isolated that suggests the existenceof an even wider diversity of Na÷-pumps (Fambrough & Bayne 1983). Thisantibody binds the Na+, K÷-ATPase in chicken neurons, muscle fibers, andkidney tubules, but not in Schwann cells, fibroblasts, or erythrocytes. Thisdistribution is not the same as that of et + in rodent tissues. The antibody bindsto nonglycosylated antigen, so the heterogeneity revealed by the antibody isunlikely to reflect posttranslational modification. The most likely explanationis molecular heterogeneity, arising either from differential expression of mem-bers of a closely related gene family or differences in subunit composition indifferent cell types. The monoclonal antibody has been used to study regu-lation of Na+ pump levels in response to demand for pumping and to studydistribution of the pump in chick neurons (Fambrough 1983). In contrast fish brain, substantial concentrations of the Na÷ pump appear to exist in theinternodal regions of chick myelinated axons.

Caz+ PUMPS Cytoplasmic Ca2+ levels regulate many facets of neuronalmetabolism, most notably the release of neurotransmitters by exocytosis.Cytoplasmic Ca2 + levels are regulated by the activity of voltage-sensitiveCa2+ channels and several Ca2+ removal systems, including a Na+:Ca2+

antiporter in the plasma membrane, a Ca2 + porter in the mitochondrial innermembrane, and several distinct ATP-dependent Ca2+ uptake systems in theplasma membrane, smooth endoplasmic reticulum, and synaptic vesicles(reviewed in Reichardt & Kelly 1983). The major neuronal ATP-dependentCa2+ translocators have been purified (Goldin et al 1983, Chan et al 1984).An Mr+ 140,000 translocator, regulated by CaZ+-calmodulin, has the samemolecular weight as the Ca2 +-calmodulin-regulated (Ca~ + + Mg2 +) ATPasesin red blood cells and many other tissues. A second, Mr+ 94,000 Ca2+ trans-locator is not regulated by Ca2 + p-calmodulin and is similar in size to the Ca2+


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



translocator in muscle sarcoplasmic reticulum. A monoclonal antibody to thisneuronal translocator, however, does not cross-react with either the sarco-plasmic reticulum or red blood cell translocators (Chan et al 1984), thussuggesting that this is a nerve-cell-specific protein. The antibodies to thistranslocator should facilitate studies on the regulation of its activity. Its ultra-structural localization will also be very interesting.ACETYLCHOLINE RECEPTOR The acetylcholine (ACh) receptor has been in-tensely studied with monoclonal antibodies, yielding much information aboutstructure and function. The aCh receptor is a pentameric protein complex thatforms a transmembrane channel that is opened by binding to ACh (reviewedin Conti-Troncini & Raftery 1982). The receptor consists of four different,but homologous, subunits that form a pentameric rosette around a centralchannel with stoichiometry ~213"~. It is the primary target in myasthenia gravisand in an animal disease model, experimental autoimmune myasthenia gravis(EAMG).

Several groups have isolated monoclonal antibodies to the ACh receptor,purified from Torpedo electroplax, Electrophorus electroplax, or mammalianskeletal muscle (Mochley-Rosen et al 1979, Tzartos & Lindstrom 1980, Conti-Troncini ct al 1981). Monoclonal antibodies that bind determinants unique foreach subunit have been identified (Tzartos & Lindstrom 1980). Among theseare a few examples of antibodies that block ion flux and snake a-toxin binding(Mochley-Rosen et al 1979, Gomez et al 1979). Antibodies that recognizehomologous determinants shared by the a and 13, or ~/and ~ subunits, respec-tively, have also been isolated and provide evidence for homologies betweenthese subunits (Tzartos & Lindstrom 1980). Previous studies using conven-tional antisera and comparative peptide analysis had not revealed these subunithomologies, which have since been confirmed by sequencing completely thefour genes that encode these subunits (e.g. Noda et al 1983). Nine antigenicdeterminants, some sequence dependent and some conformation dependent,have been recognized on the Electrophorus receptor (Tzartos et al 1981).Twenty-eight determinants have been recognized on denatured Torpedo recep-tor subunits (Gullick & Lindstrom 1983). Fab fragments of an antibody thatbinds the ot-subunit have been used in high resolution electron-microscopicstudies to map the position of the o~-subunits in Torpedo receptor dimers inwhich the ~-subunits link the two monomers (Kristofferson et al 1984). Theresults show clearly that the two ot-subunits are not adjacent to each other, butare separated by an intercalated 13 or ",/-subunit. Examination of the bindingof Fa~, fragments specific for other subunits should reveal the order in whichthe subunits surround the central ion channel. Antibodies that bind to differentantigenic determinants are being used to probe the structure of each receptorsubunit. In many cases these have been shown to bind different peptides(Gullick et al 1981, Gullick & Lindstrom 1983). Some, but not all, of the


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



epitopes in the ~- and 13-subunits have been ordered along the peptide sequenceby examining the sensitivity to limited proteolysis of in vitro synthesizedsubunits that have been co-translationally inserted into microsomes (Andersonet al 1983). Extracellular and cytoplasmic domains on the 13 subunit havebeen identified. In general, these results are consistent with models of subunitstructure derived from hydrophobicity of the amino acid sequence, but are notsufficiently refined to determine whether a fifth amphipathic o~-helix crossesthe lipid bilayer (Finer-Moore & Stroud 1984). Four and five transmembranedomain models of the acetylcholine receptor could in theory be distinguishedby determining whether antibodies specific for the C-terminus of a receptorsUbunit bind to the cytoplasmic or extracellular surface. Recently, an antibodyto a synthetic peptide corresponding to the C-terminus of the ~-subunit hasbeen shown to bind to the cytoplasmic side of the membrane, arguing per-suasively for a five trans-membrane domain model (Young et al 1984).

The acetylcholine receptor contains a major immunogenic determinant thatis located on the extracellular surface of the a-subunit and is conserved betweenspecies (Tzartos & Lindstrom 1980; Tzartos et al 1981). Antibodies to thisdeterminant do not prevent ion flux through the channel, but do induce passiveEAMG when injected into rats (Tzartos et al 1981). The ability of differentmonoclonal antibodies to induce EAMG has shown to correlate with theirability to aggregate solubilized receptor in vitro (Conti-Troncini et al 1981).As expected, only antibodies to the o~ subunit, two copies of which areincluded in each receptor, are able to aggregate receptor into complexes largerthan dimers. Only those anti-a antibodies that induce the formation of largeaggregates efficiently induce EAMG. These antibodies are also the only onesto increase the turnover of ACh receptor on the surface of cultured myotubes.Monoclonal antibodies have been used in competitive binding assays to deter-mine the specificity of anti-receptor antibodies in the serum of patients withmyasthenia gravis. Patients with myasthenia gravis produce antibodies to thesame regions on receptor as produced by animals immunized with receptorpurified from fish electric organ (Tzartos et al 1982). Most of these bind the main immunogenic region. These results suggest that antibody productionin myasthenia gravis is stimulated by receptor, not by a cross-reacting antigen.No correlation was found between the titer of antibody to a specific determi-nant and the severity of the disease state. This suggests that other factors areimportant in determining the severity of illness. Monoclonal antibodies willmake it possible to investigate these factors in detail.

RECEPTORS TO OTHER LIGANDS Monoclonal antibodies to the 13-adrenergicreceptor have been isolated and used to map its distribution in the brain, whereit is concentrated in postsynaptic densities (Strader et al 1983). Monoclonalantibodies to purified rat brain muscarinic acetylcholine receptor have been


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



isolated and have been used to show that all identified antigenic epitopes onthese receptors are conserved in all tissues and species examined, includingboth vertebrates and invertebrates (Ventner et al 1984). These data suggestthat there is only one form of the receptor and that it has been extraordinarilyconserved throughout evolution. Monoclonal antibodies to the a-adrenergicreceptor have been isolated by immunizing with rat liver plasma membranes(Ventner et al 1984). Some of these antibodies cross-react with the muscarinicacetylcholine receptor. The receptors may share antigenic sites because theymodulate common effectors. These results show that plasma membrane prep-arations can be used to obtain monoclonal antibodies specific for receptors,provided that the antibodies can be screened effectively. Plasma membraneshave been used to obtain monoclonal antibodies specific for both the epidermaland nerve growth factor receptors (Richert et al 1983, Chandler et al 1984).

Internal Organelles and Cytoskeletal Elements

The complex subcellular anatomy of neurons has been examined with con-ventional and monoclonal antibodies. The studies have revealed that specificmolecules are concentrated in anatomically distinct regions of the cell.

CYTOSKELETAL ELEMENTS Neurons contain the same cytoskeletal elementsas other cells--actin, tubulin, and intermediate filament networks. The roleof these filaments in neural function is poorly understood, although fast trans-port of membrane vesicles has been closely associated with microtubules invivo and in vitro and requires the integrity of the microtubule network (reviewedin Reichardt & Kelly 1983). Studies initiated with conventional antibodiesand extended with monoclonal antibodies have examined the distribution ofactin, tubulin, and intermediate filament-associated proteins in cultured neu-rons and in sections of the brain. Antibodies specific for each subunit of theneurofilament triplet have shown that all three subunits are found only inneurons (e.g. Shaw et al 1981a, Lee et al 1982). Some classes of neurons,for example the granule cells in the cerebellum, lack neurofilaments and donot bind antibodies to any of the neurofilament subunits (e.g. Shaw et al198 l b). In electron-microscopic examination, each antibody can be shown tostain the same filament (Sharp et al 1982). The Mr 200,000 subunit appearsto have a periodic distribution in the filaments, while the other two smallersubunits are distributed continuously (e.g. Sharp et al 1982). While the threeneurofilament subunits always appear to coexist in the same neurons, the Mr200,000 subunit does not appear to be found in neurofilaments in all parts ofsome classes of neurons (Hirokawa et al 1984). For example, a monoclonalantibody specific for the Mr 200,000 subunit does not bind to dendrites inpyramidal cells, even though these dendrites have high concentrations of theMr 68,000 subunit (Debus et al 1982). More recent work has raised the pos-


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


sibility, though, that some of the observed restrictions in distribution of theMr 200,000 subunit may reflect differences in its state of phosphorylation.Many monoclonal antibodies to the subunit have been shown to be specificfor either phosphorylatcd or nonphosphorylated forms of the subunit (Gold-stein et al 1983, Sternberger & Sternberger 1984). The two classes of anti-bodies stain different neurons and parts of neurons. Their staining patternsuggests, for example, that the Mr 200,000 subunit is more heavily phospho-rylated in axons than in dendrites of Purkinje cells.

A differential distribution of other cytoskeletal proteins has also been shownin pyramidal and Purkinje cells. A conventional antiserum to the high molec-ular weight microtubule-associated-proteins (MAP1 and MAP2) stains den-drites but not axons, whereas antibodies to tubulin bind to both regions (Matuset al 1981). More recent studies with a MAP2 monoclonal antibody suggestthat MAP2 is restricted to dendrites, while MAPI is probably not restrictedin its distribution (Caceres et al 1983, 1984, Vallee & Davis 1983, Huber Matus 1984). MAP2 has been shown to be a substrate for both cAMP-depen-dent and Ca2 +-calmodulin dependent protein kinases, and it has a binding sitefor the type II regulatory subunit (RII) of the cAMP-dependent kinase. Bindingsites for the RII subunit are concentrated in dendrites and are almost inde-tectable in axons. This is essentially the same distribution reported for MAP2(Miller et al 1982). A differential distribution of actin and 13-tubulin has alsobeen shown in the Purkinje cell dendritic shaft and dendritic spine. A mono-clonal antibody to [3-tubulin stained only the dendritic shaft, whereas anti-bodies to MAP2 and actin stained both the shaft and the spine (Caceres et al1983). One monoclonal antibody to actin, however, did not stain actin in thespines. These results suggest that there may be different functional states ofactin and that the interactions between cytoskeletal components may vary indifferent compartments of the cell.

Conventional antibodies to brain-specific and erythrocyte-specific spectrins,proteins that bind to both the membrane and cytoskeleton, have been used toshow that both forms are present in neurons, but have different distributions(Lazarides & Nelson 1983a,b, Lazarides et al 1984). The erythrocyte formappears only at a terminal state in differentiation and is restricted to the cellsoma and dendrites, while the brain-specific form, also known as fodrin,appears early in neuronal development and is distributed throughout the neu-ron. It seems likely that the spectrins function to coordinate domains in theneuronal membrane and cytoskeleton.

As illustrated above, monoclonal antibodies have the potential for identi-fying and probing the function of modified forms of proteins that are associatedwith particular regions of the neuron. As one example, injection of a mono-clonal antibody specific for the tyrosylated form of ot-tubulin has been shownto have dramatic effects on organelle movement and cytoplasmic organization


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



in fibroblasts. This suggests that addition of tyrosine to tubulin may modulateits activity within this cell (Wehland & Willingham 1983). Intriguingly, mon-oclonal antibodies specific for the tyrosylated and nontyrosylated forms oftubulin stain the axons of cercbellar granule ceils differently, depending ontheir age, in patterns that suggest that tyrosine addition to tubulin is reducedas axons mature (Cummings et al 1984). Monoclonal antibodies to differentepitopes on actin and calmodulin stain different subcellular structures in neu-rons and fibroblasts, respectively, suggesting that these proteins interact withdifferent binding proteins in different compartments (Caceres et al 1983, Par-due ct al 1983). Thus, monoclonal antibodies make it possible to study thespecific interactions of these proteins with other cellular macromolecules. Anindividual monoclonal antibody, however, cannot be assumed to reveal all thesites to which its antigen is localized.

ORGANELLES Synaptic vesicles and mitochondria are prominent organellesin nerve terminals. A monoclonal antibody directed against a neuron-specificmitochondrial protein has been isolated using rat brain as an immunogen(Hawkes et al 1982a). This antibody binds a r 23,000 protein i n neuronalmitochondria that is absent from other cell types in the brain. The antigenappears in neurons only after the termination of cell division. This suggeststhat changes in mitochondrial proteins may be important in neuronaldifferentiation.

Monoclonal procedures have been very useful tbr identifying antigens sharedby many classes of synaptic vesicles. Two monoclonal antibodies to a Mr65,000 vesicle membrane protein bind vesicles in many and possibly all typesof nerve terminals (Matthew et al 1981). The antibodies have been used suc-cessfully to purify synaptic vesicles from crude brain homogenates by immu-noprecipitation and to confirm the presence of norepinephrinc and severalpeptide transmitters in vesicle fractions. The vesicle antigen is highly con-served throughout the vertebrate phylogeny. A second vesicle-specific protein,the phosphoprotein synapsin I, was initially studied with polyclonal antibodiesand was shown also to exist in virtually all types of nerve terminals andthroughout the vertebrate phylogeny (e.g. Goelz et al 1981). More recently,monoclonal antibodies have been isolated to this phosphoprotein and theseshould make possible more detailed studies on the structure and function ofsynapsin I in the future (Nestler & Greengard 1983). The distributions of thesetwo antigens have been used to monitor differentiation of the nerve axon andnerve terminal during development in vivo or in vitro. Dramatic changes withdevelopment are seen both in the brain and culture dish (Chun & Schatz 1983;Matthew & Reichardt 1982).

Cholinergic synaptic vesicles from fish electric organs contain a specificproteoglycan that has been defined by both conventional and monoclonal anti-


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


sera (Jones et al 1982, Buckley et al 1983). Antibodies have been used show that this proteoglycan is released into the cleft by exocytosis duringstimulation and is recycled during rest (Jones et al 1982). Studies with monoclonal antibody show that the antigen is found on the surface of thecholinergic nerve terminals in the vicinity of active zones. Evidence suggeststhat a fraction remains in the cleft for extended periods during rest, possiblyimmobilized in the extracellular matrix (Buckley et al 1983).SYNAPSE CONSTITUENTS Chemical synapses exist in a diversity of morpho-logical conformations, usually containing one or more of the following spe-cializations: presynaptic vesicle clusters, presynaptic membrane densities, andpostsynaptic dense material. Conventional and monoclonal antibodies haveprovided a promising approach to identifying the molecules in the speciali-zations associated with the synapse. Few antibodies to antigens concentratedin the presynaptic nerve terminal have been found so far. The only well-definedantigens are components of synaptic vesicles (discussed under Organelles),which include neurotransmitters and neuropeptides (see next section).

Antibodies to antigens in the postsynaptic specializations are more common.The ACh receptor has been localized at the neuromuscular junction with mon-oclonal antibodies to the receptor (Z. W. Hall, personal communication), andhas also been shown by use of o~-bungarotoxin to be in the membrane at thetop and sides of the postsynaptic folds (Fertuck & Salpeter 1976). Antibodiesto a Mr 43,000 protein that copurifies with ACh receptor have been used tolocalize the protein at the neuromuscular junction (Froehner et al 1981). electron-microscopic examination, the Mr 43,000 protein appears to form adense bar of contrasted material that is coextensive with ACh receptor (Sealock1982, Scalock et al 1984). It can bc cross-linked to the acetylcholine receptorwith a heterobifunctional reagent. Monoclonal antibodies to the Mr 43,000protein and four acetylcholine receptor subunits have been used to show thatthe cross-linked product is a [~-receptor subunit-43 kDa dimer (Burden et al1983). The same anti-43 kDa protein monoclonal antibody immunoprecipi-tates a Mr 43,000 protein that binds ATP and has protein kinase activity(Gordon et al 1983, A. S. Gordon, personal communication), so this mono-clonal antibody has made it possible to show rigorously that the Mr 43,000polypeptide associated with the acetylcholine receptor has protein kinasc activ-ity. Concentrated between the folds at the neuromuscular junction are 10 nMfilaments, almost certainly intermediate filaments. An amorphous, electron-dense material is between these filaments and the membrane. Recently, amonoclonal antibody has been described that binds an antigen, related anti-genically to intermediate filaments, that is concentrated in this amorphousmaterial (Burden 1982). Other antibodies have been used to localize to theneuromuscular junction antigens related to actin and three actin-binding pro-teins--ot-actinin, vinculin, and filamin (Bloch & Hall 1983).


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Classical CNS synapses have somewhat similar postsynaptic specializa-tions. Antibodies are not available to most of the receptors in the CNS. Devel-opment of monoclonal reagents to these receptors is likely to be one of themajor contributions of modern immunology in the next few years. Wherereceptor antibodies are available, such as to the [3-adrenergic receptor, theyhave localized the receptor to the postsynaptic density (Strader et al 1983).Microfilaments and microtubules have been visualized in the lattice of thepostsynaptic density (e.g. Matus 1981). Recently, monoclonal antibodies 13-tubulin, actin, and the high molecular weight microtubule-associated-pro-tein MAP2 have localized these molecules to the postsynaptic density (Cacereset al 1983). The major postsynaptic density protein, a Mr 50 kDa polypeptideof previously unknown function, has recently been shown, by using mono-clonal antibodies specific for this kinase (Kennedy et al 1983, Kelly et al1984), to be the 50 kDa subunit of a Ca2+-calmodulin-dependent proteinkinase. Immunocytochemistry with these antibodies shows that this subunit isnot localized exclusively to the postsynaptic density, but is found also inneuronal cell bodies, axons, and terminals in all or nearly all areas of the brain(McGuinness et al 1983). Conventional antisera have been used to demonstratethe presence in the postsynaptic density of a Mr 95,000 protein (Nieto-Sampedro et al 1982), fodrin (Carlin et al 1983), calmodulin (Wood et 1980), and calcineurin, which is the Ca2 +-calmodulin regulated protein phos-phatase 2B (Wood et al 1980). Monoclonal antibodies to these and otherantigens will be crucial for delineating the structure and composition of thisorganelle.

Extracellular Matrix Components

The extracellular matrix between the pre- and postsynaptic elements of theneuromuscular junction has become a subject of intense interest. Acetylcho-linesterase is inserted into this matrix and is important in removing acetyl-choline from the synapse. Acetylcholinesterase occurs in several molecularforms: soluble or membrane bound, intra- or extracellular, with or without acollagen-like tail (reviewed in Massoulie & Bon 1982). The occurrence forms with a collagen-like tail is correlated with innervation. With the objec-tive of understanding the relationship between the different forms of esterase,Fambrough et al (1982b) isolated five monoclonal antibodies to the plasma-membrane-associated acetylcholinesterase of purified erythrocytes. All of theseantibodies recognized an antigen in the neuromuscular junction; two of thesewere shown to bind to the form of esterase with a collagen-like tail. The resultsargue that there is a high degree of homology between different forms of theesterase.

It is now clear that components in the synaptic extracellular matrix induce


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



synaptic specializations in both nerve and muscle during regeneration (reviewedin Burden 1982). Conventional antibodies to anterior lens capsule, two col-lagen extracts, and a substantially purified factor that induces aggregation ofACh receptors in cultured myotubes have been shown to distinguish synapticand nonsynaptic regions of basal lamina (Sanes & Hall 1979, Nitkin et al1983). More recently, antibodies to the major components of basal laminahave been screened for binding specificity (Sanes 1982). Although collagenIV, laminin, and fibronectin are found in both synaptic and nonsynaptic regions,collagen V, appears to be excluded from synaptic regions. Monoclonal pro-cedures have been used to generate several antibodies directed to synapse-specific components of the basal lamina (Chiu & Sanes 1982, Fambrough etal 1982a). One antigen has been identified as a heparan sulfate proteoglycansynthesized by aneural muscle cells that is concentrated within the synapticcleft at the neuromuscular junction (Anderson & Fambrough 1983). This pro-teoglycan is unlikely to be related to the proteoglycan defined by monoclonalantibodies that is found in synaptic vesicles and on the surface of cholinergicnerve terminals in fish electric organ (Buckley et al 1983). These and othersynapse-specific antigens could be important in synapse function, structure,or development.

NERVOUS SYSTEM ANATOMY

Major Cell TypesOne important application that hybridoma technology has had in neurobiologyis the generation of probes that recognize different subsets of cells in thenervous system. These range from antibodies that distinguish neurons fromglial cells, and peripheral neurons from central neurons, to those which rec-ognize very small subsets of neurons. Antibodies to cell surface antigens areparticularly important for isolation of a cell type by positive or negative selec-tion (reviewed by Basch et al 1983). Positive selection procedures includeadhesion to antibody columns or magnetic beads (e.g. Meier et al 1982) andcell sorting (Dangl & Herzenberg 1982). Selenium- and iron-conjugated mon-oclonal antibodies can be used to select antigen-positive cells by growing cellmixtures in defined media that are missing these essential nutrients (Block Bothwell 1983). Antigen-positive cells can also be selected in hydrogen-per-oxide-containing media by using peroxidase-conjugated antibodies to protectthese cells from the deleterious effects of peroxide (Basch et al 1983). Negativeselection procedures include antibody-directed complement- or toxin-mediatedkilling of unwanted cell types (e.g. Brockes et al 1979, Vitetta et al 1983).

The major cell types in the peripheral and central nervous systems can nowbe distinguished using conventional antisera. Polyclonal antisera have beenproduced that distinguish astrocytes, Schwann cells, oligodendrocytes, fibro-blasts and neurons (see Table 1 for references). Monoclonal antibodies have


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Table 1 Major cell class markers

Cell and antigen Referencesa

NeuronsTetanus toxin 1Neurofilament proteins 2, 3, 4Neuron-specific enolase 5N-CAM 6A4 738/D7 8NILE glycoprotein 9BSP-2 10GQ ganglioside 11, 12Thy-1 13, 14OligodendrocytesGalactocerebroside 13, 1501-04 16

Schwann cellsRan-1 17, 13(GFAP)b 3(GAL-C)b 18

Cell and antigen Referencesa

~,strocytesGlial fibrillaryacidic protein (GFAP) 19, 20, 13S-100 19, 21, 22a2-Glycoprotein 23Non-neuronal enolase 24M1, C1 25, 26Ran-2 27Vimentin 2, 3, 28Stage specific embryonic

antigen (SSEA- 1) 28Glutamine synthetase 29(Thy- 1)~ 30(Tetanus toxin)b 13, 31(GQ ganglioside)b 31, 11, 14

Epithelial cells, fibro-blasts, ependymal cells

Vimentin 11, 12Fibronectin 13(Thy - 1 )~ 32(GFAP)~ 2

~1. Mirsky et al (1978). 2. Shaw et al (1981b). 3. Yen & Fields (1981). 4. Shaw et al (1981a). Marangos et al (1982). 6. Rutishauser et al (1982). 7. Cohen & Selvendran (1981). 8. Vulliamy et al (1981).9. Salton et al (1983). 10. Hirn et al (1981). 11. Berg & Schachner (1982). 12. Eisenbarth et al (1979).13. Raff et al (1979). 14. Mirsky (1982). 15. Ranscht et al (1982). 16. Sommer & Schachner (1981). Brockes et al (1977). 18. Mirsky et al (1980). 19. Ludwin et al (1976). 20. Bignami & Dahl (1977). Ghandour et al (1981). 22. Haan et al (1982). 23. Langley et al (1982). 24. Langley & Ghandour (1981).25. Lagenaur et al (1980). 26. Sommer et al (1981). 27. Bartlett et al (1981). 28. Solter & Knowles (1978).29. Norenberg & Martinez-Hernandez (197911. 30. Pruss (1979). 31. Raff et al (1983). 32. Brockes (1979).

b_( )- Shzred markers.

been made to some of these cell specific markers, and some new antigens havealso been discovered such as M1 and C1 for astrocytes (Lagenaur et al 1980).The astrocyte markers MI and C1 are expressed at different times duringdevelopment, with C1 appearing at embryonic day 10 in the mouse. Cell typespecific antibodies have been used to study a wide range of other questions,including the requirements for synthesis of myelin components by Schwanncells and oligodendrocytes (Mirsky et al 1980) and the effects of neurons glial cell differentiation (Holton & Weston 1982).

For the majority of cells, the markers shown in Table 1 are restricted intheir distribution. However, some markers are found on more than one celltype, although in some cases distribution of antigen may be altered by artifacts


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



of fixation (Ludwin et al 1976, Ghandour et al 1981). It has been shown thatsome Schwann cells may express glial fibrillary acidic protein (GFAP) (Yen& Fields 1981). Also, there is a population of astrocytes in white mattercultures with a neuron-like morphology that expresses two neuronal epitopes:the A2B5 antigen (GQ ganglioside) and tetanus toxin receptor (Raft et al 1983).These oligodendrocytes and neuron-like astrocytes are derived from a commonprecursor that is an A2Bs-positive, GFAP-negative cell. Culture conditionsdetermine which phenotypc is eventually expressed by this precursor (Raft Miller 1983). These precursor cells have been purified from embryonic opticnerve by using a fluorescence-activated cell sorter to positively select A2B5 +cells (Abney et al 1983). The purified cells develop into oligodendrocytes vitro in the absence of living neurons.

Neuronal Subpopulations

Conventional and monoclonal antibodies have been used in a wide variety ofexperiments to detect, categorize, and monitor the development of antigensthat distinguish one type of neuron from another. Antibodies that distinguishneuronal subpopulations fall into two classes: (a) antibodies to neurotrans-mitters, neuropeptides, or enzymes involved in their metabolism; (b) mono-clonal antibodies to random antigens identified by screening with immuno-cytochemical procedures supernatants from large numbers of hybridoma clones.

Neurotransmitters, Neuropeptides, and Transmitter Enzymes

The reagents most widely used to distinguish different neurons have beenantibodies to neurotransmitters. During the past decade, immunocytochemicalexamination has shown that more than 30 peptides are localized within specificneurons, coexisting in many cells with other transmitters (e.g. Hrkfelt et al1980, lversen 1983). Even larger numbers of peptides have been revealedwithin the genes for these, and tissue-specific RNA splicing and prohormoneprocessing have further expanded the numbers of possible peptide transmitters.Antibodies capable of distinguishing between related peptides should be par-ticularly valuable for future work. Recently, it has become possible to visualizeseveral classical transmitters directly with antibodies raised to serotonin, glu-tamic acid, and -/-amino butyric acid coupled to protein carriers (Cuello et al1982, Storm-Mathisen et al 1983, Seguela et al 1984). These classical trans-mitters have also been found to be localized within specific subpopulations ofneurons.

The number of available monoclonal antibodies to neurotransmitters, neu-ropeptides, and transmitter-related enzymes is expanding rapidly. Monoclonalantibodies are proving particularly useful in making reagents of defined spec-ificity available in large quantities to many laboratories. We discuss only afew examples of special interest.


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Monoclonal antibodies specific for substance P and serotonin have beenused in a number of creative applications of monoclonal technology. Theyhave been internally labelled by growth of the hybridomas in the presence of[3H]-lysine. These internally labelled antibodies have been used to localizeeach transmitter in nerve terminals by high resolution autoradiography (Cuelloet al 1982). Internally labelled antibodies offer a number of advantages inimmunocytochemistry. First, the antibodies are not chemically coupled andare fully active. Second, molecules as small as Fab fragments can be used topenetrate sections, so it is possible to use these antibody fragments with tissuesthat are more thoroughly fixed for good preservation of ultrastructure than ispossible with antibody conjugates. It is also possible to combine autoradiog-raphy and immunoperoxidase to detect two antigens simultaneously using theelectron microscope. These procedures have been used to investigate the coex-istence of two putative transmitters in individual neurons, e.g. substance Pand enkephalin-containing terminals in the substantia gelatinosa (Cuello et al1982), and substance P and serotonin in the nucleus raphe magnus of the rat(Cuello et al 1982).

Another new method utilizes bispecific antibodies in which one antigen-binding site is directed against a neurotransmitter and the other is directedagainst horseradish peroxidase (Milstein & Cuello 1983). These antibodiesare synthesized in cell lines derived from the fusion of transmitter-specific andperoxidase-specific hybridomas. Their use minimizes the size of reagents thatmust diffuse into tissue sections.

Monoclonal antibodies specific for several neurotransmitter enzymes havebeen isolated, including tyrosine hydroxylase, dopamine-13-hydroxylase, andcholine acetyltransferase (CAT) (Ross et al 1981a,b, Crawford et al 1982,Levey et al 1983, Eckenstein & Thoenen 1982). The generation of monoclonalantibodies to the latter enzyme has been particularly useful, since CAT isdifficult to purify and is not very immunogenic. Recently, monoclonal anti-bodies specific for CAT have been obtained using partially purified prepara-tions of bovine and rat CAT (Levey et al 1983, Crawford et al 1982, Eckenstein& Thoenen 1982). These antibodies immunoprecipitate CAT, bind to at leastthree non-overlapping sites on the enzyme, and bind cholinergic neurons insections of brain. Although some of the antibodies are species-specific, othersbind CAT in a wide variety of species. These antibodies provide the firstunambiguous markers for cholinergic neurons.

Other Antibodies that Define Neuronal SubclassesThe most successful strategy for isolating monoclonal antibodies that bindsubclasses of neurons has been to immunize mice with material from one partof the nervous system and screen the antibodies secreted by the hybridomason sections or cultures. Antibodies specific for peripheral and central neurons


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



have been identified (Vulliamy et al 1981, Cohen & Selvendran 1981). Anti-bodies to retinal membranes have been identified that distinguish photorecep-tors, other neurons, and Muller cells from each other (Barnstable 1980,Barnstable et al 1983, Trisler et al 1983). Antibodies to other regions of themammalian CNS distinguish many of the different neurons within these regions.Striking among these are a series of antibodies, originally isolated using Dro-sophila nervous tissue as an immunogen, that cross-react with the humannervous system (Miller & Benzer 1983). Some of these antibodies stain sub-populations of neurons and others stain particular areas within individual neu-rons. When analyzed in immunoblots, the Drosophila and human antigensoften are closely similar, suggesting an extraordinary conservation of antigenicepitopes (see also Ventner et al 1984). Another striking antibody, CAT 301,isolated using cat spinal cord as an immunogen, binds in the cerebellum onlyto a surface or extracellular matrix protein associated with the cells of Lugaro,a very rare cell type (McKay & Hockfield 1982, Hockfield & McKay 1983a).This antibody also binds elsewhere in the CNS, including pyramidal cells inthe hippocampus and a variety of cells in area 17 of the visual cortex. Thisantibody exhibits particularly intriguing binding specificity to cells in the vis-ual system of monkeys and cats (Hendry et al 1984). In the monkey lateralgeniculate nucleus, binding is primarily restricted to magnocellular, not par-vocellular, layers. In area 17, patches of stained cells are seen in layers III,IVB, and VI. These patches line up radially with each other and with thecenters of ocular dominance columns. The distribution of the CAT 301 anti-body suggests that it distinguishes cell groups with particular functions in thelateral geniculate nucleus and visual cortex. Conceivably, injection of thisantibody coupled to ricin or another cytotoxic agent could be used to kill thesecell populations in order to examine in more detail their roles in processingvisual information.

Specific glycolipids, defined by antibodies, have been found to be associatedwith subpopulations of neurons (Richardson et al 1982), most notably subsetsof sensory neurons (Dodd et al 1984). As these antigens are resistant to pro-teases, they should be particularly useful for separating neurons.

Neuron-specific Antigens in Invertebrates

Monoclonal antibodies to neural antigens in invertebrates, especially the leechand insects, have revealed a large degree of antigenic diversity among neurons(e.g. Zipser & McKay 1981). Many of the antibodies directed to antigens the leech nervous system define small groups of cells, for example subsets ofsensory neurons and motoneurons. The antibodies distinguish a very largenumber of different sets of neurons, some of which are nested within others(Hogg et al 1983). The antibodies have made it possible to identify all theneurons of a particular type in the leech nervous system and have revealed


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



relationships between neurons that were not previously recognized (Zipser1982, Zipser et al 1983). Examination of the position of axons stained byindividual antibodies has shown that they have consistent positions in con-nectives (Hockfield & McKay 1983b). In some cases, antigens mark axonsclustered into fascicles that are derived from scattered cell somata; in othercases, antigens mark clusters of cell somata whose axons are dispersed intodifferent fascicles. Thus, the factors important for aggregating cell somataand nerve bundles must be different.

Monoclonal antibodies directed against Drosophila and grasshopper neuraltissues also reveal a large diversity in the antigenic properties of differentneurons (e.g. Fujita et al 1982, Aceves-Pina et al 1982, Kotrla & Goodman1982). Antigens specific for the neurophil, cortex, and nerve fibers are readilydistinguished (Fujita et al 1982). Focusing on the compound eye, Benzer& colleagues (Fujita et al 1982) isolated antibodies that labelled the lens, theunderlying crystalline cone, the secretory cone cells, and the photorcceptors.Several of these were used to study the development of the compound eye(Zipursky et al 1984). The Mr’S of many of these antigens have been success-fully estimated by immunoblot analysis.

MONOCLONAL ANTIBODIES THAT PROBEDEVELOPMENT OF THE NERVOUS SYSTEM

Determination of Neural Crest Cells

Multiple cell types, including neurons and Schwann cells in the peripheralnervous system, are derived from the neural crest (reviewed in Le Douarin etal 1981). Transplantation experiments have shown that populations of crestcells can differentiate differently depending on the environmental stimuli thatthey encounter. An important issue, now being addressed using monoclonalantibodies, is whether cells in the crest are truly pluripotent or whether sub-populations with restricted fates are generated before crest cell migration. Amonoclonal antibody, termed NC-1, that binds to a cell surface antigen onavian neural crest cells has recently been isolated and used to examine neuralcrest cell migration (Vincent et al 1983). With this antibody, it is possible examine neural crest cell migration in normal animals. Previously, this requiredexperimental manipulation, usually transplantation. Non-neural derivatives ofthe neural crest, such as melanocytes and mesenchymal cells in the branchialarches, do not bind the NC-1 antibody, but do appear to be derived from NC-1-positive cells (Vincent & Thiery 1984), a result consistent with a pluripotentprecursor population of crest cells. A complementary monoclonal antibody,E/C8, that binds mesenchymal cells in the branchial arches, but not neuralcrest cell_s, has been isolated by immunizing with avian sensory neurons (Ciment& Weston 1982). It has been possible to show that E/C8-positive mesen-


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


chymal cells from the third and fourth branchial arches can develop intoneurons, but not melanocytes in vitro (Ciment & Weston 1983). They caninvade aneural gut to form enteric ganglionic neurons in organ culture, butare not competent to form melanocytes in developing embryos. It should bepossible to determine whether loss of the NC-1 antigen or acquisition of theE/C8 antigen is related to the loss by cells in the branchial arches of compe-tence to differentiate into melanocytes. The E/C8 antibody also binds neuronalcells in both the central and peripheral nervous systems. It binds to neuronalprecursors at an earlier stage than any other known neuronal marker. Forexample, it appears in the future sites of sensory ganglia a day before con-densation of these ganglia. It should consequently be very useful in studyingearly steps in neuronal commitment and differentiation.

Although the previous results suggest that neural crest cells are homoge-neous before migration, monoclonal antibodies specific for ciliary neuronshave been isolated that reveal heterogeneity in early neural crest cell cultures(Barald 1982). Two antibodies bind a small percentage of cultured neural crestcells provided the cells come from the region of crest that gives rise to theciliary ganglion. Two different antigenic determinants have been defined bythese antibodies, increasing the probability that they are related to cell lineage.This suggests that heterogeneity arises among crest cells at an early stage andresults in the appearance of cell types that are partially restricted in thei~rdevelopmental potential. Positive and negative selections using antibodies tosurface antigens that define neural crest subpopulations should be helpful forexploring crest cell lineages.

Neuronal Cell Adhesion Molecules (N-CAM and Ng-CAM)

Both Ca2+-dependent and Ca2+-independent adhesion systems have beendetected in embryonic chick neural tissues (Brackenbury et al 1981). N-CAM,a glycoprotein that mediates ISa2 +-independent adhesion between chick neuralcells, has been purified as a molecule that neutralizes antibodies, preparedagainst neuronal membranes, that inhibit neural cell adhesion (see Edelman1983). Conventional antibodies to this glycoprotein were used to demonstratethe presence of N-CAM on the surface of the somata and processes of neurons,but not on other cell types in the chick brain. The antibodies prevented theformation of axonal bundles in sensory neuron cultures and disrupted histo-genesis of developing retina in organ culture (Rutishauser et al 1978, Buskirket al 1980). Very recently, the injection of N-CAM antibodies into the devel-oping retina has been shown to disrupt the topological organization of retinalganglion cell axons in the optic nerve and to prevent some of these axons fromcontacting their normal target in the optic yectum (Thanos et al 1984). Theseresults argue that N-CAM is important in mediating interactions among neuralcells in development. Monoclonal antibodies to N-CAM have recently been


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



isolated (Rutishauser et al 1982). Using these antibodies it has become possibleto purify milligram quantities of N-CAM for biochemical investigations,including binding studies (Rutishauser ct al 1982), and chemical characteri-zation (Hoffman et al 1982). Using affinity-purified N-CAM, it has beenpossible to detect differences in the carbohydrate attached to embryonic andadult brain N-CAM molecules and show that these differences alter the adhe-sive properties of N-CAM (Rothbard et al 1982, Hoffman & Edelman 1983).Monoclonal antibodies with cross-species reactivity have also been used todetect and purify a very similar N-CAM from rodent brain (Chuong et al1982), and to show that mice homozygous for the neurological mutation stag-gerer do not synthesize the adult form of N-CAM in the cerebellum, the siteat which this mutation affects histogenesis (Edelman & Chuong 1982).

More recently, similar strategies using monoclonal antibodies have beenused to purify and characterize other molecules that mediate cell adhesion. Apartially purified tryptic fragment of a molecule that mediates neuronal adhe-sion to glia has been used to raise monoclonal antibodies that inhibit thebinding of neuronal membranes to glia (Grumet et al 1984). The monoclonalantibodies have been used to purify the intact, unproteolyzed form of thisglycoprotein, termed Ng-CAM, which differs from N-CAM in having a Mrof 135,000 and a different pattern of peptide fragments generated by pro-teolysis. Ng-CAM can be detected by immunofluorescence on neurons, butnot on glia. It is found on the same neurons as N-CAM. Despite the chemicaldifferences between N-CAM and Ng-CAM, some monoclonal antibodies bindboth glycoproteins, and the conserved determinants do not appear to be oncarbohydrate: This suggests that there is a shared protein domain on these twodifferent cell adhesion molecules. A trypsin fragment of a cell adhesion mol-ecule, liver-CAM (L-CAM), has been purified by the same strategy withmonoclonal antibodies (Gallin et al 1983). This Ca2+-dependent adhesionmolecule has a wide but specific distribution in both the embryo and adult(Edelman et al 1983, Thiery et all 1984). It appears to be lost from cells whenthey become committed to neuronal differentiation.

Neuronal Cell Migration

Studies with conventional antibodies have demonstrated strong spatial andtemporal correlations between the appearance of fibronectin and the migrationof granule cells to form the external granule cell layer in the cerebellum (Hattenet al 1982) and migration of neural crest cells to form the variety of tissuesderived from the crest (Thiery et al 1982a,b). When cerebellar granule cellsor neural crest cells are cultured in vitro, their abilities to adhere to and migrateon fibronectin substrata correlate temporally with their migratory behaviors invivo (Hatten et al 1982, Rovasio et al 1983). In addition, N-CAM appears an appropriate time in vivo to be important in condensation of the sympathetic


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



and sensory ganglia from migrating crest cells (Thiery et al 1982b). Migrationof cerebellar granule cells from the outer to inner granule cell layer can occurin vitro in brain slices. In a promising start in identifying molecules importantin this migration, Schachner and colleagues have shown that mono- and poly-clonal antibodies to L 1, a neuronal cell surface glycoprotein, reduce the move-ment of granule cells in cultured slices of developing cerebellum (Lindner etal 1983). The polyclonal, but not monoclonal, antibodies to L1 also block Ca2 +-independent aggregation of granule cells in vitro (Rathjen & Schachner1984). L1 antisera bind to antigens of three distinct Mr, each of which cor-responds in size to an antigen detected with antisera to Ng-CAM. If these twomolecules are in fact the same, Ng-CAM is likely to guide granule cell migration.

Axon Growth and Pathfinding

A remarkable monoclonal antibody, I5, has been used in a series of experi-ments on the grasshopper nervous system to study the mechanisms by whichaxons grow along stereotypic pathways to reach their targets. This antibodystains a specific subset of neurons, including pcripheral pioneer neurons (Ho& Goodman 1982). Since it stains all parts of these neurons, even before theinitiation of axon growth, it has been possible to use this antibody to studythe pathways and contacts made by pioneer fiber growth cones. The 15 anti-body stains an array of previously unseen pioneer neurons that lay downdifferent segments of the early fiber tracts. Identification of these cells hasmade it clear that pioneer neurons and other landmark cells are quite close toeach other during initial fiber growth--within the reach of filopodia in neigh-boring growth cones. Consequently, pathways may be constructed by sequen-tial movement of growth cones from one of these landmark cells to the next.Evidence for preferential filopodial adhesion to landmark cells has been obtainedby comparing at the electron microscope level the number of filopodia adheringto landmark vs other cells, using Lucifer Yellow to fill the filopodia of indi-vidual ncurons and an antibody to Lucifer Yellow to visualize the filled processes(Taghert et al 1982). The 15 monoclonal antibody also stains a previouslyunrecognized class of mesodermal cells that arise early in development (Hoet al 1983). Their pattern of growth and development, revealed by stainingwith the 15 antibody, indicates that they form a scaffold that guides the laterdevelopment of tendons, muscles, and nerves. The nature of the antigen definedby the 15 antibody is not yet known, but it is clearly associated with a set ofcells with important roles in development.

Studies in both vertebrates and invertebrates have shown that the growthcones of different neurons will make divergent choices at branch points (e.g.Raper et al 1983a,b). A monoclonal antibody has recently been isolated thatinhibits axon growth by avian retinal ganglion cells, but not sensory neurons(Henke-Fahle & Bonhoeffer 1983). The antigen defined by this.antiserum


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



could be one member of the family of molecules that guide these disparatedecisions.

Trophic Factors

Many factors have been described that regulate the expression of differentiatedfunctions in neurons. These include substances essential for survival, prolif-eration, transmitter choice, neurite growth, and steps in synapse formation.Immunological studies will clearly be critical in establishing their biologicalrelevance.

Nerve growth factor (NGF) is a protein that is essential for normal devel-opment of both sensory and sympathetic neurons. It is the one neurotrophicfactor, of many believed to exist, that has been purified, characterized, anddemonstrated to have a role in normal development in vivo. The presence ofNGF antibodies during development results in massive neuronal death in thesensory ganglia (Gorin & Johnson 1979). Injection of NGF antibodies at anytime destroys the sympathetic nervous system (Levi-Montalcini & Booker1960). Although mouse NGF acts on fish and avian neurons, antisera to NGFshow strong species specificity (e.g. Harper et al 1983). Recently, severalgroups have isolated monoclonal antibodies to NGF and most of these preventNGF binding to its receptor and NGF-dependent neurite outgrowth in vitro(.e.g. Warren et al 1980, Zimmermann et al 1981, H. Thoenen, personal com-munication). The monoclonal antibodies also appear to show strong speciesspecificity (H. Thoenen, personal communication). Recently, these antibodieshave been used in two-step, sandwich-type antigen assays to measure for thefirst time the endogenous levels of NGF in tissues innervated by sympatheticneurons (Korsching & Thoenen 1983a). The same assay has been used show that endogenous NGF is transported retrogradely from targets and isaccumulated in sympathetic ganglia and on the distal side of nerve ligatures(Korsching & Thoenen 1983a,b). Since NGF is present at only a few nano-grams per grams of tissue, it is very difficult to demonstrate that the antigenpresent in the tissue is NGF and not a cross-reacting antigen. Different mon-oclonal antibodies to NGF give similar results. If these antibodies can beshown to bind different epitopes on NGF, it will strengthen the argument thatNGF and not a cross-reactir~g antigen is being detected.

Glial growth factor (GGF), a mitogen for Schwann cells and astrocytes,was found in pituitary and brain extracts (Brockes et al 1980). Although was initially purified over 4000-fold by biochemical fractionation, the yieldwas very low. Monoclonal antibodies to GGF, prepared using a partially pur-ified factor as an immunogen, have been invaluable for isolating larger quan-tities of the factor (Brockes et al 1981) and should facilitate future studies its distribution and function. The monoclonal antibodies do not directly blockthe mitogenic activity of GGF, either because they do not bind a site essential


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



for activity or because their affinity is lower than that of GGF receptors. Toremove activity, GGF must be precipitated with a second antibody.

Markers of Cell Position

Topological arrangements are preserved in the pattern of synapses formedbetween groups of neurons in many parts of the nervous system and areimportant for conveying spatial information. When axons from ganglion cellsin the retina form synapses in the tectum, a point-to-point representation of thevisual field is conserved. Sperry (1963) postulated that selective chemoaffinitygenerated by two orthogonal gradients of molecules provided the molecularbasis for preserving this spatial order. In an effort to test this theory,Trisler et al (1981) have searched for antibodies that define cell position in theretina. A cell surface antigen, termed TOP, was identified that is distributedin a bilaterally symmetric dorsal-ventral gradient in the retina. At least 30-fold more antigen was found in dorsal than in ventral retina. The antigen wasfound on all cell types in fresh retinal dissociates. It appears in the optic cupsof 2 day-old chick embryos, and the gradient is established before day 4. Thusa gradient is established early in retinal development, during the period ofneuroblast proliferation, and is maintained after neurogenesis is completed.TOP has now been purified by affinity chromatography and shown to be a Mr47,000 cell surface glycoprotein (Trisler et al 1983). It remains to be deter-mined whether the molecule has an important role in generating spatial orderin the connections made by retinal ganglion cells with other parts of the brain.

A monoclonal antibody that recognizes a surface antigen whose expressionis position-dependent has also been identified by using a grasshopper mem-brane fraction as an immunogen (Kotrla & Goodman 1982). The antibody,termed epi- 1, stains an antigen on a broad strip of ectodermal epithelial cellsnear the midline in the blastula stage and a narrower strip of the same cells inthe grasshopper gastrula. In later development, the antigen is restricted to twoof the seven rows of neuroepithelial cells in each segment. The antigen is alsopositionally expressed on strips of cells near the distal end of each limb bud.In summary, screens for antibodies defining antigens that mark cell positionhave produced dramatic results in both vertebrate and invertebrate species andsuggest that monoclonal antibodies to such molecules will be very useful indevelopmental studies.

STRATEGIES FOR MAKING MONOCLONALANTIBODIES

In this section, we review aspects of particular interest to neuroscientists regardingthe methods for producing, screening, and productively using hybridomas andmonoclonal antibodies. Numerous books and reviews on monoclonal antibod-


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


ies have been published in the last few years (e.g. Kennett et al 1980, Hurrell1982).

Isolation of Specific Hybridoma Cell Lines

Detailed methods for producing monoclonal antibodies have been publishedelsewhere (e.g. Fazekas de St. Groth & Scheidegger 1980, Stahli et al 1980).Differences in procedures can result in selections of antibodies with differentallotypes and specificities. It has therefore proven well worth the effort toprepare the immunogen, choose the immunization protocol, and select a screen-ing procedure to optimize the probability of obtaining antibodies of thegreatest interest and utility for future studies.

Recent work has shown that the probability of obtaining hybridoma celllines is proportional to the strength of the immune response to a particularantigen (Lake et al 1979). Therefore, the first requirement for obtaining spe-cific monoclonal antibodies is to induce a strong response to the desired anti-gen. Since inbred mouse strains differ significantly in their responsiveness todefined antigens, screening a variety of strains has proven useful when testingfor an immune response. The original immunization protocols were optimizedfor injections of cells and membranes, and typically it was found that anoptimal time for fusion of spleen and myeloma cells was three to four daysafter a final i.v. injection (Lake et al 1979). It now appears that hybridomasspecific for soluble antigens are produced more efficiently by daily i.v. injec-tions for three to four days prior to fusion, probably because soluble antigensare rapidly removed from the circulation (Stahli et al 1980).

Different allotypes are preferable for different purposes. IgM antibodies areexcellent cytotoxic and agglutinating reagents, but are not as useful as IgGantibodies for many other purposes, including immunocytochemistry, whichis crucial for many applications in neuroscience. Specific protocols for pro-ducing a high frequency of hybridoma antibodies of a specific allotype havebeen published (e.g. Colwell et al 1982). Immunization protocols of sufficientlength to generate a high frequency of IgG-secreting hybridomas are usuallypreferable to short immunization regimens. Allotype-specific reagents can alsobe used in an initial screening to select for antibodies likely to be useful infuture applications.

A major goal of neuroscientists has been to obtain cell-type-specific anti-bodies. The most successful fusions seeking these reagents have used fixedneural tissues as immunogens and have screened fixed tissue sections (Zipser& McKay 1981, McKay & Hockfield 1982). This strategy (a) minimizes diffusion of antigens that can occur in unfixed tissue sections, (b) optimizesthe morphology of cells, which may be unrecognizable in unfixed tissues, and(c) increases the probability that antibodies can be used in ultrastructuralinvestigations. Since fixation can generate epitopes not present on unfixed


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



antigens, not all antibodies generated in this way will be useful, for bindingunfixed antigens, either on living cells or in immunoblots.

Interpretation of antibody binding patterns is almost always strengthenedby identifying the molecules bound in each tissue by a particular monoclonalantibody. The most common procedures for doing this assay involve antibodybinding to denatured antigens separated by size on polyacrylamide gels (e.g.Towbin et al 1979). To increase the probability of antibodies recognizing suchantigens, denatured antigens have been injected in one step of some immu-nization protocols (Zipser & McKay 1981, McKay & Hockfield 1982, Flasteret al 1983).

Antibodies that cross species efficiently are naturally more widely appli-cable than those that bind only the species from which a particular antigenwas derived. To increase the probability of obtaining such antibodies, neuralmaterial from more than one species has been used during the sequence ofimmunizations (e.g. Aceves-Pina et al 1982).

Another problem has been to identify monoclonal antibodies to minor epi-topes in an antigen mixture. Monoclonal antibodies to the dominant antigenshave been coupled to a resin and used to deplete an antigen mixture of pre-viously identified antigens (Springer 1981). In another approach, cyclophos-phamide, an alkylating agent known to kill antigen-stimulated lymphocytespreferentially, has been injected into previously immunized mice in an effortto kill lymphocytes specific for dominant antigenic determinants. Minor anti-gens have been recognized subsequently in surviving splenic lymphocytes thatwere challenged in vitro and fused to produce hybridomas (Matthew & Pat-terson 1983).

An often challenging problem has been to obtain a strong immune responseto a minute amount of a neuronal antigen or to a functionally important andconserved part of such a molecule to which the mouse may be effectivelytolerant. Microsequencing of natural proteins and chemical synthesis of mil-ligram quantities of peptide sequences in these proteins has been one suc-cessful approach to overcoming such problems (Hunkapiller & Hood 1983,Sutcliffe et al 1983a,b). Immunization protocols in which lymphocytes arechallenged with antigens in vitro provide another promising approach becausethey often require dramatically less antigen (picogram to nanogram) and canyield hybridoma lines derived from B lymphocytes that would be suppressedin vivo (e.g. Luben & Mohler 1980, Reading 1982, Pardue et al 1983). including ",/-interferon, it may prove possible in the future to obtain hybridomaclones in vivo and in vitro using even lower amounts of antigens (Nakamuraet al 1984). In vitro protocols also have potential problems. The in vitroresponses are often primitive compared to those in animals and a high pro-portion of the antibodies are IgM’s. Too much antigen can easily result inisolation of hybridomas secreting low affinity antibodies. Prolonged in vitro


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



culture of lymphoeytes with repeated challenges with low doses of antigenhas been reported to produce a higher frequency (80%) of IgG-allotype-secret-ing hybridoma lines (Pardue et al 1983).

Identification of Specific Hybridomas

The most common screening procedures for monoclonal antibodies use enzyme-linked immunosorbent assays (ELISA) in which antibody binding to antigensadsorbed to solid supports is measured. These procedures can be modified todetect fewer than 100,000 molecules of bound IgG, a very high level ofsensitivity (Shalev et al 1980). Many ELISA assays, though, require as muchas a microgram of bound antigen, because much of the antigen is not accessibleto antibody (Kennel 1982). As a result, whereas modern immunization pro-cedurcs have made it possible to obtain hybridoma-secreting antibodies to sub-nanogram quantities of an antigen, it may be impractical to assay for antibodiesbinding such antigens in standard ELISA assays. NGF, receptors, and ion-selective channels, present typically in nanomolar concentrations, are exam-ples of molecules that are too rare in most crude preparations to be detectedin routine ELISA assays.

One way of overcoming this problem has been to precede hybridoma selec-tion with a partial or complete purification of the molecule of interest. Anti-bodies to Nerve Growth Factor, the acetylcholine receptor, beta-adrenergicreceptor, sodium channel, and choline acetyltransferase have been isolated inthis way (Zimmermann et al 1981, Tzartos & Lindstrom 1980, Strader et al1983, Moore et al 1982, Crawford et al 1982). A second approach has beento use substrates that bind antigens more efficiently and in more access-ible forms than PVC plastic. Application of neuronal antigen mixtures tonitrocellulose provides an assay that requires about tenfold less antigen thanstandard solid phase assays (Hawkes et al 1982). Assays using a beta-galactosidase-immunoglobin conjugate and a fluorogenic substrate have increasedthe sensitivity of assays for NGF and several other proteins approximately100-fold (e.g. Korsching & Thoenen 1983a).

In many cases, solid phase immune assays have been replaced with a morespecific assay. The most specific assays have involved tests of function orenzymatic activity. Antibodies to choline acetyltransferase and glial growthfactor were identified by their abilities to precipitate each molecule (Crawfordet al 1982, Brockes et al 1980). Monoclonal antibodies to the Na+ channel,muscarinic acetylcholine receptor, and oq-adrenergic receptor were identifiedby their abilities to precipitate [:~H]-saxitoxin, [3H]-propylbenzilcholine mus-tard, and [3H]-phenoxybenzamine, respectively, from partially purified prep-arations (Moore et al 1982, Ventner et al 1984). These screens are mostsuccessful when dealing with a purified antigen.

The most sensitive assays for cell-type specific monoclonal antibodies have


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



utilized histological screens of sections of nervous tissues (e.g. Zipser McKay 1981, Hockfield & McKay 1981). In adequately preserved tissues,antibodies that bind specific subsets of cells and specific organelles can berecognized in sections in the light microscope. The most successful screensin sections of mammalian nervous systems have used frozen or Vibratomesections of fixed tissues (McKay & Hockfield 1982). Although time consum-ing, these screens are the only means of recognizing antibodies directed againstspecific subsets of neurons. Discussions of immunocytochemical procedurescan be found in several reviews and are beyond the scope of this article(Sternberger 1979, Jones & Hartman 1978, Vaughn et al 1981).

SUMMARY

The preceding discussion documents the diverse ways in which monoclonalantibodies have contributed to neuroscience research. They provide highlyspecific reagents to membrane-associated proteins, such as pumps, channels,receptors, and cell-adhesion molecules, that are useful for purifying theseproteins, studying their structures at high resolution, and mapping their dis-tributions. In many cases, the specific reagents were obtained using onlypartially purified antigens. Monoclonal antibodies to cytoskeletal proteins,organelles, and protein kinases have revealed that specific molecules are con-centrated in anatomically distinct regions of the cell. A protein kinase hasbeen shown to be a major postsynaptic constituent in many synapses. Indi-vidual proteins, such as actin, tubulin, and calmodulin appear to have differentantigenic epitopes shielded in different parts of the cell.

Monoclonal antibodies have provided a diversity of cell-type-specific reagentsin both vertebrate and invertebrate nervous systems. They seem likely to beuseful in identifying functionally related subpopulations of neurons and describingneural cell lineages. They will also serve to identify molecules that are impor-tant in regulating cell migration in the cerebellum, in marking cell position inthe retina, and directing axon growth.

This review also documents many purposes for which monoclonal antibod-ies are poorly suited or must be used with caution:

1. A monoclonal antibody to a protein does not always reveal every placewhere that molecule is located. Pre- or post-translational microhetero-geneity can expose different epitopes on the protein, such as may occur onthe Na +-channel. Other proteins within the cell may shield antigenic siteson proteins such as calmodulin.

2. Monoclonal antibodies can bind to epitopes on unrelated molecules (Nigget al 1982, Lane & Koprowski 1982). This is revealed in some cases asmultiple bands on immunoblots. Some cross-reactivity, however, may have


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



a functional basis. For example, structural homology is clearly the basisfor the antigenic epitopes that are shared among the five classes of inter-mediate filaments (Pruss et al 1981). The epitope that appears to be sharedbetween the muscarinic and oq-adrenergic receptors may be conservedbecause the two receptors modulate common effectors. The cross-reactivitybetween these receptors was only recognized because very specific andsensitive assays exist for each. It is quite possible that these same antibodiesalso bind sites on many other types of receptors. Mapping the distributionof this epitope may therefore have little relationship to the actual distri-bution of the muscarinic receptor. (Antibody binding to proteins in poly-acrylamide gels is probably not sensitive enough to detect these additionalreceptors.) If two antibodies to different epitopes on the same protein showthe same pattern of binding, it greatly strengthens the interpretation ofhistochemical data. Antibodies to different peptides in the same proteinhave been used for this type of analysis (Sutcliffe et al 1983a,b). With thissame objective, antigens identified by monoclonal antibodies on polyacry-lamide gels have been excised and used to generate additional monoclonalantibodies with the same binding specificity (Flaster et al 1983).

3. Many molecules in the nervous system can be anticipated to be detectedby immunocytochemistry, but not by other currently available procedures,

so it may not be possible to identify the antigens defined by many mono-clonal antibodies. Many other molecules of importance, such as the Ca2 +-

dependent K + channel and NGF, exist at such low concentrations that thebest current immunocytochemical procedures do not have the specificityand sensitivity needed to discern their distributions in the nervous system.

Literature CitedAbney, E. R., Williams, B. P., Raft, M. C.

1983. Tracing the development of oligoden-drocytes from precursor cells using mono-clonal antibodies, fluorescence-activated cellsorting and cell culture. Dev. Biol. 100:16671

Aceves-Pina, E., Barbel, S., Evans, L., Jan,Y. N., Jan, L. Y. 1982. Immunocytochem-ical studies of neural development in Dro-sophila. Soc. Neurosci. Abstr. 8:15

Anderson, D. J., Blobel, G., Tzartos, S., Gul-lick, W., Lindstrom, J. 1983. Transmem-brane orientation of an early biosynthetic formof acetylcholine receptor delta subunit deter-mined by proteolytic dissection in conjunc-tion with monoclonal antibodies. J. Neu-rosci. 3:1773-84

Anderson, M. J., Fambrough, D. M. 1983.Aggregates of acetylcholine receptors areassociated with plaques of a basal laminaheparan sulfate proteoglycan on the surfaceof skeletal muscle fibers. J. Cell Biol.97:1396-1411

Barald, K. E 1982. Monoclonal antibodies toembryonic neurons, cell-specific markers forchick ciliary ganglion. In Neural Development, ed. N. C. Spitzer, pp. 101-9. NewYork: Plenum

Barnstable, C. J. 1980. Monoclonal antibodieswhich recognize different cell types in therat retina. Nature 286:231- 35

Barnstable, C. J., Akagawa, K., Hofstein, R.,Horn, J. P. 1983. Monoclonal antibodies thatlabel discrete cell types in the mammaliannervous system. Cold Spring Harbor Symp.Quant. Biol. 48:863-76

Bartlett, P. E, Nobel, M. D., Pruss, R. M.,Raft, M. C., Rattray, S., Williams, C. A.1981. Rat neural antigen-2 (RAN-2): A cellsurface antigen on astrocytes, ependymalcells, Muller ceils and leptomeninges definedby a monoclonal antibody. Brain Res.204:339-51

Basch, R. S., Berman, J. W., Lakow, E. 1983.Cell separation using positive immunoselec-tion. J. lmmunol. Methods 56:269-80


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Berg, G. J., Schachner, M. 1982. Electronmicroscopic localization of A2B5 cell sur-face antigen in monolayer cultures of murinecerebellum and retina. Cell Tissue Res.224:637-45

Bignami, A., Dahl, D. 1977. Specificity of theglial fibrillary acidic protein for astroglia. J.Histochem. Cytochem. 25:466-99

Bloch, R. J., Hall, Z. W. 1983. Cytoskeletalcomponents of the vertebrate neuromuscularjunction: Vinculin, alpha-actinin and fila~nin.J. CellBiol. 97:217 23

Block, T., Bothwell, M. 1983. Use of iron- orselenium-coupled monoclonal antibodies tocell-surface antigens as a positive selectionsystem for cells. Nature 301:342-44

Bloom, G. S., Schoenfeld, T. A., Vallee, R.B. 1984. Widespread distribution of the majorpolypeptide component of MAP1 (Microtu-bule-associated protein) in the nervous sys-tem. J. Cell Biol. 98:320-30

Brackenbury, R., Rutishauser, U., Edelman,G. 1981. Distinct calcium independent andcalcium-dependent adhesion systems ofchicken embryo cells. Proc. Natl. Acad. Sci.USA 78:387-91

Brockes,J. P., Lemke, G., Balzer, D. R. Jr.1980.Purification and preliminary charac-terization of a glial growth factor frown thebovine pituitary. J. Biol. Chem. 255:8374-77

Brockes, J. P., Fields, K. L., Raff, M. C. 1977.A surface antigenic marker for rat Schwannceils. Nature 266:364-66

Brockes, J. P., Fields, K. L., Raft, M. C. 1979.Studies on cultured rat Schwann cells. I.Establishment of purified populations fromcultures of peripheral nerve. Brain Res.165:105-18

Brockes, J. P., Fryxell, K., Lernke, G. E. 1981.Studies on cultured Schwann cells--Theinduction of myelin synthesis and the controlof their proliferation by a new growth factor.In Glial-Neurone Interactions, ed. J. E.Treherne. London/New York: CambridgeUniv. Press

Buckley, K. M., Schweitzer, E. S., Miljanich,G. P., Clift-O’Grady, L., Kushner, P. D. etal. 1983. A synaptic vesicle antigen isrestricted to the junctional region of the pre-synaptic plasma membrane. Proc. Natl. Acad.Sci. USA 80:7342-46

Burden, S. J. 1982. Identification of an intra-cellular postsynaptic antigen at the frog neu-romuscular junction. J. Cell Biol. 94:521-30

Burden, S. J., DePalma, R. L., Gottesman, G.S. 1983. Crosslinking of proteins in acetyl-choline receptor-rich membranes: Associa-tion between the beta-subunit and the 43 kdsubsynaptic protein. Cell 35:687- 92

Buskirk, D. R., Thiery, J.-P., Rutishauser, U.,Edelman, G. M. 1980. Antibodies to a neuralcell adhesion molecule disrupt histogenesis

in cultured chick retinae. Nature 285:488-89

Caceres, A., Binder, L. I., Payne, M. R.,Bender, P., Rebhun, L., Steward, O. 1984.Differential subcellular localization of tub-ulin and the microtubule associated proteinMAP2 in brain tissue as revealed by immu-nocytochemistry. J. Neurosci. 4:394-410

Caceres, A., Payne, M. R., Binder, L. I.,Steward, O. 1983. Immunocytochemicallocalization of actin and microtubule-asso-ciated protein MAP2 in dendritic spines. Proc.Natl. Acad. Sci. USA 80:1738-42

Carlin, R. C., Bartelt, D. C., Siekevitz, P. 1983.Identification of fodrin as a major calmo-dulin binding protein in post synaptic densitypreparations. J. Cell Biol. 96:443-81

Chan, S. Y,, Hess, E. J., Rahaminoff, H., Gol-din, S. M. 1984. Purification and immuno-logical characterization of a calcium pumpfrom bovine brain synaptosomal vesicles. J.Neurosci. 4:1468-78

Chandler, C. E., Parsons, L. M., Hosang, M.,Shooter, E. M. 1984. A monoclonal anti-body modulates the interaction of nervegrowth factor with PCI2 cells. J. Biol. Chem.259:6882- 89

Chiu, A. Y., Sanes, J. R. 1982. Differentiationof basal lamina is an early event in the devel-opment of the neuromuscular junction in vivo.Soc. Neurosci. Abstr. 8:128

Chun, J. J. M., Schatz, C. J. 1983. Immuno-histochemical localization of synaptic vesi-cle antigens in developing cat cortex. Soc.Neurosci. Abstr. 9:692

Chuong, C.-M., McClain, D. A., Streit, P.,Edelman, G. M. 1982. Neural cell adhesionmolecules in rodent brain isolated by mon-oclonal antibodies with cross-species reac-tivity. Proc. Natl. Acad. Sci. USA 79:4234-38

Ciment, G., Weston, J. A. 1982. Early appear-ance in neural crest and crest-derived cellsof an antigenic determinant present in avianneurons. Dev. Biol. 93:355-67

Ciment, G., Weston, J. A. 1983. Enteric neu-rogenesis by neural crest-derived branchialarch mesenchymal cells. Nature 305:42427

Cohen, J., Selvendran, S. Y. 1981. A neuronalsurface antigen is found in the CNS but notin peripheral neurons. Nature 291:421- 23

Colwell, D. E., Gollahon, K. A., McGhec, J.R., Michalek, S. M. 1982. IgA hybridomas:A method for generation in high numbers.J. lmmunol. Methods 54:259-60

Conti-Troncini, B., Raftery, M. 1982. Thenicotinic cholinergic receptor. Ann. Rev.Biochem. 51:491-50

Conti-Troncini, B., Tzartos, S., Lindstrom, J.1981. Monoclonal antibodies as probes ofacetylcholine receptor structure. 2. Bindingto native receptor. Biochemistry 20:2181-91


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


226 VAEENTINO, WINTER & REICHARDT

Crawford, G. D., Correa, L., Salvaterra, P. M.1982. Interaction of monoclonal antibodieswith mammalian choline acetyltransferase.Proc. Natl. Acad. Sci. USA 79:7031-35

Cuello, A. C., Priestley, J. V., Milstein, C.1982.Immunocytochemistry with internallylabeled monoclonal antibodies. Proc. Natl.Acad. Sci. USA 79:665-69

Cummings, R., Burgoyne, R. D., Lytton, N.A. 1984. Immunocytochemical demonstra-tion of alpha-tubulin modification duringaxonal maturation in the cerebellar cortex.J, Cell Biol. 98:347-51

Dangl, J. L., Herzenberg, L. A. 1982. Selec-tion of hybridomas and hybridoma variantsusing the fluorescence activated cell sorter.J. lmmunol. Methods 52:1-14

Debus, E., Flugge, G., Weber, K., Osborn,M. 1982. A monoclonal antibody specificfor the 200K polypeptide of the neurofila-merit triplet. EMBO J. 1:41-45

Dodd, J., Solter, D., Jessel, T, M. 1984. Mon-oclonal antibodies against carbohydrate dif-ferentiation antigens identify subsets of pri-mary sensory neurones. Nature 311: 469-72

Eckenstein, E, Thoenen, H. T. 1982. Produc-tion of specific antisera and monoclonal anti-bodies to choline acetyltransferase: Charac-terization and use for identification ofcholinergic neurons. EMBO J. 1:363 - 68

Edelman, G. M. 1983. Cell adhesion mole-cules. Science 219:450-57

Edelman, G. M., Chuong, C.-M. 1982.Embryonic to adult conversion of neural celladhesion molecules in normal and staggerermice. Proc. Natl. Acad. Sci. USA 79:7036-40

Edelman; G. M., Gallin, W. J., Delouvee, A.,Cunningham, B. A., Thiery, J.-P. 1983. Earlyepochal maps of two different cell adhesionmolecules. Proc. Natl. Acad. Sci. USA80: 4384- 88

Eisenbarth, G. S., Walsh, E S., Nirenberg, M.1979. Monoclonal antibody to an plasmamembrane antigen of neurons. Proc. Natl.Acad. Sci. USA 76:4913-17

Ellisman, M. H., Levinson, S. R. 1982.Immunocytochemical localization of sodiumchannel distribution in the excitable mem-branes of Electrophorus electricus. Proc. Natl.Acad. Sci. USA 79:6707-11

Fambrough, D. M. 1983. Studies of the Na÷-K+ ATPase of skeletal muscle and nerve.Cold Spring Harbor Symp. Quant. Biol.48:297-304

Fambrough, D. M., Bayne, E. K., Gardner, J.M., Anderson, M. J., Wakshull, E., Rotundo,R. 1982a. Monoclonal antibodies to skeletalmuscle cell surface. Neuroimmunolog); ed.J. Brockes, pp. 49-89. New York: Plenum

Fambrough, D. M., Engel, A. G., Rosenberg,

T. L. 1982b. Acetylcholinesterase of humanerythrocytes and neuromuscular junctions:Homologies revealed by monoclonal anti-bodies. Proc. Natl. Acad. Sci. USA 79:1078-82

Fambrough, D. M., Bayne, E. K. 1983. Mul-tiple forms of (Na* +K+)-ATPase inchicken. Selective detection of the majornerve, skeletal muscle, and kidney form bya monoclonal antibody. J. Biol. Chem.258:1926- 35

Fazekas de St. Groth, S., Sheidegger, D. 1980.Production of monoclonal antibodies: Strat-egy and tactics. J. Immunol. Methods 35:1-21

Fertuck, H. C., Salpeter, M. M. 1976. Quan-titation of junctional and extrajunctional ace-tylcholine receptors by electron microscopeautoradiography after I25-alpha-bungaro-toxin binding at mouse neuromuscular junc-tions. J. Cell Biol. 69:114-58

Finer-Moore, J., Stroud, R. M. 1984. Amphi-pathic analysis and possible formation of theion channel in acetylcholine receptor. Proc.Natl. Acad. Sci. USA 81:155-59

Flaster, M. S., Schley, C., Zipser, B. 1983.Generating monoclonal antibodies againstexcised gel bands to correlate immunocyto-chemical and biochemical data. Brain Res.277:196- 99

Fritz, L. C., Brockes, J. P. 1983. Immunocy-tochemical properties and cytochemicallocalization of the voltage-sensitive sodiumchannel from the electroplax of the eel (Elec-trophorus electricus). J. Neurosci. 3:2300-9

Froehner, S. C., Gulbrandsen, V., Hyman, C.,Jeng, A. Y., Neubig, R. R., Cohen, J. B.1981. Immunofluorescence localization at themammalian neuromuscular junction of theMr 43,000 protein of Torpedo postsynapticmembranes. Proc. Natl. Acad. Sci. USA78:5230-34

Fujita, S. C., Zipursky, S. L., Benzer, S., Fer-rus, A., Shotwell, S. L. 1982. Monoclonalantibodies against the Drosophila nervoussystem. Proc. Natl. Acad. Sci. USA 79:7929-33

Gallin, W. J., Edelman, G. M., Cunningham,B. A. 1983. Characterization of L-CAM, amajor cell adhesion molecule from embry-onic liver cells. Proc. Natl. Acad. Sci. USA80:1038-42

Ghandour, M. S., Langley, O. K., Labour-dette, G., Vincedon, B., Gombos, G. 1981.Specific and artifactual cellular localizationsof Sl00 protein: An astrocyte marker in ratcerebellum. Dev. Neurosci. 4:68-78

Goelz, S. E., Nestler, E. J., Chehrazi, B.,Greengard, P. 1981. Distribution of ProteinI in mammalian brain as determined by adetergent-based radioimmune assay. Proc.


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Natl. Acad. Sci. USA 78:2130-34Goldin, S. M., Chan, S. Y., Papazian, D. M.,

Hess, E. J., Rahamimoff, H. 1983. Purifi-cation and characterization of ATP-depen-dent calcium pumps from synaptosomes. CoMSpring Harbor Symp. Quant. Biol. 48:287-96

Goldstein, M. E., Sternberger, L. A., Stern-berger, N. H. 1983. Microheterogeneity("Neurotypy’) of neurofilament proteins.Proc. Natl. Acad. Sci. USA 80:3101-5

Gomez, C. M., Richman, D. P., Berman, P.W., Burrcs, S. A., Arnason, B. G. A., Fitch,F. W. 1979. Monoclonal antibodies againstpurified nicotinic acetylcholine receptor.Biochem. Biophys. Res. Commun. 88:575-82

Gordon, A. S,, Millay, D., Diamond, I. 1983.Identification of a molecular weight 43,000protein kinase in acetylcholinc receptor-enriched membranes. Proc. Natl. Acad. Sci.USA 80:5862- 65

Gorin, P. D., Johnson, E M. Jr. 1979. Exper-imental autoimmune model of nerve growthfactor deprivation: Effect on developingperipheral sympathetic and sensory neurons.Proc. Natl. Acad. Sci. USA 76:5382-86

Grumet, M., Hoffman, S., Edelman, G. M.1984. Two antigenically related neuronal celladhesion molecules of different specificitiesmediate neuron-neuron and neuron-gliaadhesion. Proc. Natl. Acad. Sci. USA 81:267 -71

Gulliek, W. J., Lindstrom, J. M. 1983. Map-ping the binding of monoclonal antibodiesto the acetylcholine receptor from Torpedocalifornica. Biochemistry 22:3312-20

Gullick, W. J., Tzartos, S., Lindstrom, J. 1981.Monoclonal antibodies as probes of acetyl-choline receptor structure. I. Peptide map-ping. Biochemistry 20:2173- 80

Haan, E. A., Boss, B. D., Cowan, W. M. 1982.Production and characterization of mono-clonal antibodies against the "brain-spe-cific" proteins 14-3-2 and S-100. Proc. Natl.Acad. Sci. USA 79:7585 89

Harper, G. P., Barde, Y.-A., Edgar, D., Gan-ten, D., Hefti, E, et al. 1983. Biological andimmunological properties of the nerve growthfactor from bovine seminal plasma: Com-parison with the properties of mouse nervegrowth factor. Neuroscience 8:375- 87

Hatten, M. E., Furie, M. B., Rifkind, D. B,1982. Binding of developing mouse cere-bellar cells to fibronectin: a possible mech-anism for the formation of the external gran-ular layer. J. Neurosci. 2:1195-1206

Hawkes, R., Niday, E., Gordon, J. 1982. Adot-irnmunobinding assay for monoclonal andother antibodies. Analyt. Biochem. 119:142-47

Hawkes, R., Niday, E., Matus, A. 1982a. MIT-

23: A mitochondrial marker for terminalneuronal differentiation defined by a mono-clonal antibody. Cell 28:253-58

Hendry, S. H. C., Hockfield, S., Jones, E. G.,McKay, R. 1984. Monoclonal antibody thatidentifies subsets of neurones in the centralvisual system of monkey and cat. Nature307:267-69

Henke-Fahle, S., Bonhoeffer, E 1983. Inhi-bition of axonal growth by a monoclonalantibody. Nature 303:65- 67

Hirn, M., Pierres, M., Deagostini-Bazin, H.,Hirsch, M., Goridis, C. 1981. Monoclonalantibody against cell surface glycoprotein ofneurons. Brain Res. 214:433-39

Hirokawa, N., Glicksman, M. A., Willard, M.B. 1984. Organization of mammalian neu-rofilament polypeptides within the neuronalcytoskeleton. J. Cell Biol. 98:1523-36

Ho, R. K., Goodman, C. S. 1982. Peripheralpathways are pioneered by an array of centraland peripheral neurons in grasshopperembryos. Nature 297:404-6

Ho, R. K., Ball, E. E., Goodman, C. S. 1983.Muscle pioneers: Large mesodermal cells thaterect a scaffold for developing muscles andmotoneurones in grasshopper embryos.Nature 301:66- 69

Hockfield, S., McKay, R. 1983a. A surfaceantigen expressed by a subset of neurons inthe vertebrate central nervous system. Proc.Natl. Acad. Sci. USA 80:5758-61

Hockfield, S., McKay, R. 1983b. Monoclonalantibodies demonstrate the organization ofaxons in the leech. J. Neurosci. 3:369-75

Hoffman, S., Sorkin, B. C., White, P. C.,Brackenbury, R., Mailhammer, R., Rutis-hauser, U., Cunningham, B. A., Edelman,G. M. 1982. Chemical characterization of aneural cell adhesion molecule purified fromembryonic brain membranes. J. Biol. Chem.257:7720- 29

Hoffman, S., Edelman, G. M. 1983. Kineticsofhomophilic binding by embryonic and adultforms of the neural cell adhesion molecule.Proc. Natl. Acad. Sci. USA 80:5762 66

Hogg, N., Flaster, M., Zipser, B. 1983. Crossreactivities of monoclonal antibodies betweenselect leech neuronal and epithelial tissues.J. Neurosci. Res. 9:445-57

Hrkfelt, T., Johansson, O., Ljungdahl, A.,Lundberg, J. M., Schultzberg, M. 1980.Peptidergic neurons. Nature 284:515 - 21

Holton, B., Weston, J. A. 1982. Analysis ofglial cell differentiation in peripheral ner-vous tissue. II. Neurons promote SI00 syn-thesis by purified glial precursor cell popu-lations, Dev. Biol. 89:72-81

Huber, G., Matus, A. 1984. Differences in thecellular distributions of two microtubuleassociated proteins, MAP~ and MAP2, in ratbrain. J. Neurosci. 4:151- 60


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Hunkapiller, M. W., Hood, L. E. 1983. Proteinsequence analysis: Auto~nated inicro-sequencing. Science 219:650-59

Hurrell, J. G. R. 1982. Monoclonal Hybri-doma Antibodies: Techniques and Applica-tions. Boca Raton, Florida: CRC Press

Iversen, L. L., 1983. Neuropeptides--Whatnext? Trends Neurosci. 6:293 - 94

Jones, E. G., Hartman, B. K. 1978. Recentadvances in neuroanatomical methodology.Ann. Rev. Neurosci. 1:215-96

Jones, R. T., Walker, J. H., Stadler, H., Whit-taker, V. P. 1982. Further evidence that gly-cosaminoglycan specific to cholinergic ves-icles recycles during electrical stimulation ofthe electric organ of Torpedo marmarata. CellTissue Res. 224:685- 88

Kelly, P. T., McGuinness, T. L., Greengard,P. 1984. Evidence that the major postsyn-aptic density protein is a component of a Ca ÷calmodulin-dependent protein kinase. Proc.Natl. Acad. Sci. USA 81:945-49

Kennedy, M, B., Bennett, M. K., Erondu, N.E. 1983. Biochemical and immunochemicalevidence that the "major postsynaptic den-sity protein" is a subunit of a calmodulin-dependent protein kinase. Proc. Natl. Acad.Sci. USA 80:7357-61

Kennel, S. J. 1982. Binding of monoclonalantibody to protein antigen in fluid phase orbound to solid supports. J. hnmunol. Meth-ods 55:1-12

Kennett,R. H., McKearn, T. J., Bechtol, K.1980.Monoclonal Antibodies. New York:Plenum

Korsching, S., Thoenen, H. 1983a. NGF insympathetic ganglia and corresponding tar-get organs of the rat: Correlation with den-sity of sympathetic innervation. Proc. Natl.Acad. Sci. USA 80:3513-16

Korsching, S., Thoenen, H. 1983b. Quantita-tive demonstration of the retrograde axonaltransport of endogenous NGF. Neurosci. Lett.39:1-4

Kotrla, K., Goodman, C. S. 1982. Positionalexpression of a surface antigen on cells inthe neuroepithelium and appendages ofgrasshopper embryos. Soc. Neurosci. Abstr.8:256

Kristofferson, D., Desmeules, P., Lindstrom,J., Stroud, R. M. 1984. Acetylcbolinereceptor alpha subunits located usingmonoclonal antibody Fab fragments.(In preparation)

Lagenaur, C., Sonmaer, I., Schachner, M. 1980.Subclass of astroglia in mouse cerebella rec-ognized by monoclonal antibody. Dev. Biol.79:367-78

Lake, P., Clark, E. A., Khorshidi, M., Sun-shine, G. H. 1979. Production and charac-terization of cytotoxic Thy-1 antibody-secreting hybrid cell lines. Detection of Tcell subsets. Eur. J. Immunol. 9:875-86

Lane, D., Koprowski, H. t982. Molecular rec-ognition and the future of monoclonal anti-bodies. Nature 296:200-2

Langley, O. K., Ghandour, M. S.. 1981. Animmunocytochemical investigation of non-neuronal enolase in cerebellum: A new astro-cyte marker. Histochem. J. 13:137-48

Langley, O. K., Ghandour, M. S., Vincendon,G,, Gombos, G,, Warecka, K. 1982. Immu-noelectron microscopy of alpha2-glycopro-tein: An astrocyte-specific antigen. J. Neu-roimmunol. 2:131-43

Lazarides, E., Nelson, W. J. 1983a. Erythro-cyte and brain forms of spectrin in cerebel-lum: Distinct membrane cytoskeletal domains.Science 200:1295 - 96

Lazarides, E., Nelson, W. J. 1983b. Erythro-cyte form of spectrin in cerebellum: Appear-ance at a specific stage in the terminal dif-ferentiation of neurons. Science 222:931- 33

Lazarides, E., Nelson, W. J., Kasamatsu, T.1984. Segregation of two spectrin forms inthe chicken optic system: A mechanism forestablishing restricted membrane-cytoskele-tal domains in neurons. Cell 36:269-78

LeDouarin, N. 1982. The Neural Crest. Cam-bridge: Cambridge Univ. Press

Le Douarin, N. M., Smith, J., LeLievre, C.S. 1981. From the neural crest to the gangliaof the peripheral nervous system. Ann. Rev.Physiol. 43:653-71

Lee, V., Wu, H. L., Schlaepfer, W. W. 1982.Monoclonal antibodies recognize individualneurofilament triplet proteins. Proc. Natl.Acad. Sci. USA 79:6089-92

Levey, A. I., Armstrong, D. M., Atweh, S.E, Terry, R. D., Wainer, B. H. 1983. Mon-oclonal antibodies to choline acetyltransferase: Production, specificity, and immuno-histochemistry. J. Neurosci. 3:1- 9

Levi-Montalcini, R. M., Booker, B. 1960.Destruction of the sympathetic ganglia inmammals by an antiserum to nerve growthfactor. Proc. Natl. Acad. Sci. USA 46:384-91

Lindner, J., Rathjen, E, Schachner, M. 1983.L1 mono- and polyclonal antibodies modifycell migration in early postnatal mouse cer-ebellum. Nature 305:427- 30

Luben, R. A., Mohler, M. A. 1980. In vitroimmunization as an adjunct to the productionof hybridomas producing antibodies againstthe lymypbokine osteoclast activating factor.Mol. lmmunol. 17:635-39

Ludwin, S. K., I~ _sek, J. C., Eng, L. E 1976.The topographical distribution of S-100 andGFA protein in the adult rat brain: An immu-nohistochemical study using horseradishperoxidase-labelled antibodies. J. Comp.Neurol. 165:197- 208

McGuinness, T. L., Kelly, P. T., Ouimet, C.C., Greengard, P. 1983. Studies on the sub-cellular and regional distribution of calmo-


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


dulin-dependent protein kinase II in rat brain.Soc. Neurosci. Abstr. 9:1029

McKay, R. D. G, Hockfield, S. J. 1982. Mon-oclonal antibodies distinguish antigenicallydiscrete neuronal types in the vertebrate cen-tral nervous system. Proc. Natl. Acad. Sci.USA 79:6747-51

Marangos, P. J., Polak, J. M., Pearse, A. G.E. 1982. Neuron-specific enolase. A probefor neurons and neuroendocrine cells. TrendsNeurosci. 5:193 - 96

Massoulie, J., Bon, S. 1982. The molecularforms or cholinesterase and acetylcholines-terase in vertebrates. Ann. Rev. Neurosci.5:57-106

Matthew, W. D., Patterson, P. H. 1983. Theproduction of a monoclonal antibody whichblocks the action of a neurite outgrowth pro-moting factor. Cold Spring Harbor Syrnp.Quant. Biol. 48:625-31

Matthew, W. D., Reichardt, L. E 1982. Mon-oclonal antibodies used to study the inter-action of nerve and muscle cell lines. Prog.Brain Res. 58:375-81

Matthew,W. D., Tsavaler, L., Reichardt, L. F.1981. Identification of a synaptic vesicle-specific membrane protein with a wide dis-tribution in neuronal and neurosecretory tis-sue. J. Cell Biol. 91:257-69

Matus, A., 1981. The postsynaptic density.Trends Neurosci. 4:51- 53

Matus, A., Bernhardt, R., Hugh-Jones, T. 1981.High molecular weight microtubule-associ-ated proteins are preferentially associated withdendritic microtubules in brain. Proc. Natl.Acad. Sci. USA 78:3010-14

Meier, D. H., Lagenauer, C., Schachner, M.1982. Immunoselection of oligodendrocytesby magnetic beads. I. Determination of anti-body coupling parameters and cell bindingconditions. J. Neurosci. Res. 7:135-45

Miller, C. A., Benzer, S. 1983. Monoclonalantibody cross-reactions between Droso-phila and human brain. Proc. Natl. Acad.Sci. USA 80:7641-45

Miller, P., Walter, U., Theurkauf, W. E., Val-lee, R. B., DeCamilli, P. 1982. Frozen tis-sue sections as an experimental system toreveal specific binding sites for regulatorysubunit of type II cAMP-dependent proteinkinase in neurons. Proc. Natl. Acad. Sci.USA 79:5562- 66

Milstein, C., Cuello, A. C. 1983. Hybridhybridomas and their use in immunohisto-chemistry. Nature 305:537-40

Mirsky, R. 1982. The use of antibodies to defineand study major cell types in the central andperipheral nervous system. In Neuroimmu-nology, ed. J. Brockes, pp. 141-82. NewYork: Plenum

Mirsky, R., Wendon, L. M. B., Black, P.,Stolkin, C., Bray, D. 1978. Tetanus toxin:A cell surface marker for neurones in cul-

ture. Brain Res. 148:251-59Mirsky, R., Winter, J., Abney, E. R., Pruss,

R. M., Gavrilovic, J., Raft, M. C. 1980.Myelin-specific proteins and glycolipids inrat Schwann ceils and oligodendrocytes inculture. J. Cell Biol. 84:483-94

Mochley-Rosen, D., Fuchs, S., Estflmr, Z. 1979.Monoclonal antibodies against defined de-terminants of acetylcholine receptor. FEBSLett. 106:389-92

Moore, H.-P. H., Fritz, L. C., Raftery, M. A.,Brockes, J. P. 1982. Isolation and charac-terization of a monoclonal antibody againstthe saxitoxin-binding component from theelectric organ of the eel Electrophorus elec-tricus. Proc. Natl. Acad. Sci. USA 79:1673-77

Nakamura, M., Manser, T., Pearson, G. D. N.,Paley, M. J., Gefter, M, L. 1984. Effect ofIFN-gamma on the immune response in vivoand on gene expression in vitro. Nature306:381-82

Nakayama, H., Withy, R. M., Raftery, M. A.1982. Use of a monoclonal antibody to purifythe tetrodotoxin binding component from theelectroplax of Electrophorus electricus. Proc.Natl. Acad. Sci. USA 79:7575-79

Nestler, E. J., Greengard, P. 1983. Unpub-lished results

Nieto-Sampedro, M., Bussineau, C. M., Cot-man, C. W. 1981. Postsynaptic density anti-gens: Preparation and characterization of anantiserum against postsynaptic densities. J.Cell Biol. 90:675 - 86

Nigg, E. A., Walter, G., Singer, S. J. 1982.On the nature of crossreactions observed withantibodies directed to defined epitopes. Proc.Natl. Acad. Sci. USA 79:5939-43

Nitkin, R. M., Wallace, B. G., Spira, M. E.Godfrey, E. W., McMahan, U. J. 1983.Molecular components of the synaptic basallamina that direct differentiation of regen-erating neuromuscular junctions. Cold SpringHarbor Syrup. Quant. Biol. 48:653-65

Noda, M., Takahashi, H., Tanabe, T., Toyos-ato, M., Kikyotani, S., et al. 1983. Struc-tural homology of Torpedo californica ace-tylcholine receptor subunits. Nature 302:528-32

Norenberg, M. D., Martinez-Hernandez, A.1979. Fine structural localization of gluta-mine synthetase in astrocytes of rat brain.Brain Res. 161:303-10

Pardue, R. L., Brady, R. C., Perry, G. W.,Dedman, J. R. 1983. Production of mono-clonal antibodies against calmodulin by invitro immunization of spleen cells. J. CellBiol. 96:1149- 54

Pruss, R. 1979. Thy-1 antigen on astrocytes inlong term cultures of rat central nervous sys-tem. Nature 280:688-90

Pruss, R. M., Mirsky, R., Raff, M. C., Thorpe,R., Dowding, A. J., Anderton, B. H. 1981.


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



All classes of intermediate filaments share acommon antigenic determinant defined by amonoclonal antibody. Cell 27:419-28

Raft, M. C., Abney, E. R., Cohen, J., Lindsay,R., Noble, M. 1983. Two types of astrocytesin cultures of developing rat white matter:Differences in morphology, surface ganglio-sides and growth characteristics. J. Neu-rosci. 3:1289-1300

Raff, M. C., Fields, K. L., Hakamori, S., Mir-sky, R., Pruss, R. M., Winter, J. 1979. Cell-type-specific markers for distinguishing andstudying neurons and the major classes ofglial cells in culture. Brain Res. 174:283-308

Raft, M. C., Miller, R. H. 1983_ A glial pro-genitor cell that develops in vitro into anastrocyte or an oligodendrocyte dependingon the culture medium. Nature 303:390-96

Ranscht, B., Clapshaw, P. A., Price, J., Noble,M., Seifert, W. 1982. Development of oli-godendrocytes and Schwann cells studied witha monoclonal antibody against galactocere-broside. Proc. Natl. Acad. Sci. USA 79:270913

Raper, J. A., Bastiani, M., Goodman, C. S.1983a. Pathfinding by neuronal growth conesin grasshopper embryos. I. Divergent choicesmade by the growth cones of sibling neu-rons. J. Neurosci. 3:20-30

Raper, J. A., Bastiani, M., Goodman, C. S.1983b. Pathfinding by neuronal growth conesin grasshopper embryos. II. Selective fas-ciculation onto specific axonal pathways. J.Neurosci. 3:31-41

Rathjen, E G., Schachner, M. 1984. Mono-clonal antibody LI recognizes neuronal cellsurface glycoproteins mediating cellularadhesion. In Neuroimmunology, ed. P. O.Behan, E Spreafico, pp. 79 88. New York:Raven

Reading, C. L. 1982. Theory and methods forimmunization in culture and monoclonalantibody production. J. lmmunol. Methods53:261-91

Reichardt, L. E, Kelly, R. B. 1983. A molec-ular description of nerve terminal function.Ann. Rev. Biochem. 52:871-926

Richardson, P. J., Walker, J. H., Jones, R. T.,Whittaker, V. P. 1982. Identification of acholinergic-specific antigen Chol-1 as a gan-glioside. J. Neurochem. 38:1605 - 14

Richert, N. D., Willingham, M. C., Pastan, I.1983. Epidermal growth factor characteri-zation of a monoclonal antibody specific forthe receptor of A431 cells. J. Biol. Chetn.258:8902-7

Ross, M. E., Reis, D. J., Joh, T. H. 1981a.Monoclonal antibodies to tyrosine hydrox-ylase: Production and characterization. BrainRes. 208:493-98

Ross, M. E., Baetze, E. E., Rees, D. J., Joh,

T. J. 1981b. Monoclonal antibodies tocatecholamine - neurotransmitter - synthesiz-ing enzymes can be used for immunocyto-chemistry and immunohistochemistry. InMonoclonal Antibodies to Neural Antigens,ed. R. McKay, M. C. Raff, L. F. Reichardt,pp. 101 - 108. Cold Spring Harbor, NY: ColdSpring Harbor Lab.

Rothbard, J. B., Brackenbury, R., Cun-ningham, B. A., Edelman, G. M. 1982. Dif-ferences in the carbohydrate structures ofneural cell-adhesion molecules from adult andembryonic chicken brains. J. Biol. Chem.257:11064- 69

Rovasio, R. A., Delouvee, A., Yamada, K.M., Timpl, R., Thiery, J.-P. 1983. Neuralcrest migration: Requirements for exoge-nous fibronectin and high cell density. J. CellBiol. 96:462-73

Rutishauser, U., Gall, W. E., Edelman, G. M.1978. Adhesion among neural cells of thechick embryo. IV. Role, of the cell surfacemolecule CAM in the formation of neuritebundles in cultures of spinal ganglia. J. CellBiol. 79:382-93

Rutishauser, U., Hoffman, S., Edelman, G.M. 1982. Binding properties of a cell adhe-sion molecule from neural tissue. Proc. Natl.Acad. Sci. USA 79:685-89

Salton, S. R. J., Richter-Landsberg, C., Greene,L. A., Shelanski, M. J. 1983. Nerve growthfactor-inducible large external (NILE) gly-coprotein: Studies of a central and peripheralneuronal marker. J. Neurosci. 3:441-54

Sanes, J. R. 1982. Laminin, fibronectin andcollagen in synaptic and extrasynaptic por-tions of muscle fiber basement membrane.J. Cell Biol. 93:442-51

Sanes, J. R., Hall, Z. W. 1979. Antibodies thatbind specifically to synaptic sites on musclefiber basal lamina. J. Cell Biol. 83:357-70

Sealock, R. 1982. Visualization at the mouseneuromuscular junction of a submembranestructure in common with Torpedo postsyn-aptic membranes. J. Neurosci. 2:418-23

Sealock, R., Wray, B. E., Froehner, S. S. 1984.Ultrastructural localization of the Mr 43,000protein and the acetylcholine receptor in Tor-pedo postsynaptic membranes using mono-clonal antibodies. J. Cell Biol. 98:2239-44

Seguela, P., Gefford, M., Buijs, R. M., LeMoal,M. G. 1984. Antibodies against gamma-aminobutyrie acid. Specificity studies andimmunocytochemical results. Proc. Natl.Acad. Sci. USA 81:3888-92

Shalev, A., Greenberg, A. H., McAlpine, P.J. 1980. Detection of attograms of antigenby a high sensitivity enzyme-linked immu-noadsorbent assay (HS-ELISA) using a fluo-rogenic substrate. J. Immunol. Methods38:125-39

Sharp, G. A., Shaw, G., Weber, K. 1982.


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Immunoelectronmicroscopical localizationof the three neurofilament triplet proteinsalong neurofilaments of cultured dorsal rootganglion neurons. Exp. Cell Res. 137:403-13

Shaw, G., Osborne, M., Weber, K. 1981a.Arrangement of neurofilaments, microtu-bules and microfilament-associated proteinsin cultured dorsal root ganglia ceils. Eur. J.Cell Biol. 24:20-27

Shaw, G., Osborne, M., Weber, K. 1981b. Animmunofluorescence microscopical study ofthe neurofilament triplet proteins, vimentinand glial fibrillary acidic protein within theadult rat brain. Eur. J. Cell Biol. 26:68-82

Solter, D., Knowles, B. B. 1978. Monoclonalantibody defines a stage-specific mouseembryonic antigen (SSEA-1). Proc. Natl.Acad. Sci. USA 75:5565-69

Sommer, I., Lagenaur, C., Schachncr, M. 1981.Recognition of Bcrgmann glial and epen-dymal cells in the mouse nervous system bymonoclonal antibody. J. Cell Biol. 90:448-58

Sommer, I., Schachner, M. 1981. Monoclonalantibodies (01 to 04) to oligodendrocyte cellsurfaces: An immunocytological study in thecentral nervous system. Dev. Biol. 83:311-27

Sperry, R. W. 1963. Chemoaffinity in the orderlygrowth of nerve fiber patterns and connec-tions. Proc. Natl. Acad. Sci. USA 50:703-10

Springer, T. A. 1981. Monoclonal antibodyanalysis of complex biological systems:Combination of cell hybridization andimmunoadsorbents in a novel cascade pro-cedure and its application to the macrophagecell surface. J. Biol. Chem. 256:3833-39

Stahli, C., Staehelin, T., Miggiano, V., Schmidt,J., Haring, P. 1980. High frequencies ofantigen specific hybridomas: Dependence onimmunization parameters and prediction byspleen cell analysis. J. lmmunol. Methods32:297-304

Sternberger, L. A. 1979. lmmunocytochemis-try. New York: Wiley. 2nd ed.

Ster~berger, L. A., Sternberger, N. H. 1983.Monoclonal antibodies distinguish phospho-rylated and non-phosphorylated forms ofneurofilaments in situ. Proc. Natl. Acad. Sc;USA 80:6126-30

Storm-Mathisen, J., Leknes, A. K., Bore, A."1\, Vaaland, J. L., Edminson, P., Huag, M. S., Ottersen, O. P. 1983. First visuali-zation of glutamate and GABA in neuronsby immunocytochemistry. Nature 301:917-2O

Strader, C. D., Pickel, V. M., Joh, T. H.,Strohsacker, M. W., Shorr, R. G. L., et al.1983. Antibodies to the beta-adrenergicreceptor: Attenuation of catecholamine-sen-

sitive adenylate cyclase and demonstrationof postsynaptic receptor localization in brain.Proc. Natl. Acad. Sci. USA 80:1840-44

Sutcliffe, J. G., Milner, R. J., Shinnick, T. M.,Bloom, E E. 1983a. Identifying the proteinproducts of brain-specific genes using anti-bodies to chemically synthesized peptides.Cell 33:671- 82

Sutcliffe, J. G., Shinnick, T. M., Green, N.,Lerner,R. A. 1983b. Antibodies that reactwith predetermined sites on proteins. Sci-ence 219:660 - 65

Sweadner, K. J. 1979. Two molecular formsof (Na+ + K+)-stimulated ATPase in brain.J. Biol. Chem. 254:6060-87

Taghert, P. H., Bastiani, M. J., Ho, R. K.,Goodman, C. S. 1982. Guidance of pioneergrowth cones: Filopodial contacts and cou-pling revealed with an antibody to Luciferyellow. Dev. Biol. 94:391-99

Thanos,S., Bonhoeffer, E, Rutishauscr, U.1984.Fiber-fiber interaction and tectal cluesinfluence the development of the chickenretinotectal projection. Proc. Natl. Acad. Sci.USA 81:1906 10

Thiery, J.-P., DeLouvee, A., Gallin, W. J.,Cunningham, B. A., Edelman, G. M. 1984.Ontogenetic expression of cell adhesionmolecules: L-CAM is found in epitheliaderived from the three primary germ layers.Dev. Biol. 102:61-78

Thiery, J.-P., Duband, J. L., DeLouvee, A.1982a. Pathways and mechanisms of aviantrunk neural crest cell migration and locali-zation. Dev. Biol. 93:324-43

Thiery, J.-P., Duband, J.-L., Rutishauser, U.,Edelman, G. M. 1982b. Cell adhesion mol-ecules in early chick embryogenesis. Proc.Natl. Acad. Sci. USA 79:6737-41

Yowbin, H., Staehelin, T., Gordon, J. 1979.Electrophoretic transfer of proteins frompolyacrylamide gels to nitrocellulose sheets:Procedure and some applications. Proc. Natl.Acad. Sci. USA 76:4350-54

Yrisler, D., Grunwald, G. B., Moskal, J., Dar-veniza, P., Nirenberg, M. 1984. Moleculesthat identify cell type or position in the ret-ina. In Neuroimmunology and Neural Dis-ease, ed. P. Behan, E Spreafico, pp. 89-97. New York: Raven

Trisler, G. D., Schneider, M. D., Nirenberg,M. 1981. A topographic gradient of mole-cules in retina can be used to identify neuronposition. Proc. Natl. Acad. Sci. USA78:2145-49

Tzartos, S. J., Lindstrom, J. M. 1980. Mon-oclonal antibodies used to probe acetylcho-line receptor structure. Localization of themain immunogenic region and detection ofsimilarities between subunits. Proc. Natl.Acad. Sci. USA 77:755-59

Tzartos, S. J., Rand, D. E., Einarson, B. L.,


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.



Lindstrom, J. M. 1981. Mapping of surfacestructures of Electrophorus acetylcholinereceptor using monoclonal antibodies. J. Biol.Chem. 256:8635-45

Tzartos, S. J., Seybold, M. E., Lindstrom, J,M. 1982. Specificities of antibodies to ace-tylcholine receptors in sera from myastheniagravis patients measured by monoclonalantibodies. Proc. Natl. Acad. Sci. USA79:188 - 92

Vallee, R. B., Davis, S. B. 1983. Low molec-ular weight microtubule-associated proteinsare light chains of microtubule-associatedprotein (MAPI). Proc. Natl. Acad. Sci, USA80:1342- 46

Vaughn, J. E., Barber, R. P., Ribak, C. E.,Houser, C. R. 1981. Methods for the immu-nocytochemical localization of proteins andpeptides involved in neurotransmission. Curt.Trends Morphol. Tech. 3:33-70

Ventner, J. C., Eddy, B., Hall, L. M., Fraser,C. M. 1984. Monoelonal antibodies detectthe conservation of muscarinic cholinergicreceptor structure from Drosophila to humanbrain and detect possible structural homol-ogy with alpha~-adrenergic receptors. Proc.Natl. Acad. Sci. USA 81:272-76

Vitetta, E. S., Cushley, W., Uhr, J. W. 1983.Synergy of ricin A chain-containing immu-notoxins and ricin B chain-containing immu-notoxins in in vitro killing of neoplastic humanB cells. Proc. Natl. Acad. Sci. USA 80:6332-35

Vulliamy, T., Rattray, S., Mirsky, R. 1981. Cell-surface antigen distinguishes sensory andautonomic peripheral neurones from centralneurones. Nature 291:418-20

Warren, S. L., Fanger, M., Neet, K. E. 1980.Inhibition of biological activity of mouse-beta-nerve growth factor by monoclonalantibody. Science 210:910-12

Wehland, J., Willingham, M. C. 1983. A ratmonoclonal antibody reacting specificallywith the tyrosylated form of alpha-tubulin.II. Effects on cell movement, organization

of microtubules, and arrangement of Golgielements. J. Cell Biol. 97:1476-90

Wood, J. G., Jean, D. H., Whitaker, J. N.,McLaughlin, B. J., Albers, R. W. 1977.Immunocytochemical localization of the so-dium, potassium activated ATPase in knife-fish brain. J. Neurocytol. 6:571-81

Wood, J. G., Wallace, R. W., Whitaker, J. N.,Cheung, W. Y. 1980. Immunocytochemicallocalization of calmodulin and a heat-labilecalmodulin-binding protein (CaM-BP 80) basal ganglia of mouse brain. J. Cell Biol.84:66-76

Yen, S.-H., Fields, K. L. 1981. Antibodies toneurofilament, glial filament and fibroblastintermediate filament proteins bind to dif-ferent cell types in the nervous system. J.Cell Biol. 88:115-26

Young, E. E, Blake, J., Ramachandran, J.,Ralston, E., Hall, Z. W., Stroud, R. M. 1984.Localization of the C-terminus of the ace-tylcholine delta subunit on the membrane.Implications for the ion channel. Proc. Natl.Acad. Sci. USA. In press

Zimmermann, A., Sutter, A., Shooter, E. 1981.Monoclonal antibodies against nerve growthfactors and their effects on receptor bindingand biological activity. Proc. Natl. Acad. Sci.USA 78:4611-15

Zipser, B. 1982. Complete distribution pat-terns of neurons with characteristic antigensin the leech central nervous system. J. Neu-rosci. 2:1453-64

Zipser, B., McKay, R. 1981. Monoclonal anti-bodies distinguish identifiable neurons in theleech. Nature 289:549-54

Zipser, B., Stewart, R., Flanagan, T., Flaster,M., Macagno, E. R. 1983. Do monoclonalantibodies stain sets of functionally relatedlccch ncurons. Cold Spring Harbor Syrup.Quant. Biol. 48:551-56

Zipursky, S. L., Venkatesh, T. R., Teplow, D.B., Benzer, S. 1984. Neuronal developmentin the Drosophila retina: Monoclonal anti-bodies as molecular probes. Cell 36:15-26


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.


Ann

u. R

ev. N

euro

sci.

1985

.8:1

99-2

32. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

nive

rsity

of

Cal

ifor

nia

- Sa

n Fr

anci

sco

on 1

0/15

/06.

For

per

sona

l use

onl

y.

Applications of Monoclonal Antibodies to Neuroscience Research

Documents

Transcript of Applications of Monoclonal Antibodies to Neuroscience Research