Expression of Nerve Growth Factor,Brain-Derived Neurotrophic Factor, andNeurotrophin-3 in the Somatosensory

Cortex of the Mature Rat:Coexpression With High-Affinity

Neurotrophin Receptors

ANDREW F. PITTS1,2,3AND MICHAEL W. MILLER1,2,4*

1Research Service (151), Veterans Affairs Medical Center, Iowa City, Iowa 52246-22082Department of Psychiatry, University of Iowa College of Medicine, Iowa City,

Iowa 52242-10003Psychiatry Service (116A), Veterans Affairs Medical Center, Iowa City, Iowa 52246-2208

4Department of Pharmacology, University of Iowa College of Medicine, Iowa City,Iowa 52242-1109

ABSTRACTNeurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor

(BDNF), and neurotrophin-3 (NT-3), are critical for the maintenance and plasticity of centralnervous system (CNS) neurons. We tested the hypothesis that cortical neurons participate inredundant autocrine/paracrine systems. Three sets of studies determined the distribution ofNGF-, BDNF-, and NT-3-expressing neurons, the frequency of neurons coexpressing NGF andBDNF, and the frequency of neurons expressing a neurotrophin and its associated high-affinityreceptor. The distribution of NGF-, BDNF, and NT-3-immunoreactive neurons was identical.Neurotrophin-positive cells were parceled throughout the cortex, although the labeling frequencywas not the same in all layers. More than 30% of the neurons in layers II/III, V, and VI werelabeled, whereas only 5–10% of the neurons in layer IV was immunopositive for a neurotrophin.Some glia were also neurotrophin positive, particularly BDNF-positive glia. About 70% of theneurons in layers II/III and V coexpressed NGF and BDNF or coexpressed NGF and NT-3.Ligand-receptor colabeling was also common among cortical neurons. For example, nearly 70% ofthe NGF-, BDNF-, and NT-3-positive neurons in layer V colabeled with their respective high-affinity receptors, i.e., trkA, trkB, and trkC, respectively. Thus, (a) neurons express multipleneurotrophins and (b) cortical neurons (e.g., layer V neurons) contain the components requiredfor autocrine/paracrine and/or anterograde communication (e.g., neurons in layer II/III supportlayer V neurons). These systems mean that the cortex is capable of regulating itself autono-mously. J. Comp. Neurol. 418:241–254, 2000. Published 2000 Wiley-Liss, Inc.†

Indexing terms: anterograde; autocrine; immunohistochemistry; neocortex; neurotrophins;

paracrine; trk receptors

The neurotrophins are a group of proteins that are crit-ical for the maintenance of neuronal integrity, plasticity,and survival within the central nervous system (CNS;Lindsay, 1993; Lindholm et al., 1994; Thoenen, 1995;Prakash et al., 1996). The family consists of several mem-bers, nerve growth factor (NGF), brain-derived neurotro-phic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), and neurotrophin-6 (NT-6; Thoenen andBarde, 1980; Levi-Montalcini, 1987; Leibrock et al., 1989;Hohn et al., 1990; Maisonpierre et al., 1990; Rosenthal etal., 1990; Halbook et al., 1991; Berkmeier et al., 1991,

1992; Ibanez et al., 1992; Ip et al., 1992; Altin and Brad-shaw, 1993; Lindsay, 1993; Goetz et al., 1994). The syn-

Grant sponsor: National Institutes of Health; Grant numbers: AA06916,AA07568, AA09616, and DE07734; Grant sponsor: Department of Veter-ans Affairs.

*Correspondence to: Michael W. Miller, Department of Psychiatry-M.E.B., University of Iowa College of Medicine, Iowa City, IA 52242-1000.E-mail: michael-w-miller@uiowa.edu

Received 10 June 1999; Revised 22 September 1999; Accepted 1 October1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 418:241–254 (2000)

PUBLISHED 2000 WILEY-LISS, INC. †This article is a USGovernment work and, as such, is in the public domain in theUnited States of America.

thesis of neurotrophin mRNA is controlled by a variety ofstimuli. This includes the up-regulation of the mRNA withlong-term potentiation, during a seizure, following sen-sory stimulation, and by stimulation with glutamate oracetylcholine (Ach; Gall and Isackson, 1991; Zafra et al.,1991; Patterson et al., 1992; Castren et al., 1992; 1993;Lindsay, 1993; Lindholm et al., 1994; Isackson, 1995;Kang and Schuhman, 1995). In contrast, inducible neuro-trophins are down-regulated by the activity of g-amino-butyric acid.

According to classical description, the neurotrophinsfunction in a retrograde manner. That is, growth factorsare released from a dendrite and/or a soma of a postsyn-aptic neuron and taken up by a presynaptic axon bearingthe appropriate receptor (Hendry et al., 1974; Johnson etal., 1978, 1987; Dreyfus et al., 1989; DiStefano et al., 1992;Hefti et al., 1993; Morse et al., 1993; Sobreviela et al.,1996). Although this process may occur at a low, constitu-tive pace, afferent stimulation can induce mRNA synthe-sis and a larger neurotrophin release (Lindholm et al.,1994; Blochl and Thoenen,1995).

An example of a retrograde neurotrophin system is thecholinergic pathway arising in the basal forebrain andterminating in the cortex (Mesulam et al., 1983; Saper,1984). Cholinoceptive targets (neocortex, cingulate cortex,and hippocampus) strongly express neurotrophin recep-tors (Large et al., 1986; Shelton and Reichardt, 1986;Maisonpierre et al., 1990). The neurotrophins are takenup by the cholinergic afferents and transported to thesomata in the basal forebrain nuclei (Seiler and Schwab,1984; Johnson et al., 1987; DiStefano et al., 1992; Morse etal.,1993). Interestingly, the release of ACh from the basalforebrain afferents enhances neurotrophin synthesis inthe cortical targets (Lindholm et al., 1994; Isackson, 1995;Thoenen, 1995) and maintains a functionally plastic statewithin the cholinoceptive targets, such as the neocortexand hippocampus (Bear and Singer, 1986; Juliano et al.,1991; Jacobs et al., 1991, 1994). In turn, the neurotrophinsmaintain the viability, phenotypic markers, and appropri-ate morphology of the ascending projections (Mobley et al.,1985; Buck et al., 1988; Dreyfus et al., 1989; Takei et al.,1989).

Recent data show that neurotrophins can supportpostsynaptic neurons (i.e., via an anterograde trophic sup-port system) and act locally through an autocrine/paracrine mechanism (Thoenen, 1995; Pitts and Miller,1995, 1996; Altar and DiStefano, 1998). After all, an elec-tron microscopic analysis shows that the most commonelements expressing neurotrophin receptors are dendritesand somata (and not axons as would be expected with aretrograde system; Pitts and Miller, 1995). The presentstudy provides immunohistochemical evidence that corti-cal neurons can coexpress multiple neurotrophins andneurotrophin-receptor pairs. That is, cortical neurons pos-sess the anatomic scaffold for redundant autocrine/paracrine regulation.

MATERIALS AND METHODS

Animals

Ten adult, male Long-Evans rats (Harlan-Sprague-Dawley, Indianapolis, IN) were examined; four in thesingle-label studies and six in the double-label analyses.In all studies, animals were treated humanely according

to the practices described in the Public Health ServicePolicy on Humane Care and Use of Laboratory Animals.All protocols were reviewed and approved by the AnimalCare and Use Committees at the University of Iowa andthe Iowa City Veterans Affairs Medical Center.

All rats were prepared by the following procedure. Ratswere anesthetized by the intraperitoneal injection of acocktail of ketamine and xylazine (60 and 7.5 mg/kg, re-spectively). They were then euthanized by transcardialperfusion with 150–200 ml of 4.0% paraformaldehyde in0.10 M phosphate buffer (pH 7.4; PBS) followed immedi-ately by an equal volume of 10% sucrose (w/v) in PBS. Thebrains were removed and immersed in 10% sucrose in PBSfor 4–6 hours and cryoprotected in 30% sucrose and0.010% sodium azide in PBS for at least 3 days. Afterfreezing, each brain was cut into a set of 30-mm coronalsections through the primary somatosensory cortex at thelevel of the rostral limit of the dorsal hippocampus.

Single-label immunohistochemistry

Processing. Sections for neurotrophin labeling wereprocessed by an indirect immunoperoxidase method (Hsuet al., 1981; Pitts and Miller, 1995). Free- floating sectionswere washed in PBS, treated with 0.030% hydrogen per-oxide for 30 minutes, and immersed overnight in 10%evaporated milk in PBS at 4°C to block nonspecific activ-ity.

A section was incubated with a primary antibody di-rected against NGF, BDNF, or NT-3. The anti-NGF pri-mary was a polyclonal goat antibody IgG (generously sup-plied by Patricia Osborne and Eugene Johnson, Jr.,Washington University School of Medicine, St. Louis,MO). The antibody has a titer of 1:200,000 in an enzyme-linked immunosorbent assay (ELISA). It exhibits no cross-reactivity with BDNF or NT-3. In utero injections of thisantibody have selective effects on NGF-dependent first-order neurons of the dorsal root ganglia (Ruit et al., 1992).The anti-BDNF and anti-NT-3 primary antibodies wereboth polyclonal guinea pig IgG (kindly supplied by FranzHefti, Genentech, San Francisco, CA) with titers of1:7,000 and 1:10,000, respectively (Dugich-Djordjevic etal., 1995). Neither antibody exhibits neurotrophin cross-reactivity. The working dilutions were 1:2,500 for the anti-NGF and 1:500 for the anti-BDNF and anti-NT-3 antibod-ies, respectively. The floating sections were exposed to theprimary antibodies overnight at 4°C in a solution of 1.0%evaporated milk and 0.010% Tween-20 in PBS (pH 7.4;TPBS).

The antigen-antibody distribution was determined byusing an avidin-biotin complex. The sections were incu-bated in either 1:200 biotinylated anti-goat antibody (Vec-tor, Burlingame, CA; for the anti-NGF-reacted tissue) or1:200 biotinylated anti-guinea pig antibody (Vector; forthe anti-BDNF- and NT-3-reacted sections) in TPBS over-night at 4°C with continuous agitation. Subsequently, thesections were incubated with a 1.8% avidin-bound perox-idase complex (Vector) in PBS for 1 hour at room temper-ature. The immunoreaction product was visualized by ahistochemical reaction of the peroxidase with 0.030% hy-drogen peroxide in the presence of diaminobenzidine(DAB). The sections were mounted on slides, dehydratedwith graded alcohols, cleared with xylene, and cover-slipped.

Alternate sections were stained with cresyl violet. These

242 A.F. PITTS AND M.W. MILLER

sections were used for areal and laminar identificationsand for the stereological analyses.

Controls. Three controls for the immunohistochemi-cal reactions were performed. Sections were treated in anidentical fashion to those described above with the excep-tion that either the primary or secondary antibody wasomitted. Additional sections were processed without beingincubated with both the primary and secondary antibod-ies; thus, these sections were exposed only to the DAB andserved as a control for endogenous peroxidase activity.The results of the three controls were consistently nega-tive; no specifically labeled cells were identified.

Analysis. The distributions of the labeled cells in thedorsal primary somatosensory cortex (area 3) were exam-ined. Identification of the areal and laminar features ofthe somatosensory cortex were based on the the criteria ofMiller and Vogt (1984). The density of immunostainedcells in each layer was determined by using the stereologi-cal procedure of Smolen et al. (1983), Miller and Potempa(1990), and Pitts and Miller (1995). This procedure cor-rects for biases resulting from counting cell fragments aswhole cells. The density of cresyl violet-stained neuronswas determined similarly. The ratio of the density of im-munolabeled neurons to the density of cresyl violet-stained neurons was used as a labeling index.

Double-labeling fluorescenceimmunohistochemistry

Processing. Studies of the incidence of coexpression ofNGF and BDNF and for neurotrophin- receptor pairs wereconducted. The somatosensory cortex was cut into a seriesof 30- mm coronal sections. Nonspecific activity wasblocked by an overnight immersion in TPBS.

The colabeling for NGF and BDNF used the goat andguinea pig antibodies, respectively, described above. Sec-tions were washed with PBS and then incubated for 8hours at 4°C with the anti-NGF antibody diluted 1:2,500in TPBS. Unbound primary antibody was removed bywashing the sections with PBS. The sections were incu-bated with a biotinylated rabbit anti-goat IgG in TPBS for8 hours at 4°C, washed in PBS, and then treated withstreptavidin conjugated to fluorescein isothiocyanate(FITC; Vector) at a concentration of 1:100 in TPBS for 8hours at 4°C. Following washes in PBS and in a solution of0.15 M NaCl, 0.10 M NaHCO3 (pH 8.2), the BDNF immu-nolabeling was performed. Sections were incubated with aTexas Red-conjugated anti-guinea pig secondary antibody(Vector) for 8 hours at 4°C. This secondary antibody wasdiluted 1:100 in TPBS. The sections were rinsed threetimes with PBS, mounted on slides, coverslipped withVectastain (Vector), and examined immediately for FITC(NGF) or Texas Red (BDNF) fluorescence.

Coexpression of NGF with trkA and BDNF with trkBwas examined serially by using a dual immunofluores-cence approach in which the receptor immunolabeling wasperformed before the neurotrophin immunohistochemis-try. The sections were incubated with an anti-receptorantibody. The anti-trkA and anti-trkB antibodies (SantaCruz Biotechnology, Santa Cruz, CA) were rabbit poly-clonal antibodies. Each was directed against a uniquesequence in the C-terminal portion of the receptor. Theanti-trkB antibody only recognized the full-length protein;thus, it did not bind to the truncated, 110-kDa isoform.The selective immunostaining pattern and frequency withthese anti-trk isoform antibodies has been confirmed with

other anti-trk isoform antibodies (Jacobs and Miller, 1996;Miller and Pitts, 2000).

The incubation with a primary antibody was followed byapplication of Texas Red- conjugated anti-rabbit second-ary antibody (Vector) at a concentration of 1:100 in TPBSfor 8 hours at 4°C. After completing the receptor immu-nolabeling, the sections were probed with an anti-neurotrophin antibody (see above). The sections werewashed with PBS, rinsed with 0.15 M NaCl and 0.10 MNaHCO3 (pH 8.2), and then incubated with a biotinylatedanti-goat IgG (for the NGF labeling; 1:200 in TPBS) or abiotinylated anti-guinea pig (for the BDNF labeling; 1:200in TPBS) for 8 hours at 4°C. The immunoreaction wascompleted by incubating the sections with streptavidin-FITC for 8 hours at 4°C. After being washed with PBS, thesections were mounted with Vectastain (Vector), cover-slipped, and examined for Texas Red (trkA or trkB) orFITC (NGF or BDNF).

The coexpression of a neurotrophin with the low-affinityneurotrophin receptor, p75, was examined with dual-labelimmunofluorescence. p75 immunolabeling was performedfirst. Sections were incubated with an anti-p75 antibody(Oncogene, Manhasset, NY) diluted 1:500 in TPBS over-night at 4°C. Subsequently, the sections were processedfor neurotrophin immunoreactivity by using the proceduredescribed above. The p75 was combined with a rat-adsorbed anti-mouse secondary antibody, conjugated tobiotin (Vector). Neurotrophin staining was carried outwith an anti-rabbit secondary coupled to Texas Red. La-beling of p75 was completed by using streptavidin-conjugated FITC. The remaining steps were identical tothe above methods for trk double labeling.

Controls. Three elimination controls were used forthe dual-label fluorescence studies. (1) Sections were in-cubated without either the anti-neurotrophin or the anti-trk primary antibody. In each of these controls, the sec-tions were incubated with both secondary antibodies. (2)In the second control, the sections were incubated withboth primary antibodies, but they were exposed to onlyone secondary antibody. (3) In some cases, the fluorescentconjugates were swapped. That is, biotinylated anti-rabbitstaining (for the neurotrophin receptor/FITC) was per-formed with an anti-goat or anti-guinea pig antibody con-jugated with Texas Red. There was no immunolabelingdetected in any of the elimination controls and no changein labeling frequency when the fluorescent conjugateswere swapped.

Analysis. The fraction of coexpressing neurons wascalculated. Each slide was examined with epifluorescenceillumination for Texas Red and FITC fluorescence. Thenumber of cells staining with only one or both fluoro-phores was determined. No stereological corrections wereperformed. Analyses of variance were used to discern sta-tistically significant differences.

RESULTS

NGF expression

NGF-positive cells were distributed throughout the so-matosensory cortex (Fig. 1). Based on the size and shape ofthe cell bodies, the amount of perikaryon to nucleoplasm,and the morphology of the processes, it appeared thatvirtually all of the immunolabeled cells were neurons (Fig.2). Typically, labeled neurons exhibited dense immunore-

243NGF, BDNF, AND NT-3 EXPRESSION IN CORTEX

action product in the perikaryon and in proximal den-drites. Many of the neurons boasted a large process aris-ing from the apex of the cell body, likely an apical dendriteof a pyramidal neuron. In addition, the nuclei of manyneurons were immunolabeled.

NGF-positive neurons were not evenly distributed in allcortical laminae (Fig. 3). About one-fourth of the neuronalsomata in layers II/III, Va, and VI were NGF immunore-active. In layer Vb, the labeling frequency was nearlydoubled; four of every nine layer Vb neurons was NGFpositive. This contrasts with layer IV where only one inevery 16 neurons expressed NGF.

Rarely, a labeled glial cell body was identified in thesubcortical white matter (Fig. 4).

BDNF expression

Like NGF, BDNF was expressed by neurons throughthe full depth of the cortex (Fig. 1). Morphologically, therewere no distinguishable differences between the neuronalstaining with the anti-NGF and anti-BDNF antibodies(Fig. 2). Furthermore, the distribution of BDNF-positiveneurons was heterogeneous in a pattern that mimickedthe NGF immunolabeling (Fig. 3). That is, the lowestamount of BDNF immunolabeling was detected in layerIV and the highest was in layer Vb.

In contrast to NGF, BDNF was conspicuously expressedby glia. Although many were distributed in the gray mat-ter (Fig. 2), the most noticeable ones were in the whitematter (Fig. 4). All of the labeled glia appeared to bemacroglia, however, it was not possible to determinewhether the labeled glia were astrocytes or oligodendro-cytes. That said, the polygonal somata and the architec-ture of the processes suggested that most were astrocytes.Furthermore, parallel processes in the white matter werealso labeled. Apparently, these processes were axons be-cause they could be quite long (as long as 200 mm), con-tinuous, and solid; three features that eliminate the pos-sibility of labeled internodes of myelin.

NT-3 expression

NT-3-positive neurons were evident in each layer of thesomatosensory cortex. Many of these could be identified aspyramidal neurons (Fig. 2). Each had a prominent apicaldendrite and an array of smaller caliber dendrites arisingfrom the base of the cell body. A small number of immu-nolabeled neurons had the somatodendritic morphologycharacteristic of cortical local circuit neurons (e.g.,bitufted and chandelier neurons; Peters and Jones, 1984).The laminar distribution of NT-3-positive neurons washeterogeneous (Fig. 3). Layer IV contained significantlyfewer NT-3-immunolabeled neurons than did the otherlayers, most obviously layer Vb, where two of every fiveneurons expressed NT-3.

Apparently, there were no glia within the cortical pa-renchyma that were NT-3 positive. On the other hand, asmall number of glia in the subcortical white matter wereintensely labeled with the NT-3 antibody (Fig. 4). Thesewere less common and more poorly labeled than were theBDNF-labeled glia.

Neurotrophin coexpression

The quantitative data of the laminar neurotrophin-labeling frequencies were compared. No significant differ-ences were detected. For example, no statistically signifi-cant differences were detected among the low

neurotrophin-labeling frequencies in layer IV nor wereany differences discerned for layer Vb where neurotrophinexpression was highest. These quantitative data, particu-larly for layer Vb, provide compelling evidence that anindividual neuron expresses more than one neurotrophin.After all, if each of the three neurotrophin is expressed bymore than one-third of the neurons in a specific layer (asthey are in layer Vb), then numerically at least a smallsubset of the neurons must express at least two neurotro-phins. This possibility was explored by using a dual fluo-rescence technique.

The coexpression of two pairs of neurotrophins (NGF/BDNF and NGF/NT-3) was explored using a double-labeling immunofluorescence technique. Neurotrophindouble labeling was common throughout all layers of thecortex (Fig. 5). This was interpreted as coexpression inas-much as control sections labeled with only a single pri-mary antibody showed that this dual labeling was not dueto bleed-through (data not shown). For example, FITClabeling was not detected when Texas Red filters were inplace. Quantitative data were generated for two laminae,layers II/III and Vb. Of the neurons that expressed NGFand/or BDNF, three-fourths expressed both neurotrophins(Fig. 6). Only a small number expressed exclusively NGF.Thus, after reconfiguring the data, it was evident that 93%of the NGF-positive neurons (single- plus double-labeledneurons) expressed BDNF (double-labeled neurons). Thesame pattern was evident in layers II/III and Vb.

As for the neurons that expressed NGF and/or NT-3,more than two-thirds coexpressed the two neurotrophins.More neurons in this pairing expressed only NGF. Never-theless, the majority (75%) of the NGF-positive neuronscoexpressed NT-3. By combining the results of the twoexperiments with the data from the single-immunolabelstudies, two conclusions can be drawn. (1) More thantwo-thirds of all NGF-expressing neurons coexpressBDNF and NT-3. (2) Since three-eighths of all layer Vbneurons are NGF positive, then one-fourth or more of alllayer Vb neurons must express all three neurotrophins.

Neurotrophin-neurotrophin receptorcoexpression

The somata of neurons expressing a neurotrophin re-ceptor were most common in layer V, and specifically inlayer Vb (Pitts and Miller, 1995; Miller and Pitts, 2000).This pattern was evident for the low-affinity (p75) andhigh-affinity (trk isoforms) receptors. Two batteries ofneurotrophin receptor coexpression studies were per-formed.

The coexpression of each neurotrophin with p75 wasexamined. More than two-thirds of the p75-positive layerVb neurons coexpressed a neurotrophin (Figs. 7 and 8). Asingle-label study showed that more than one-third of alllayer V neurons were p75 positive (Pitts and Miller, 1995).Since p75 is apparently expressed solely by pyramidal

Fig. 1. Neurotrophin expression in the somatosensory cortex of themature rat. Neurons expressing nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3) wereimmunolabeled with a specific antibody. All cortical layers hadneurotrophin-positive neurons. The various cortical laminae are iden-tified by Roman numerals on the cresyl violet-stained tissue on theleft. wm, white matter. Scale bars 5 100 mm.

244 A.F. PITTS AND M.W. MILLER

Figure 1

Figure 2

neurons and pyramidal neurons constitute seven-eighthsof all layer V neurons (Peters et al., 1985), then, of all layerVb pyramidal neurons, (1) nearly 30% (i.e., 1

34 8

7) ex-

pressed p75 and (2) one in every five Vb pyramidal neu-rons (i.e., 1

34 8

73 2

3) expressed both a neurotrophin and

p75.The expression of a neurotrophin (NGF, BDNF, and

NT-3) and its associated high- affinity receptor (trkA,trkB, and trkC, respectively) was examined in layer Vb(Fig. 7). Of the neurons that expressed NGF and/or trkA,three-fourths expressed both proteins (Fig. 8). Interpret-ing the data differently, 80% of the NGF-positive neuronscoexpress trkA. Similarly, 76% and 81% of BDNF- andNT-3-positive neurons, respectively, coexpressed theircognate receptor. It is noteworthy that regardless of whichpairing was examined, the frequency of neurons that ex-pressed only the neurotrophin was consistently and sig-nificantly (P , 0.05) greater (1.7- to 5-fold) than the fre-quency of neurons expressing only the receptor.

DISCUSSION

Neurotrophin expression

Neurons expressing NGF-, BDNF-, or NT-3 have sev-eral common features. (1) The distribution of immunola-beled neurons is similar for all three neurotrophins. (2)The density of neurotrophin-expressing neurons in eachlayer is similar. Regardless of the neurotrophin, aboutone-fourth of all neuronal somata in each layer of thesomatosensory cortex are neurotrophin positive. The no-table exceptions are layer Vb where more than 40% of theconstituent neurons express a neurotrophin and layer IVwhere fewer than one in eight neurons exhibit neurotro-phin immunoreactivity. (3) Both pyramidal (projection)

and stellate (local circuit) neurons express the neurotro-phins.

The largely pancortical distribution of neurotrophin-expressing somata does not match an individual hodologi-cal pattern. Rather, this distribution encompasses the so-mata of neurons participating in all major corticalprojection systems. The cell bodies of corticocortical pro-jection neurons (including callosal projection neurons) areprimarily in layers II/III and Va (Wise and Jones, 1976;Ivy and Killackey, 1981; Miller and Vogt, 1984; Izraeli andPorter, 1995; Porter, 1996). Corticofugal projection neu-rons to the brainstem and thalamus are predominantlylocated in layer Vb and layer VIa, respectively (Price andWebster, 1972; Hicks and D’Amato, 1977; Wise and Jones,1977; Miller, 1987).

The pattern of cortical neurotrophin expression fails tomatch the distribution of a specific cortical afferent sys-tem(s). Corticocortical and thalamic afferents primarilyterminate in layers I–III and V and in layers I, IV, andVIa, respectively. Each of these layers is replete withneurotrophin-positive neurons, with the notable exceptionof layer IV. A first inclination would be to conclude thatthe thalamocortical system is NGF independent. Such aconclusion is contradicted by two findings. First, neuronsin the ventrobasal thalamus express at least NGF (unpub-lished data). Second, neurons with cell bodies in deeplayer II/III and in layers V and VIa have dendrites thatpass through layer IV. Therefore, the possibility thatthalamocortical afferents rely on neurotrophin systemscannot be eliminated.

Neurotrophins within a cell body may be synthesized denovo. The data supporting de novo synthesis are quitecompelling. First, cortical neurons can elaborate mRNAfor the neurotrophins (Korsching et al., 1985; Shelton andReichardt, 1986; Gall and Isackson, 1991; Hofer et al.,1989, 1990; Lauterborn et al., 1991; Valenzuela et al.,1993; Lindholm et al., 1994; Blochl and Thoenen, 1995).Second, in other levels of the trigeminal-somatosensorysystem, virtually all first- and second-order trigeminalneurons that express NGF protein also elaborate NGFmRNA (Jacobs and Miller, 1999). Third, the frequency ofneurotrophin-positive neurons in every layer is greater

Fig. 3. Laminar distribution ofneurotrophin-immunoreactive neu-rons. The labeling ratio for neuronsthat express a particular neurotro-phin is described for each layer ofthe cerebral cortex. These ratioswere calculated as the quotient ofthe density of neurotrophin-positiveneurons and the density of cresylviolet-stained neurons. Each barrepresents the mean of five animalsand the T-bars signify the standarderrors of the means.

Fig. 2. Neurotrophin-positive neurons in layer V. Layer V is re-plete with neurons expressing nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3). Mostimmunoreactive cells are neurons (solid arrows), however, a smallnumber of BDNF-positive cells are glia (open arrows). Scale bars 5 50mm.

247NGF, BDNF, AND NT-3 EXPRESSION IN CORTEX

Figure 4

than the frequency of neurons expressing the neurotro-phin trk receptors (Pitts and Miller, 1995; Miller andPitts, 2000). In fact, there is as much as an eightfolddifference in layers II/III and VI.

Coexpression of multiple neurotrophins

The high incidence of expression of a single neurotro-phin in various cortical laminae mathematically necessi-tates that there be some coexpression. For example, thetotal of the labeling indices for each neurotrophin for layerVb is 130%. This value exceeds 100%, the maximalamount allowing no coexpression. In fact, the double-labeling studies show that there is considerable coexpres-sion in layer Vb (.70%). Even in layers where the overlapis not mathematically required (e.g., layer II/III), coex-pression is the rule, not the exception. Furthermore, be-cause coexpression of NGF and BDNF and of NGF andNT-3 is so high, expression of three neurtrophins in asingle neuron is a foregone conclusion.

The expression of multiple neurotrophins within a sin-gle neocortical neuron implies redundant systems. Thereare several mechanisms by which this may be actualized.First, a neurotrophin may be incorporated from an exter-nal source (delivered via a retrograde, anterograde, orparacrine mechanism). Second, alternatively, a neuronmay release one of multiple neurotrophins. These possi-bilities are not mutually exclusive.

Retrograde support systems

The commonly considered mode of neurotrophin actionis through a retrograde system (Loughlin and Fallon,1993). Accordingly, a growth factor is generated somati-cally, transported to somatodendritic synaptic sites wherethe neurotrophin is released, and impacts an afferenttrans-synaptically. In other words, a retrograde supportsystem goes in a direction counter to that for a classicalneurotransmitter.

There is an anatomical basis for retrograde supportwithin the trigeminal system. In at least two cranial nervenuclei, the principal sensory nucleus of the trigeminalnerve and the trigeminal motor nucleus, more than three-fourths of the neurons are NGF positive (Jacobs andMiller, 1999). These nuclei are innervated by cortical pro-jections arising from layer Vb pyramidal neurons (Mizunoet al., 1983; Travers and Norgren, 1983; Yasui et al.,1985). Layer Vb has the highest concentration of neuronsexpressing low- and high-affinity neurotrophin receptors

Fig. 4. Neurotrophin expression in the white matter. The whitematter contains glia that are neurotrophin positive. Of the neurotro-phins, brain-derived nerotrophic factor (BDNF) is most commonlyexpressed by these cells. Only a few are nerve growth factor (NGF) orneurotrophin-3 (NT-3) immunoreactive. Each photograph is orientedso that the cortex is to the left and the lateral ventricle is to the right.Scale bars 5 50 mm.

Fig. 5. Dual immunofluorescence for nerve growth factor (NGF) and brain-derived nerotrophic factor(BDNF). A double-labeling technique shows that many layer V neurons expressing NGF (arrows in left)are also BDNF positive (arrows in right). Scale bars 5 20 mm.

249NGF, BDNF, AND NT-3 EXPRESSION IN CORTEX

(Pitts and Miller, 1995; Miller and Pitts, 2000). With thisorganization, it is likely that the NGF elaborated by theneurons in the cranial nerve nuclei retrogradely supportthe large corticofugal projection neurons in layer Vb.

Anterograde communication

Another possibility for local signaling within the neor-tex is via anterograde communication. Defined in refer-ence to the more classical model of retrograde communi-cation, anterograde support involves the release ofneurotrophin at the synapse and binding on the postsyn-aptic side by high-affinity receptors. The net result may bean alteration in short-term signaling properties. Thus, thedirection of anterograde support parallels that for neuro-transmission.

Although the incidence of anterograde support may befrequent throughout the cortex, it is difficult to discrimi-nate from other neurotrophin mechanisms. The mostreadily identifiable pathway involves supragranular syn-aptology. Neurotrophin-positive somata are common inlayer II/III. Likewise, the supragranular neuropil is in-tensely receptor immunoreactive (Pitts and Miller, 1995;Miller and Pitts, 2000). This immunoreactivity largelyresides in dendritic profiles. Presumably, most of thesedendrites are parts of the apical trees of layer V pyramidal

neurons. Thus, the neurotrophins produced by the neu-rons in layer II/III apparently support layer V pyramidalneurons by an anterograde mechanism.

In addition to the evidence above, other data supportthe conclusion that the cortex relies on anterograde neu-rotrophin communication. These include a short-termneurotrophin-initiated signaling system that may under-lie the subtle events associated with synaptic plasticity(Berninger and Poo, 1996). Further support for an antero-grade neurotrophin system within layer II/III comes fromexperiments using colchicine (Altar et al., 1997; Altar andDiStefano, 1998). Application of colchicine increases anti-neurotrophin immunoreactivity within layers II/III andIV, with the latter showing the vastly greater increase.

Autocrine/paracrine regulation

Many layer V projection neurons express both a neuro-trophin and the cognate receptor or p75. Interestingly, therates of coexpression for a neurotrophin with either thehigh- or low-affinity receptor are quite similar. This wouldsuggest that the coexpression of a trk isoform and p75 iscommon. In fact, empirical data from a double-labelingstudy show that most layer V neurons do coexpress a trkisoform and p75 (Pitts and Miller, 1996). We can concludethat most neurotrophin-expressing layer V neurons also

Fig. 6. Neurotrophin coexpression. The in-cidence of neurons that express nerve growthfactor (NGF) and brain-derived nerotrophicfactor (BDNF) or NGF and neurotrophin-3(NT-3) is high. More than two-thirds of thelayer V neurons coexpress two neurotrophins.Each bar is the mean (6 the standard error ofthe mean) of five animals.

250 A.F. PITTS AND M.W. MILLER

elaborate the associated high-affinity receptor and p75.Furthermore, the layer V neurons express the componentsrequired to form high/low- affinity receptor heterodimers.

Presumably, the ligand-receptor coexpression meansthat the layer V neurons can both produce and bind aneurotrophin. Thus, these neurons must use an autocrine/paracrine mechanism. This is not specific to the cortex fora similar organization is evident in lower trigeminal cen-ters (Jacobs and Miller, 1999). Neurotrophins releasedfrom either constitutive or inductive pathways (Blochl andThoenen, 1995; Lindholm et al., 1994) could bind to high-affinity receptors expressed by the neuron’s own cell bodyand dendrites (i.e., autocrine stimulation) or it may affectneighboring neurons via a paracrine mechanism. The ef-fectiveness of these autocrine/paracrine systems is limitedby the diffusion distance for the neurotrophins. BDNF,which may be bound and cleared by trkB receptors ex-pressed by glia, has a shorter diffusion distance thanNGF, and possibly NT-3 (Morse et al., 1993). Thus, al-though BDNF is more abundant with regard to mRNAsynthesis (Lindsay, 1993), NGF may have broader effects.Locally directed effects can coexist with retrograde and/oranterograde systems (Altar and DiStefano, 1998).

CONCLUSIONS

The cortex contains various neurotrophin systems thatregulate intracortical activity (e.g., autocrine/paracrineand anterograde systems) and mediate interactions be-tween the cortex and distant structures (e.g., via retro-grade support). Various data suggest that neurotrophinrelease is inducible (Gall and Isackson, 1991; Zafra et al.,1991; Patterson et al., 1992; Castren et al., 1992, 1993;Lindsay, 1993; Lindholm et al., 1994; Isackson, 1995;Kang and Schuhman, 1995) and can modify synaptic ac-tivity (e.g., Zafra et al., 1991; Lo, 1995; Berninger and Poo,1996; Wang et al., 1998). Such interpretations rely on theprinciple that neurotrophins act in a retrograde manner.This conceptualization of neurotrophin activity is limitedand needs to be broadened. In light of the present datathat cortical neurotrophins can participate in autocrine/paracrine and anterograde systems, we hypothesize thatneurotrophins have more global effects on cortical activitythan just through retrograde mechanisms. Neurotrophinsmay be transmitter-like effectors in autocrine/paracrineand anterograde systems. In fact, electron microscopicdata suggest that neurotrophins are used more often in

Fig. 7. Dual immunofluorescence for nerve growth factor (NGF) and trkA. Arrows indicate theNGF-positive layer V neurons that also express its high-affinity receptor, trkA. Scale bars 5 20 mm.

251NGF, BDNF, AND NT-3 EXPRESSION IN CORTEX

these roles than in retrograde systems (Pitts and Miller,1995).

The double-labeling studies show that cortical neuronscan use multiple neurotrophins. Such data are intriguingbecause each neurotrophin has a different effective rangeand regulates different activities (Zafra et al., 1990; Gall andIsackson, 1991; Isackson et al., 1991; Rocamora et al., 1992;Lindsay, 1993; Morse et al., 1993; Kim et al., 1994; Lindholmet al., 1994; Berninger and Poo, 1996; Prakash et al., 1996).Therefore, we posit that these inducible, overlapping neuro-trophin systems function in more complex ways than on-off

responses and that they permit subtle, graded regulation ofcortical activity. As such, they may subserve normal corticalactivities such as learning and memory as well as beingrecruitable during damage-induced repair.

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