Neurotrophin receptors in the somatosensory cortex of the mature rat: co-localization of p75, trk, isoforms and c-neu

Ž .Brain Research 852 2000 355–366www.elsevier.comrlocaterbres

Research report

Neurotrophin receptors in the somatosensory cortex of the mature rat:co-localization of p75, trk, isoforms and c-neu

Michael W. Miller a,b,c,), Andrew F. Pitts a,b,d

a Research SerÕice, Veteran Affairs Medical Center, Iowa City, IA 52246-2208, USAb Department of Psychiatry, UniÕersity of Iowa College of Medicine, Iowa City, IA 52242-1000, USA

c Department of Pharmacology, UniÕersity of Iowa College of Medicine, Iowa City, IA 52242-1109, USAd ( )Psychiatry SerÕice 116A , Veterans Affairs Medical Center, Iowa City, IA 52246-2208, USA

Accepted 28 September 1999

Abstract

Trk immunoreactivity is expressed by a discrete population of cortical neurons, primarily those with cell bodies in layer Vb anddendrites in supragranular cortex. We tested the hypothesis that neurons co-express multiple isoforms of trk receptors. The distribution ofneurons expressing specific high affinity neurotrophin receptors was determined immunohistochemically. Multiple antibodies directedagainst each trk isoform and an antibody directed against an epitope shared by all three trk isoforms were used. The distribution ofneurons expressing each of the three receptors was virtually identical. Each anti-trk antibody primarily labeled neurons with cell bodies in

Ž .layer V. More than one-third of layer V neurons was positive for a high affinity trk receptor. Few immunoreactive somata 1%–5% werein the other layers. In addition, the neuropil in the supragranular laminae was immunopositive for each trk isoform. Recent data show thatlayer V neurons in the mature somatosensory cortex express the tyrosine kinase receptor c-erbB2, also known as c-neu. Immunofluores-cence double labeling shows that ;80% of the c-neu-immunolabeled neurons in layer V co-expressed pan-trk immunoreactivity andtwo-thirds of all c-neu-positive neurons expressed a specific trk isoform. We concluded from these data that there is significantco-expression of trk isoforms in layer V neurons. In summary, trkA, trkB, trkC, and c-neu were primarily expressed by corticalprojection neurons in layer V and co-expression among these receptors was common. This implies that cortical growth factor systems areredundant and that cortical neurons are responsive to more than one growth factor. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Autocrine; c-erbB2; Cerebral cortex; Glia; Local circuit neuron; Neurotrophin; Pyramidal neuron; White matter

1. Introduction

Neurotrophins mediate their growth promoting activitiesthrough high and low affinity receptors. The family of high

w xneurotrophin receptors includes trkA, trkB and trkC 17 .Each trk isoform is a 140-kDa tyrosine kinase. Binding aneurotrophin induces receptor autophosphorylation whichin turn initiates a complex signal transduction cascadew x16,17,28–32,45 . Each trk receptor has a preference for aparticular neurotrophin: trkA for nerve growth factorŽ . Ž .NGF , trkB for brain-derived neurotrophic factor BDNF ,

Ž . w xand trkC for neurotrophin-3 NT-3 28,29,31,32,37,60,62 .Various studies show that cerebral cortex richly expresses

) Corresponding author. Department of Psychiatry-M.E.B., Universityof Iowa College of Medicine, Iowa City, IA 52242-1000, USA. Fax:q1-319-353-3003; e-mail: [email protected]

high affinity receptors. Cortical tissue expresses not onlyw xthe protein of the high affinity receptors 53,69 , but also

w xtrk mRNA 23,35,65 .The low affinity receptor, p75, reportedly binds each

w xneurotrophin with equal affinity 56 . Some data, however,w xsuggest that p75 is more specific for NGF 11,58 or that

p75 may interact with trkA specifically to narrow itsw xspecificity 9,27,43 . Regardless, p75 usually forms a dimer

w xwith a high affinity receptor 2,3,19,43 .Immunohistochemical studies show that neurotrophin

receptors are commonly expressed in cerebral cortex byw xpost-synaptic profiles, somata and dendrites 53,69 . Anti-

bodies that recognize either p75 or an epitope shared bythe three trk isoforms, so-called pan-trk, consistently labelthe cell bodies of layer V pyramidal neurons and theneuropil in the supragranular laminae of motor–somato-sensory cortex. It is unclear how many of these pan-trk-

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 02176-9

( )M.W. Miller, A.F. PittsrBrain Research 852 2000 355–366356

positive neurons express a specific trk isoform and whetherneurons co-express two or more receptors.

Layer V neurons in the somatosensory cortex of maturerats express receptors in the epidermal growth factor recep-

Ž . w xtor EGFr family 15,36,68 . One of these receptors is thetranslation product of the c-neu proto-oncogene. This on-coprotein is a 185-kDa protein and can form heterodimers

w xwith EGFr 36,61 . Like trk, c-neu is a tyrosine kinase andit mediates activities that promote the survival and differ-

w xentiation of cortical neurons 49 .We hypothesize that neurotrophin receptors and c-neu

do not label discrete subpopulations of layer V neurons;rather that layer V neurons co-express multiple receptors.To test this hypothesis, we used double-labeling immuno-histochemistry and determined the incidence of growthfactor receptor co-expression.

2. Methods

2.1. Animals

ŽAdult, male Long–Evans rats Harlan–Sprague–Daw-.ley, Indianapolis, IN were used. Rats were anesthetized

Žwith a cocktail of ketamine and xylazine 60 and 7.5.mgrkg, respectively , and killed by transcardial perfusion

with 150–200 ml of 4.0% paraformaldehyde in 0.10 MŽ .phosphate buffer PB; pH 7.4 and then with 100–150 ml

of 10% sucrose in PB. The brains were removed from theskulls, immersed in 10% sucrose in PB for 4–5 h, and thenin a solution of 30% sucrose in PB for at least 3 days.After freezing, each brain was cut into a complete set of30-mm coronal sections.

2.2. Single label immunochemistry

2.2.1. Tissue processingSections from the brains of eight rats were processed by

an immunolabeling method routinely used in our labora-w xtory 46,48,53 . Free floating sections were incubated

overnight in a solution of 10% evaporated milk and 0.10%Ž .Tween 20 in phosphate-buffered saline TPBS at 48C to

minimize non-specific immunostaining. The tissue wasŽ .washed in TPBS containing 1.0% milk MTPBS and

incubated overnight at 48C in a solution of MTPBS withŽone of an assortment of primary antibodies described in

.Section 2.2.2 .An indirect immunolabeling method was used to visual-

ize the elements that bound an anti-neurotrophin receptorw xprimary antibody 21 . The sections were incubated in a

Žsolution of a biotinylated secondary antibody an anti-rab-bit antibody for the polyclonal antibodies and a rat-ad-sorbed anti-mouse antibody for the monoclonal antibodies;

.1:200 in MTPBS; Vector, Burlingame, CA for 8 h at 48C.

Subsequently, the sections were serially washed in anŽ .avidin-bound alkaline phosphatase solution Vector for 3

Žh at room temperature, a solution of levamisole 1:2500 in. ŽMTPBS , and a phosphatase substrate solution 5-bromo-

4-chloro-3-inoyl phosphate, nitroblue tetrazolium, magne-Ž .sium chloride, and levamisole 1:2500 in 0.10 M Tris

.buffer; pH 9.5 for 4 min. C-neu immunolabeling wasvisualized using a peroxidase reaction in the presence ofthe chromogen diaminobenzidine. The reacted sectionswere washed in distilled water, mounted on slides, dehy-drated, cleared and coverslipped.

A final series of sections was stained with Cresyl violet.These sections were used for cytoarchitectonic analyses.

2.2.2. Primary antibodiesOne antibody, referred to as pan-trk, was a rabbit

Žpolyclonal directed against an amino acid sequence re-.sidues 777–790 that was common to the C-terminals of

Ž . w xall three trk isoforms Oncogene, Manhasset, NY 38 .This antibody was used at a concentration of 1:250 inMTPBS.

Various antibodies against the specific trk isoformswere used. Two anti-trkA antibodies were used: both werepolyclonal antibodies generated in rabbits against residues

Ž .763–777 Santa Cruz Biotech., Santa Cruz, CA or againstŽthe extracellular domain Louis Reichardt, University of

. w xCalifornia, San Francisco, CA 9 . Neither antibodycross-reacted with trkB or trkC and the latter antibodyspecifically blocked NGF binding to trkA and induced trkautophosphorylation. The working concentrations for thetwo antibodies were 1:250 and 1:1000 in MTPBS, respec-tively.

Three anti-trkB antibodies were used. A rabbit poly-Žclonal generated against residues 794–808 a segment of

. w xthe tyrosine kinase sequence 38 was obtained from SantaCruz Biotech. Two rabbit polyclonal antibodies that recog-

Žnized full-length trkB were used Stuart Feinstein, Univer-sity of California, Santa Barbara, CA; David Kaplan,

. w xMcGill University, Montreal, Quebec, Canada 13,69 .The antibody from Santa Cruz Biotech. was used at adilution of 1:250 in MTPBS whereas those from Feinsteinand Kaplan were used at concentrations of 1:1000 and1:500 in MTPBS, respectively. None of the anti-trkBantibodies had demonstrable cross-reactivity with other trk

w xisoforms 13,69 .Two antibodies were used to identify trkC immuno-

reactivity. One anti-trkC antibody was generated in rabbitsŽagainst amino acid residues of 798–812 Santa Cruz

.Biotech. . The other was a rabbit polyclonal antibodyŽdirected against the intracellular domain of trkC David

.Kaplan . Neither antibody exhibited cross-reactivity withtrkA or trkB. Both antibodies were used at dilutions of1:500 in MTPBS.

Ž .The antibody directed against c-neu Ab-3 was a mo-Ž .noclonal antibody Oncogene that had no cross-reactivity

( )M.W. Miller, A.F. PittsrBrain Research 852 2000 355–366 357

w xwith EGFr 36,42,64 . The working dilution for this anti-body was 1:500 in MTPBS.

2.2.3. ControlsThree controls for the immunohistochemical reactions

were performed. In two of these controls, sections weretreated according to the methods described above exceptthat either the primary or secondary antibody was omitted.In a third control, the amount of endogenous staining wasassessed; both the primary and secondary antibodies wereeliminated from the processing.

2.2.4. AnalysesThe distribution of the immunolabeled cells in the

Ž .dorsal primary somatosensory cortex area 3 was exam-ined. Anatomical criteria were used to identify somatosen-

w xsory cortex 50 . The densities of immunolabeled andCresyl violet-stained cells were determined using a stereo-logical procedure that corrected for biases resulting from

w xcounting cell fragments as whole cells 59 . Quantitativedata were obtained and compiled with a Bioquant Image

Ž .Analysis System R&M Biometrics, Nashville, TN . Thequotient of the density of immunolabeled neurons and thedensity of Cresyl violet-stained neurons in a particularlayer was calculated as a laminar labeling ratio. Thenumber of neurons in two or three adjacent high power

Ž .fields per section was counted and the mean of at leastfive sections was used to generate a value for each animal.

2.3. Double-labeling fluorescence immunohistochemistry

2.3.1. Tissue processingSix animals were used in each of the double-labeling

studies. The procedure for the double-labeling was similarto the staining with a single probe except that the tissuewas sequentially processed through two batteries of mutu-ally exclusive immunolabeling.

The processing began by blocking non-specific activityin the free floating sections with an overnight wash in asolution of 10% milk in TPBS at 48C. Following a quickrinse in MTPBS, the sections were incubated with a pri-mary anti-rabbit antibody for 8 h at 48C. These antibodiesincluded pan-trk antibody and the anti-trkA, anti-trkB, andanti-trkC antibodies from Santa Cruz; diluted to 1:150,1:150, 1:150 and 1:100, respectively, in MTPBS. Afterthree washes with TPBS, the sections were incubated witha 1:200 dilution of the anti-c-neu antibody in MTPBS for 8h at 48C. The remaining steps in the sequence wereperformed in the dark. Sections were washed with PBSand the secondary antibodies were applied in sequence. A

Žbiotinylated, rat-adsorbed anti-mouse IgG Vector; diluted.1:100 in MTPBS was applied, followed by an anti-rabbit

Žsecondary antibody conjugated with Texas red Vector;.diluted 1:100 in MTPBS . The sections were rinsed three

times in a solution of 0.10 M bicarbonate buffer and 0.15Ž .M NaCl pH 8.2 and then incubated with streptavidin

Ž .fluorescein isothiocyanate FITC conjugate in bicarbonatebuffer for 8 h at 48C. Finally, the sections were mounted

Ž .with Vectashield Vector , coverslipped, and examinedŽ . Ž .immediately for Texas red trk and FITC c-neu fluores-

cence.Sections were also processed for p75–trk double label-

ing. The antibody used to identify p75-immunoreactiveŽneurons was an IgG mouse monoclonal antibody IgG-192;

.Oncogene . This antibody was previously used to showthat p75 was expressed by layer V neurons in rat motor

w xand somatosensory cortices 53 . For the double-labeling,the sections were initially incubated in the anti-p75 anti-body at a concentration of 1:100 in MTPBS, and subse-

Žquently, with one of the rabbit anti-trk isoforms Santa.Cruz Biotech. . The remaining steps were performed as

described above.

2.3.2. ControlsThree controls were used for the dual-label fluorescence

Ž .studies. 1 Sections were processed without either c-neuor one of the four anti-trk antibodies. In each of thesecontrols, the tissue was subsequently processed with bothsecondary antibodies. The sections were examined through

Ž .filters selective for the opposite fluorophores. 2 In thesecond control, the sections were incubated with bothprimary antibodies, but they were exposed to only one

Žsecondary antibody either the anti-mouse, rat adsorbed or. Ž .the anti-rabbit . 3 In some cases, the fluorophores were

swapped, i.e., the Texas red was used as a label for c-neuand FITC was used as a label for a trk antibody. Thiscontrolled for possible sensitivity differences between thesecondary antibody–fluorophore combinations.

2.3.3. AnalysesImmediately after mounting the sections, each sample

was examined with a Zeiss AxioSkop microscope fittedwith epifluorescence illumination to visualize Texas redand FITC labeling. The number of single- and double-labeled cells was determined by direct microscopic analy-ses. These counts were confirmed in photographic images.Five fields were examined on each of five sections. Amean representing each individual animal was calculated.The grand mean number of labeled cells for all animalsŽ Ž .."standard error of the means S.E.M. was then com-puted.

2.4. Statistical analyses

A one-way, repeated measures analysis of varianceŽ .ANOVA was used to test differences in the labelingindices. Analyses were for comparisons of the expressionof specific trk isoforms with c-neu, pan-trk and p75


expression. In situations in which the ANOVA identified aŽsignificant difference, post-hoc t-tests Bonferroni’s

.method were performed to examine specific variationsbetween pan-trk expression and the expression of a spe-cific trk receptor.

3. Results

3.1. Single label studies

3.1.1. Pan-trk and p75The pattern of neurotrophin receptor expression in pri-

mary somatosensory cortex was typified by pan-trk- andp75-immunostaining. Immunostaining was most evident inthe somata of layer V pyramidal neurons and in theneuropil of the supragranular laminae. Weak immuno-staining was also evident within the neuropil of layer V.

Ž .Most 84.4% and 69.1% of the pan-trk- and p75-positivecells were in layer V. The distributions of pan-trk- and

Ž w x.p75-immunopositive somata detailed in Ref. 53 aredescribed in Fig. 1. The labeling frequency in each layerfor pan-trk-immunolabeled cells was not statistically dif-ferent from that for the p75-positive cells.

3.1.2. TrkAThe immunostaining with the anti-trk isoform antibod-

ies was consistent with the pan-trk labeling. In specific,trkA-positive cells were mostly distributed in a tangential

Ž .band through layer V of somatosensory cortex Fig. 2B .In fact, this band was evident throughout the cortical

Žmantle, from cingulate to perirhinal cortex data not. Žshown . The trkA-labeled cells boasted large somata Fig.

.3A ; the mean somatic diameter was 16.9"2.0 mm. TheseŽcell bodies were among the largest of the layer V cells cf.,

.cells stained with Cresyl violet . Furthermore, each cellbody had a pronounced process arising from its apex; insome cases, an apical process could be traced into layer I.Based on these features, it appeared that trkA-expressingcells were pyramidal neurons.

The labeling frequency of trkA-positive somata wasŽ .determined for each cortical layer Fig. 1 . More than

three-fourths of all labeled neurons were in layer V, andgreater than two-thirds of the layer V neurons were inlayer Vb. No significant differences between the immuno-labeling obtained with the two anti-trkA antibodies weredetected.

In addition to layer V, trkA immunostaining was evi-dent in a dense zone within the neuropil of layers I andIIrIII, a less intensely stained neuropil of layer V, and asmall number of neuronal cell bodies in layers IIrIII and

Ž .VI Fig. 2B . This pattern mirrors the dendritic arboriza-tion of layer V pyramidal neurons. In fact, a previousstudy of pan-trk immunostaining showed that neuropil

w xlabel was largely labeled dendritic profiles 53 . The sub-cortical white matter was noticeably unlabeled, except for

Fig. 1. Laminar distribution of receptor-expressing somata. The series ofhistograms describes the labeling frequency of receptor-positive neurons

Ž .in each cortical sub layer. The labeling frequency was calculated as thepercentage of the number of immunoreactive neurons relative to thenumber of Cresyl violet-stained neurons. Each bar represents the mean ofeight animals and the T-bars signify the S.E.M. The vast majority ofreceptor-positive neurons were in layer V. Multiple antibodies were usedto examine the distribution of trk isoform-immunolabeled neurons. Nosignificant differences were detected among the similarly directed anti-bodies.

a small number of scattered puncta and the rare glial orŽ .endothelial cell body Fig. 4A .

3.1.3. TrkBAs with trkA, trkB was expressed principally by layer

Ž .V neurons Figs. 1 and 2C . Whereas fewer than 5% of theneurons in layers I–IV and VI were trkB-positive, one-fourth to one-third of all layer V neurons were labeledwith an anti-trkB antibody. It is important to note that thesame immunostaining pattern was evident with each of thethree anti-trkB antibodies; no statistically significant dif-ferences were detected.

The trkB-positive neurons were rather homogeneousŽ .Fig. 3B . For example, the mean maximal diameter of theimmunolabeled neurons in layer Vb was 16.5"1.8 mm.This compares to the mean of 15.4"3.2 mm for thesomata of Cresyl violet-stained layer Vb neurons. Theslight difference in somatic size may reflect the contribu-


Ž . Ž .Fig. 2. Receptor immunolabeling in cortex. The lamination of rat somatosensory cortex was identified in Cresyl violet-stained tissue A . TrkA- B , trkB-Ž . Ž . Ž .C , trkC- D , and c-neu- E positive elements were labeled with antibodies from Santa Cruz Biotech. The labeling pattern for the various receptors wasremarkably similar; somata in layer V and the supragranular neuropil were immunolabeled. Roman numerals denote the laminae and sublaminae in cortex.Scale bars are 100 mm.

tion of the local circuit neurons which are smaller andfewer than the pyramidal neurons. Note that the variation

for the size of the trkB-labeled neurons was considerablysmaller than it was for the Cresyl violet-stained neurons,


Ž .Fig. 3. Receptor immunolabeling in layer V. Large neurons in layer V were immunolabeled with specific anti-trk isoform antibodies, trkA-SC A ,Ž . Ž . Ž .trkB-SC B and trkC-SC C , and with an anti-c-neu antibody D . Scale bars are 50 mm.

further supporting the notion that the trkB-positive neu-rons was a homogeneous population.

Although there was no notable glial immunolabeling inthe gray or white matter, there was conspicuous fibrous


Ž . Ž . Ž . Ž .Fig. 4. Receptor immunolabeling in the subcortical white matter. Elements in the white matter expressed trkA- A , trkB- B , trkC- C , and c-neu- DŽ . Žimmunoreactivity. In general, these were punctate profiles arrowheads , however, trkB-positive elements were identified as parallel arrays of axons solid

. Ž .arrows . Occasionally, immunoreactive glial cell bodies open arrows were evident. Scale bars are 50 mm.

Ž .trkB-staining in the white matter Fig. 4B . Most of thefibers formed arrays of tangentially oriented fibers. Since

glial staining was rare, it is likely that these immunola-beled profiles were axons rather than internodes of myelin.


3.1.4. TrkCAntibodies directed against trkC generated the weakest

Ž .labeling on a per cell basis of all the anti-trk isoformŽ .antibodies used in the present study Fig. 2D, Fig. 3C .

Nevertheless, a consistent labeling pattern emerged withboth anti-trkC antibodies. Most trkC expressing neuronsŽ73.9"4.3% for the trkC-K and 76.2"3.3% for the

. Ž .trkC-SC were in layer V Fig. 1 . The white matterŽ .contained few trkC-expressing profiles Fig. 4C . These

included scattered puncta and isolated glial cell bodies.

3.1.5. C-neuMany c-neu-positive neurons were characterized by

Ž .Golgi-like labeling Fig. 2E, Fig. 3D . The most com-monly labeled cell type was pyramidal neurons in layer V.These neurons had prominent apical processes that couldoften be traced into layer I and basal dendrites that branchedwithin layer V. In addition, the supragranular neuropil, theanti-c-neu antibody labeled the somata of a few layerIIrIII pyramidal neurons, and a small number of somata oflocal circuit neurons, e.g., a bitufted or bipolar neuron, inlayers II–VI. The relative laminar and cell type hetero-

Žgeneity of the c-neu-positive cells in comparison to the.trk-expressing neurons is reflected by the lower frequency

of c-neu-positive cells in layer V; of the c-neu-labeled cell

bodies distributed throughout the cortical laminae, only53.3"5.2% were in layer V. This labeling frequency was

Ž . Ž .significantly p-0.05 less two-thirds than the labelingwith any of the antibodies directed against a specific trk

Ž .isoform p-0.05 . Virtually no c-neu immunoreactivityŽ .was evident in the white matter Fig. 4D .

3.1.6. ControlsNegative results were generated in each of the three

control studies. No labeling was obtained in preparationsin which the primary, secondary, or both primary andsecondary antibodies were eliminated. Thus, the labelingwith each anti-receptor antibody was specific.

3.2. Double label immunofluorescence

Many neurons expressed both the low and high affinityneurotrophin receptors. About two-thirds of the layer Vneurons expressing either p75 or a trk isoform co-ex-

Ž .pressed both Figs. 5 and 6, upper panel . Similarly, nearlyall of the layer V neurons that expressed c-neu-immuno-

Žreactivity also expressed pan-trk-immunoreactivity Fig. 6,.lower panel . The incidence of co-expression c-neu with a

particular trk isoform was lower than it was with c-neuand pan-trk. Such data were not surprising since the

Ž . Ž .Fig. 5. P75–trkA double labeling. Neurons in layer V were p75- left and trkA-positive right . Arrows indicate neurons that are p75–trkAdouble-labeled. Scale bars are 50 mm.


Fig. 6. Incidence of receptor double-labeling in layer V. The frequency ofdouble-labeling was determined by counting all neurons in a field thatwere immunolabeled with one or two anti-receptor antibodies. The label-ing indices were calculated as the percentage of the numbers of neuronsthat expressed only one or both pairs of receptors out of the total numberof immunolabeled neurons. Each triad of bars totals 100%. Bars signifythe means of six animals and T-bars represent the S.E.M.

pan-trk antibody recognizes a domain shared by all threetrk isoforms.

3.2.1. ControlsThe results of the three controls for the dual labeling

Ž .studies were negative. 1 Sections processed with only asingle primary antibody resulted in single labeling only.That is, when only the anti-c-neu antibody was usedŽ .FITC , there was no Texas red immunofluorescence.Likewise, when only an anti-trk primary antibody was

Ž .used Texas red , FITC labeling was negative. These sec-tions also verified that there was no cross-fluorescence

Ž .between filters. 2 For sections processed with only asingle secondary antibody, only the fluorescence associ-

Ž .ated with that antibody was detected. 3 After the fluoro-phores were swapped, the amount of cell labeling wasunchanged.

4. Discussion

4.1. Receptor expression

Cortex expresses various neurotrophin receptors, includ-ing p75, three trk isoforms, and c-neu. The present studyshows that neurotrophin receptors are expressed by post-

Ž .synaptic profiles cell bodies and dendrites . This patternof trk isoform expression is remarkably consistent amongthe various receptors. The consistency of the data is furtherbolstered by generating similar data with multiple antibod-ies against each trk isoform.

Data from the present study agree and conflict withthose from previously published reports. The present studybuilds on previous investigations of p75 and pan-trk ex-

w xpression in rat cortex 53 by showing the expressionpatterns for the three trk receptors is identical. Such dataconcur with immunohistochemical studies of trkB expres-

w xsion in mature cortex 55,69 . In situ hybridization studiesshow that cortical neurons in the rat express the mRNA for

w xtrkA and the other isoforms 23,35,65 . Furthermore, thedistributions of p75- and pan-trk-immunoreactivity in the

w xrat directly parallel the patterns for the macaque 52 .The distribution of c-neu-positive neurons is strikingly

similar to that for trk expressing neurons. It is uncertainwhat function c-neu plays in the mature cortex, however,evidence shows that c-neu does play a critical role in thedeveloping nervous system. It is involved in cell prolifera-

w xtion, neuronal migration and areal determination 12,36 .Although the ligand for c-neu is unknown, c-neu can formheterodimers with other members of its receptor family,

w xe.g., the EGFr, in cortical neurons 36 and in dorsal rootw xganglion neurons 33,66 . Our preliminary data show that

heregulin, a candidate ligand for c-neu, can affect neuronaldifferentiation and survival in a manner similar to NGFw x49 .

C-neu immunolabeled cells present a Golgi-like imageof layer V pyramidal neurons with their apical dendritesextending well into the supragranular laminae and an arrayof basal dendrites ramifying within layer V. The pattern oftrk and p75 expression is consistent with this pattern. Theimmunolabeled cell bodies have the morphology typical ofpyramidal neurons with conspicuous apical processes. Thedense neurotrophin receptor immunoreactivity within thesupragranular neuropil arises primarily from the dendritesw x53 and weaker neuropil labeling is evident in layer V.Moreover, the vast majority of c-neu-positive neuronsco-express a trk isoform. Thus, we conclude that theneurotrophin receptors are expressed by a unique subset oflayer V pyramidal neurons.

The layer V neurons are particularly important becausethey are the gatekeepers of cortical activity. Neurons inlayer Va project callosally to the contralateral hemispherew x25,34,51,67 , and neurons in layer Vb project to the

w xbrainstem and spinal cord 18,20,22,39,47,57,67 . A recentw xstudy 14 shows that the mRNA for trkB is expressed by

at least some corticospinal neurons. Thus, the maintenanceŽ .and activity of the layer V pyramidal neurons are aŽ .critical for the passage of cortical information and b

responsive to neurotrophin regulation.

4.2. Redundant receptor systems

In each layer of cortex, a particular neurotrophin recep-tor is expressed by a minority of the constituent neurons.This includes layer V although receptor expression is

Ž .notably greater as high as 49.0% than in other layers.Nevertheless, the similarity of the cortical distribution of


the neurotrophin- and c-neu-expressing neurons compelledus to explore the possibilities that layer V neurons co-ex-press low and high affinity neurotrophin receptors and thatc-neu-positive neurons also express a trk isoform. Al-though a considerable number of p75-positive neurons donot express a trk isoform, most neurons that express trkA,

ŽtrkB, or trkC are also p75-positive 94.3%, 86.4%, or.89.9%, respectively . These in vivo data complement data

from cell culture studies showing that functional neu-w xrotrophin receptors require p75–trk heterodimers 8,19 .

Likewise, c-neu and trk isoform co-expression is alsocommon. These data are particularly interesting in light of

Žpreliminary data from our laboratory Luo and Miller,.unpublished results showing that NGF can affect the

amount of c-neu expression. Thus, there appears to becross-talk between the neurotrophin receptors and c-neu.

The high incidence of p75–trk isoform co-labeling andc-neu-trk isoform provided additional insight into corticalreceptor systems. Since all of the anti-trk isoform antibod-ies currently available are rabbit polyclonal antibodies,direct determination of trk isoform co-expression is greatlyhampered. Nevertheless, using the amount of p75 co-ex-pression with a specific trk isoform and the amount ofc-neu-trk isoform co-expression, we can infer that manylayer V cortical neurons express two, and likely all three,

Ž .trk isoforms. For example, virtually every 98.8% layer Vneuron that is c-neu-positive also expresses a trk isoformŽ .i.e., they express pan-trk immunoreactivity . Of the trkA-,

ŽtrkB- or trkC-positive neurons, most 89.0%, 84.0% or.93.3%, respectively co-express c-neu. Using these data of

co-expressed pairs, we can estimate that between 69.8%and 84.0% of the trk-positive neurons co-express all threeisoforms. Such receptor co-expression is not restricted tocortex; extensive co-expression has been described forneurons in the trigeminal ganglion, mesencephalic trigemi-nal nucleus, principal sensory nucleus, and trigeminal mo-

w xtor nucleus 26 .The expression of multiple trk isoforms by individual

layer V neurons suggests that these neurons can respond tomultiple neurotrophins. Similar conclusions have beendrawn from studies of knockout mice that are incapable of

w xexpressing a particular trk isoform 1,40 . Cortical neuronsin the such animals can respond to the preferred neu-rotrophin of an isoform other than the eliminated trk. Also,neuronal survival is not compromised in these mice. Thesituation in the adult contrasts to that in developing neu-rons. Immature neurons express or respond maximally to a

w xparticular neurotrophin 10,58 . The redundancy may be-stow a stability upon the mature neurons that is neitherevident nor desired in the developing nervous system thatis actively being shaped by ‘‘regressive’’ processes.

4.3. Neurotrophin regulation in the subcortical white mat-ter

An interesting example of one mode of growth factorregulation is seen in the subcortical white matter. It con-

tains trk-positive components. Although all trk isoformsare expressed in the white matter, trkB immunolabeling ismost common. Interestingly, trkB knockout mice havereduced numbers of axons in the subcortical white matterw x44 . The trkB expression could result from the antero-grade transport of the receptors to the axonal terminals.This possibility is unlikely since neurotrophin receptors are

w xonly rarely expressed by intracortical axons 53 . An alter-native, and more appealing explanation involves the glialsupport of axons. The ligand for trkB, BDNF is richly

w xexpressed by glia in the white matter 54 . It is appealingto speculate that glia-derived BDNF regulatesrsupportsthe activities of the subcortical axons via a paracrinemechanism.

4.4. Retrograde Õs. autocrinerparacrine regulation

Classically, neurotrophin receptors have been associatedwith retrograde systems. A large body of data shows thatsuch retrograde mechanisms play an important role inmaintaining and modifying the activity of cholinergic pro-

w xjection neurons in the basal forebrain 5–7,24,41,63,70 .Nevertheless, various data suggest that retrograde systemsmay be only a minor role for cortical neurotrophins. Afterall, most neurotrophin receptor-positive profiles are post-

w xsynaptic dendrites 53 , only the rare axon expresses a trkneurotrophin receptor. Thus, cortical neurons possess anetwork that more likely serves anterograde and au-

w xtocrinerparacrine regulation 4,54 .Many layer V pyramidal neurons have the anatomical

substrates required for redundant autoregulatory systems.Ž .1 Co-expression of multiple neurotrophins is common

w x Ž .among layer V cortical neurons 54 . 2 Cortical neuronsw xco-express a neurotrophin and their cognate receptor 54 .

Ž .3 Most trk expressing neurons in layer V co-express allŽ .three isoforms see above . The implication from these

findings is that many layer V pyramidal neurons co-ex-press three neurotrophin ligands and the three associatedtrk isoforms. That is, these neurons possess the compo-nents for multiple, redundant autocrinerparacrine regula-tion. Furthermore, most neurotrophins appear to be in-volved in autocrinerparacrine systems rather than theirsmaller role in classic retrograde systems.

Acknowledgements

We thank Stuart Feinstein, David Kaplan, and LouisReichardt for their generous gifts of the anti-trk antibodiesand Julie Jacobs for processing some tissue and helpfuldiscussions. This study has been supported by grants fromthe Department of Veterans Affairs and from the National

ŽInstitutes of Health AA06916, AA07568, AA09611, and.DE07734 .


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Neurotrophin receptors in the somatosensory cortex of the mature rat: co-localization of p75, trk, isoforms and c-neu