314 Developmental Brain Research, 13 (1984) 314-318 Elsevier

BRD 60011

MAP2 is localized to the dendrites of hippocampal neurons which develop in culture

ALFREDO CACERES l,*, GARY BANKER 1, OSWALD STEWARD 2, LESTER BINDER 3 and MICHAEL PAYNE 4

1Department of Anatomy, Albany Medical College, Albany, NY 12208; 2Departments of Neurosurgery and Physiology, University of Virginia School of Medicine, Charlottesville, VA 22908; 3Department of Biology, University of Virginia, Charlottesville, VA 22903; and 4Department of Anatomy, New York Medical College, Valhalla, NY10595 (U.S.A.)

(Accepted December 20th, 1983)

Key words: microtubule-associated proteins - - cytoskeleton - - hippocampal neurons - - tissue culture - - dendrites - - neuronal development - - immunocytochemistry - - monoclonal antibodies

The distribution of the microtubule-associated protein MAP2 in cultured hippocampal neurons was studied using immunocyto- chemistry with monoclonal antibodies. MAP2 was preferentially localized to dendritic, but not axonal, processes even in single isola- ted cells which developed without making intercellular contacts. Hence regional differences in the molecular composition of the neu- ronal cytoskeleton can develop independently of cell interactions. The presence of MAP2 may be a useful marker for identifying den- drites in cell culture.

Neurons are unique among all cells of the body for

the remarkably complex shapes a t ta ined by their ax-

onai and dendri t ic processes. Neurona l shape is of

critical functional impor tance because it de termines

the synaptic connect ions which a cell can establish

and because it is inextr icably l inked with a selective

distr ibution of the molecular const i tuents of the cell

to part icular regions of its axon or dendri tes . The re-

gional localization of such const i tuents as neurotrans-

mit ter receptors and ion channels gives the nerve cell

a functional polar i ty and de termines the detai led

functional proper t ies which distinguish one type of

nerve cell from another .

Both intrinsic and extrinsic factors have been im-

plicated in determining the deta i led shape of neu-

rons 7A4.23.25. The informat ion present ly available

strongly suggests that the cytoskele ton is one of the

important endogenous de te rminants of cell

shapel4, 25. Recent ly it has been shown that the two

fundamental ly different classes of neuronal process-

es, axons and dendri tes , differ in the molecular com-

posit ion of their microtubules. Cer ta in of the high

molecular weight microtubule-associa ted prote ins

(MAPs) 20, including in par t icular MAP211,12,26, are

preferent ial ly associated with dendri t ic microtu-

bules. MAPs can control the rate of microtubule

polymerizat ion in vitro, and are thought to influence

the stabili ty of microtubules 22 and their interactions

with other e lements of the cytoskeleton9,13,18 in living

cells. Axons and dendri tes also differ in the molecu-

lar composi t ion of their neurofilaments15, 24.

We have chosen to study the cytoskeletal organiza-

tion of neurons in culture, where the distr ibution of

specific cytoskeletal const i tuents can be s tudied in in-

dividual cells and where the extent of cellular interac-

tions can be control led. It has been establ ished that

neurons from the rat h ippocampus e labora te process-

es in culture which can be dist inguished as axons or

dendri tes based on their light microscopic appear-

ance 3, their ul t ras t ructural features 4 and their synap-

tic polarityS. In the present s tudy we have used im-

munocytochemis t ry , to de te rmine the distr ibution of

MAP2 in h ippocampal neurons in culture in o rder to

answer the following questions. Does MAP2 become

preferent ial ly localized to the dendri t ic compar tment

of the cell even when the spatial and temporal pat-

* Permanent address: Instituto de Investigacion Medica Mercedes y Martin Ferreyra, Casilla de Correo 389, 5000 Cordoba, Argenti- na, Correspondence: G. Banker, Department of Anatomy, Albany Medical College, Albany, NY 12208, U.S.A.

0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.

tern of axonal and dendri t ic deve lopment is dis-

rupted, as it is when cells are dissociated and grown

in culture? Does innervat ion or interact ion with af-

ferent fibers play a role in de termining the selective

localization of MAP2?

Nerve cell suspensions were p repa red from the

h ippocampi of 17-19-day-old rat fetuses by trypsini-

zation and were p la ted onto polylys ine- t rea ted cov-

erslips at densit ies ranging from 1500 to 15,000

cells/cm 2, as descr ibed previously 2. The coverslips,

with neurons a t tached, were added to a l ready con-

fluent cultures of astroglial cells I and mainta ined in

Eagle ' s Minimum Essential Medium supplemented

with the N2 mixture of Bot tenste in and Sato ~0 and

0.1% ovalbumin. For immunocytochemis t ry the cul-

tures were fixed for 30 min in 4% formaldehy-

de -0 .1% glutara ldehyde in phospha te buffer, then

permeabi l ized with 50% ethanol for 20 min. In some

exper iments cytoskeletons were p repared by extract-

ing the cells with 0.2% Tri ton X-100 in a microtu-

bule-stabil izing buffer19 before fixation. Following

fixation the cells were incubated for 2 h at 37 °C with

monoclonal ant ibodies which reac ted specifically

with MAP2 (clones AP9, 0.39 mg/ml, or AP13, 0.40

mg/ml) or with fl-tubulin (clones Tu9B, 0.32 mg/ml or

Tu l2 , 0.37 mg/ml). The cultures were then incubated

with rabbi t ant i -mouse IgG (1:40) fol lowed by mouse

perox idase -an t ipe rox idase complex (1:40), then re-

acted with d iaminobenzid ine and H20 2 (ref. 11,12).

Controls t rea ted with normal mouse serum ra ther

than pr imary ant ibody, or p re t rea ted with excess pu-

rified antigen, showed little or no staining.

Al l of the ant ibodies used in this s tudy were pre-

pared against microtubules purif ied from Chinese

hamster brain, as descr ibed in detail elsewhere8,tH 2.

Each hybr idoma cell line was rec loned by limiting di-

lution 4 times. Ant ibodies were obta ined as culture

supernatants and purif ied by repea ted centrifuga-

tions to remove cellular debris; the anti- tubulin anti-

bodies were further purif ied by Protein A - S e p h a r o s e

affinity chromatography. The binding specificity of

each clone was examined by solid phase enzyme-

l inked immunoassay, and cross reactivi ty to o ther

proteins was tested by react ing the ant ibodies with

e lec t rophoret ic blots of rat h ippocampus or cerebel-

lum. In all cases the ant ibodies reac ted only with their corresponding antigens11.

Fig. 1 shows a h ippocampal culture p repa red at a

® 315

/

Fig 1. The distribution of MAP2 and tubulin in hippocampal neurons after development for 2 weeks in culture. The thicker. tapering processes which arise from these cells are dendrites (arrows). The dense plexus of finer processes consists almost entirely of axons which, because they are much longer than the dendrites, arise principally from cells outside the fields shown. MAP2 is present throughout the dendritic tree, but is not de- tectable in the axons (B). All of the neuronal processes, axons as well as dendrites, contain fl-tubulin (D). In these prepara- tions sister cultures were treated with Triton X-100 (0.2%) un- der conditions which stabilize microtubules but extract unpoly- merized subunits, then fixed in formaldehyde and glutaralde- hyde. They were exposed to monoclonal antibodies against either fl-tubulin (clone Tul2, diluted 1:15) or MAP2 (clone AP13, diluted 1:100) followed by rabbit-antimouse IgG and peroxidase-antiperoxidase, then reacted with diaminobenzi- dine. The diaminobenzidine reaction product also increases the density of the stained processes as seen by phase contract mi- croscopy; the size of individual axons and the density of the ax- onal plexus are in fact comparable in the two cultures. The bright-field photomicrographs (B and D) illustrate the pattern of staining whereas the phase contrast photomicrographs (A and C) show all of the processes, unstained as well as stained. Bar, 40 am.

high cell density and mainta ined for 2 weeks. The rel-

atively thick processes (arrows) arising from the cells

that t aper in d iameter with distance and branch in

316

Y-shaped angles are dendritic in form 2,~ and are ex-

clusively post-synaptic 5. The distal dendritic branches

are intermingled within a dense plexus of fine fibers

which have the light and electron microscopic fea-

tures of axons 4,5. lmmunocytochemistry using mono-

clonal antibodies against MAP2 produces heavy

staining of the dendritic processes, but no detectable

staining of the axonal plexus (Fig. 1A, B). Reaction

product is present even in the finest dendritic

branches so that for the first time the full extent of the

dendritic tree can be appreciated. The difference in

axonal and dendritic staining is not due to a differ-

ence in the density of microtubules in axons and den-

drites; electron microscopy of such cultures has

shown that the microtubule density in axons is equal

to or greater than that in dendrites 4,5. Neither is it be-

cause this method is insufficiently sensitive to detect

microtubule proteins in the somewhat thinner axons;

reaction with antibodies against fl-tubulin produces

uniformly intense staining of all axonal and dendritic

processes (Fig. 1C, D). It is likely that much of the

MAP2 present within the dendrites is bound to mi-

crotubules since identical staining patterns are seen

whether or not the cells are extracted with Triton

X-100 before fixation, a t reatment which removes

i

/ . ib,

/

soluble proteins but leaves polymerized cxloskeletat

elements largely intact.

To determine if the expression of MAP2 or ~ts cel-

lular localization was dependent on synaptogenesis.

or on other contact-mediated cellular interactions.

we studied the distribution of MAP2 in very low den-

sity hippocampal cultures. Under such culture condi-

tions, some of the cells develop arborizatious without

contacting any neighboring cells or their processes.

Fig. 2 shows an example of a portion of such an isola-

ted cell after development for ~ days m culture.

MAP2 is present within the cell body and the thick.

tapering dendrites, but cannot be detected in the axo-

nal processes which arise from this cell. When c{ma-

parable isolated cells were stained with antibody

against fl-tubulin all of their processes were uniform-

ly and darkly stained (data not shown).

The restriction of immunoreactivc MAP2 to the

somatodendritic compar tment of hippocampal neu-

rons which develop in culture exactly mirrors the lo-

calization of MAP2 in hippocampal pyramidal cells in

situ 1~. Previous work has demonstrated that brain

cells in culture exhibit immunoreactivity for MAP2 >,

but the axonal or dendritic nature ol the processes in

such cultures has not been established and a differen-

f

J

1

! "x

/

/ /

Fig. 2. The distribution of MAP2 in a single, isolated hippocampal neuron after development in culture for 6 days in the absence of any contacts with other cells. MAP2 is present in the dendrites, which at this stage are relatively short and unbranched. There is little or no staining of the axonal branches which arise from this cell (arrows). The photomicrograph illustrates all of the dendritic branches of this cell, but includes only about one-fourth of the axonal arborization. Because the axonal processes loop back on themselves and sometimes run along the dendrites, it is impossible to locate their origin(s). This preparation was fixed without extraction with deter- gent, permeabilized in 50% ethanol, then reacted with antibody (clone AP9, diluted l:l 5) as described above. Comparable cells reac- ted with anti-tubulin showed uniform staining of all processes, axons as well as dendrites. Bar. 50 am.

317

tial localization of MAP2 was not observed. It seems

probable from our results that MAP2 is absent or is

present in only small amounts in axons which develop

in culture, as has been shown by biochemical meth-

ods for axons in the brain 26. We cannot , however , ex-

clude the possibili ty that MAP2 is present in axons

but is unreact ive with the ant ibodies used in this

study, perhaps because interact ions with o ther mole-

cules block the antigenic sites. In ei ther case our re-

sults show that there are molecular differences be-

tween the microtubules present in the axonal and

dendri t ic processes which develop in culture.

These observat ions represent the first direct evi-

dence that the regional compar tmen ta t ion of nerve

cells into molecular ly distinct domains is an intrinsic

feature of neurons which can be expressed in the ab-

sence of innervat ion or any o ther cell-to-cell contact.

In situ the growing dendri tes of pyramida l neurons

are precisely aligned within a geometr ical ly-orga-

nized f ramework composed of neuronal and glial

processes. Because of this geometry , incoming affer-

ent fibers contact pyramida l cell dendri tes at precise-

ly-defined loci and at precisely def ined times. Clearly

none of these features is essential for the appropr ia te

compar tmenta t ion of MAP2 to occur.

It is impor tant to note that some h ippocampal neu-

rons have begun to extend processes at the stage they

are taken for culture 2. These processes are lost or re-

tract upon dissociation, but a residual cytoplasmic or-

ganization might persist that could influence process

outgrowth in culture 25. However this may be, our re-

sults show quite clearly that the growing axons and

dendri tes need not contact o ther cells for the localiza-

tion of MAP2 to occur appropr ia te ly .

These results also demons t ra te that immunocyto-

chemical staining for MAP2 can provide a conven-

ient means for identifying dendri tes in culture and for

revealing their arborizat ion pat tern. This has pre-

viously been achieved only by intracel lular injections

of dyes into individual cells 17,21. This new approach

based on immunocytochemis t ry may be of considera-

ble value for studies of dendri t ic deve lopment and

the control of dendri t ic form in culture.

Because MAPs , and MAP2 in par t icular , are be-

l ieved to influence the proper t ies of cellular microtu-

bules, and because they are present at early stages of

dendrit ic outgrowth (ref. 6 and this study), they

could play an impor tant role in regulat ing the pat tern

of growth which dendri tes undergo. MAPs could also

be involved in directing the intracel lular t ransport of

molecules, such as t ransmit ter receptors , which are

synthesized in the cell body and are specifically des-

tined for insertion into dendri t ic membranes . It

should now be possible to study the role MAPs play

in dendrit ic growth and in the es tabl ishment of neu-

ronal compar tmenta t ion using cell cultures such as

these.

This work was suppor ted by NIH Grants NS17112

(to G.B. ) and NS12333 (to O.S. ) and by Fogar ty In-

ternat ional Fel lowship FSTW02910A to A.C. who

was on leave from the D e pa r tme n t of Neurosurgery,

University of Virginia. We thank Ann Lohr and Lee

Snavely for technical assistance.

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