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Developmental Brain Research, 70 (1992) 53-63 tO 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00 53 BRESD 51525 Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex G.W. Huntley a,,, D.L. Benson a,**, E.G. Jones a and P.J. Isackson a,b,*** Departments of a Anatomy and Neurobiology, and b Biological Chemistry, University of California at Irvine, Ircine, CA 92717 (USA) (Accepted 23 June 1992) Key words: Growth factor; Gene expression; Cortical development; In situ hybridization; Primate In situ hybridization histochemistry with labeled cRNA probes complementary to monkey brain derived neurotrophic factor (BDNF) mRNAs has been used to study the cellular localization and expression of this neurotrophin in the prefrontal cerebral cortex of fetal and adult monkeys. Expression could not be detected in prefrontal cortex before the 121st fetal day. Thereafter, in fetal life and in adulthood BDNF mRNA could be detected primarily in large, putative pyramidal cells of layers III and VI throughout the prefrontal cortex. The temporal course and cellular localization of BDNF expression suggests its association with the development and stabilization of specific connections in regions of cortex that display marked functional plasticity. INTRODUCTION The development of the nervous system requires the appropriate neurochemical and morphological differ- entiation and survival of populations of synaptically connected neurons. Neuronal survival and the mainte- nance of functional integrity in the nervous system is thought to depend in large part on specific target-de- rived neurotrophic factors, a number of which have been identified (for reviews, see refs. 79 and 83) Of these, nerve growth factor (NGF) is the archetype and has dominated concepts regarding the structure, func- tion and localization of neurotrophic factors. However, relatively limited populations of neurons respond to NGF, especially in the central nervous system, and a number of other neurotrophic factors that are mem- bers of a larger neurotrophin family have been identi- fied in the central nervous system79. The structural and functional similarity between NGF and two other target-derived neurotrophic fac- tors, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), has confirmed the existence of a family of related neurotrophins 31'47'59. BDNF a appears to be predominantly expressed within brain, with highest mRNA levels in hippocampus and neocortex ~3,~s,3°'3s'6s'86. There is an approximately 10- 50.fold greater abundance of BDNF mRNA in brain in comparison with NGF mRNA, suggesting that a larger and more functionally diverse population of cells is dependent on BDNF 13`15'3°'3s't's's6. NT3 mRNA is also widely expressed and is most abundant in the hippo- campus31,40,58,59, 73. Levels of BDNF and NGF mRNAs in hippocampus and neocortex may be regulated by neural activity i¢,,as possibly through the non-NMDA class of glutamate receptor %, and developmental studies in rats have shown that peak BDNF mRNA expression commences in neocortex after neuronal genesis, differentiation and migration, at a stage when connections are presumably being established 15'5a. Given the localization of BDNF in diverse sets of neurons, these data suggest that BDNF expression may be related to the development Correspondence: E.G. Jones, Department of Anatomy and Neurobiology, University of California at Irvine, Irvine, CA 92717, USA. Fax: (I) (714) 725-2932. * Present address: Department of Neurobiology, Box 1065, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. ** Present address: Department of Neuroscience, Universi*y of Virginia, School of Medicine, Box 230 Medical Center, Charlottesville, VA 22908, USA. ** * Present address: Department of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, FL 32224, USA.

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Page 1: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

Developmental Brain Research, 70 (1992) 53-63 tO 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00

53

BRESD 51525

Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

G.W. Huntley a, , , D.L. Benson a,** , E.G. Jones a and P.J. Isackson a,b,***

Departments of a Anatomy and Neurobiology, and b Biological Chemistry, University of California at Irvine, Ircine, CA 92717 (USA)

(Accepted 23 June 1992)

Key words: Growth factor; Gene expression; Cortical development; In situ hybridization; Primate

In situ hybridization histochemistry with labeled cRNA probes complementary to monkey brain derived neurotrophic factor (BDNF) mRNAs has been used to study the cellular localization and expression of this neurotrophin in the prefrontal cerebral cortex of fetal and adult monkeys. Expression could not be detected in prefrontal cortex before the 121st fetal day. Thereafter, in fetal life and in adulthood BDNF mRNA could be detected primarily in large, putative pyramidal cells of layers III and VI throughout the prefrontal cortex. The temporal course and cellular localization of BDNF expression suggests its association with the development and stabilization of specific connections in regions of cortex that display marked functional plasticity.

INTRODUCTION

The development of the nervous system requires the appropriate neurochemical and morphological differ- entiation and survival of populations of synaptically connected neurons. Neuronal survival and the mainte- nance of functional integrity in the nervous system is thought to depend in large part on specific target-de- rived neurotrophic factors, a number of which have been identified (for reviews, see refs. 79 and 83) Of these, nerve growth factor (NGF) is the archetype and has dominated concepts regarding the structure, func- tion and localization of neurotrophic factors. However, relatively limited populations of neurons respond to NGF, especially in the central nervous system, and a number of other neurotrophic factors that are mem- bers of a larger neurotrophin family have been identi- fied in the central nervous system 79.

The structural and functional similarity between NGF and two other target-derived neurotrophic fac- tors, brain-derived neurotrophic factor (BDNF) and

neurotrophin-3 (NT3), has confirmed the existence of a family of related neurotrophins 31'47'59. BDNF a appears to be predominantly expressed within brain, with highest mRNA levels in hippocampus and neocortex ~3,~s,3°'3s'6s'86. There is an approximately 10- 50.fold greater abundance of BDNF mRNA in brain in comparison with NGF mRNA, suggesting that a larger and more functionally diverse population of cells is dependent on BDNF 13`15'3°'3s't's's6. NT3 mRNA is also widely expressed and is most abundant in the hippo- campus31,40,58,59, 73.

Levels of BDNF and NGF mRNAs in hippocampus and neocortex may be regulated by neural activity i¢,,as possibly through the non-NMDA class of glutamate receptor %, and developmental studies in rats have shown that peak BDNF mRNA expression commences in neocortex after neuronal genesis, differentiation and migration, at a stage when connections are presumably being established 15'5a. Given the localization of BDNF in diverse sets of neurons, these data suggest that BDNF expression may be related to the development

Correspondence: E.G. Jones, Department of Anatomy and Neurobiology, University of California at Irvine, Irvine, CA 92717, USA. Fax: (I) (714) 725-2932.

* Present address: Department of Neurobiology, Box 1065, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

** Present address: Department of Neuroscience, Universi*y of Virginia, School of Medicine, Box 230 Medical Center, Charlottesville, VA

22908, USA. ** * Present address: Department of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, FL 32224, USA.

Page 2: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

54

of particular neural circuits, However, to date, most

studies of BDNF m R N A localization in rodents have

emphasized regional distributions rather than the cell

types expressing the B D N F gene, and studies in higher

primates have generally been restricted to hippocam- pus 6s'69. No studies have yet examined B D N F m R N A

expression in the higher primate neocortex. The present study uses in situ hybridization histo-

chemistry to examine the expression and developmen-

tal regulation of B D N F m R N A in monkey prefrontal

cortex. The prefrontal cortex in primates is composed

of several structurally and functionally distinct areas,

its integrity is essential for normal cognitive function,

and diminished prefrontal activity in humans appears

to be a concomitant of major thought disorders such as

schizophrenia (e.g. refs. 8 and 85). The protracted

development of the prefrontal cortex, allied with the

abundance of information regarding its cellular, con-

nectional and functional organization (for review, see

ref. 19) makes the monkey prefrontal cortex an ideal

one in which to examine the developmental regulation

of BDNF expression and the nature of the cells in

which it is expressed.

MATERIALS AND METHODS

Tissue preparation Portions of the frontal lobes containing the prefrontal cortical

areas were taken from 6 fetal and 2 adult rhesus monkeys (Macaca mulatta) all of which had bt~en used in previous studies '~'~''~t'. The fetal animals, whose ages were l l0 days post-conception (Ell0), El21, El31, ElM, ElS0 and EI55, were delivered by caesarian section, deeply anesthetized with Nembutal and perfused transear- dimly with normal saline followed by a mixture of 2% paraformalde- hyde and 0.1¢~ glutaraldehyde in 0,1 M phosphate buffer (pH, 7.4). Gestation in this species of monkey, is approximately 165 days. The adult monkeys were given an overdose of Nembutal and perfused transcardially with normal saline and then with cold 4% paraformaldehyde in 0.1 M phosphate buffer. All brains were blocked, infiltrated with 30% sucrose and stored frozen at -70°C. Frozen 20 pm thick sections were cut on a sliding microtome and stored for 72 h in 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C. Sections were subsequently washed in sterile, 0.1 M phos- phate buffer and mounted onto poly-L-lysine coated slides.

Construction of cRNA probes RNA probes were prepared from a plasmid construction,

pMkl 112-22, which contains a 384 bp insert of the coding region of BDNF that was obtained following polymerase chain reaction (PCR) amplification of monkey genomic DNA "~'~. This region of monkey BDNF sequence has 5 nucleotide changes compared to human BDNF eDNA TM. Antisense and sense strand RNA probes were transcribed from Pvull-linearized pMkll12-22 in the presence of In-3~S]UTP with T3 and T7 RNA polymerase, respectively.

ht situ hybridization histochemistry The method used was derived from that of Gall and Isackson t6.

Slide-mounted sections were washed in 0.1 M glycine in 0.1 M phosphate buffer, then sequentially in proteinase K (1 t~g/ml; pH 8); 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8); and two washes of 2 × saline sodium citrate (SSC). Slides were air dried and then covered with h~bridization solution composed of: 50% deion- ized formamide, 10% a.extran sulfate, 35× Denhardt's solution, 0.15

Fig. 1. Darkfield photomicrograph of a section through area 9 taken from an E135 monkey and hybridized with the BDNF sense probe

showing only background ~t')eling.

mg/ml yeast tRNA; 0.33 mg/ml denatured herring sperm DNA, 40 mM dithiothreitol (DTT) and i x 10 4 cpm//~l of the ['~'~S]-Iabeled antisense riboprobe. The sections were then covered by sterile cover- slips and subsequently incubated for 24 h in a humid chamber at 60°C. Following hybridization, coverslips were gently removed and the slides were washed in 4 × SSC, treated with ribonuclease A (20 pg/ml; pH 8), then washed through a series of SSC washes of descending concentrations to a final stringency of 0.1 × SSC for I h at 60°C.

Sections were exposed to Amersham Beta-max film for 1-4 days, then lipid extracted in chloroform and dipped in Kodak NTB2 emulsion (diluted 1 : 1 in water), exposed for 4-6 weeks at 4°C, then developed, fixed and stained through the emulsion with thionin,

Control procedures consisted of treating sections in the exact manner as described above with the exception of either replacing the ['~SS]-Iabeled antisense riboprobe with the [3SS]-Iabeled sense ribo- probe or by pretreating sections with ribonuclease A prior to hy- bridization. In both cases, no specific hybridization to BDNF mRNA was detected (Fig. 1).

Laminar and areal boundaries of cortical areas were determined from a separate series of sections adjacent to those used for in situ hybridization and stained with thionin or for cytochrome oxidase 93 or acetylcholinesterase activity ~4. Autoradiographically labeled cells were plotted with the aid of a camera lucida. The nomenclature for delineating the prefrontal cortical areas is that of Walker 84.

RESULTS

Distribution o f BDNF mRNA in adult monkey prefrontal cortex

Cells labeled by in situ hybridization of the [3ss]-

labeled probe complementary to B D N F m R N A were

Page 3: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

55

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i ~ , ! : i ~ , ! ., :, ~ . •

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Fig. 2. Distribution and localization of BDNF mRNA in adult monkey prefrontal cortex. Darkfield photomicrographs show differences in the density of labeled cells in areas 9 (A) and 46 (B). Paired, brightfield photomicrographs of underlying Nissl-stained sections in this and subsequent figures show lamination patterns. C,D: higher power photomicrographs show grain clusters overlying pale, Nissl-stained nuclei (straight arrows) in layers III (C) and VI (D). Some of the grain clusters show linear extensions (small arrows in C). Many other neurons (curved arrows in D) a,ld

glial cells (g in C) are not labeled. Bars = 100 ~m (A,B); 10 ~m (C,D).

Page 4: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

56

A , . .¢ ~ ? , . . . . . , . . , . , . ~

'~ II 4 ¢ . I . ~ ,

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' ¢ ~ , ~ - ~ ~ :" "~'~',Z'~,"- ~ ~ U k : • ; . , ~ , ~ , - .~, , . . ~%', • ..".,.~:-.- V : ~ . ' : , . ; . ~ , . . ~-,o .~ ~ , . '~ . ~ . . : "? • , , ; • . • ~,,~,

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Fig. 3. A,B: paired brightfield and darkfield photomicrographs from sections through dorsolateral prefrontal cortex of fetal animals aged El l0 (A) and E!35 (B) showing developmental changes in cytoarchitecture and in BDNF expression, At El10, a thin cortical plate is still present (asterisk in A). No probe hybridization is evident. By E135 (B,D), cytoarchitecture similar to the adult is present. Cells hybridizing to the BDNF probe become evident at this stage. C: autoradiogram of a section from an E135 monkey brain showing areal differences in the density of labeling in layers Ill and VI already present at this stage. D: higher power photomicrograph showing grain cluster indicating a labeled cell and several unlabeled cells. Grain density is similar to that found in adults. CS, cingulate sulcus; LO, lateral orbital sulcus; Me, medial orbital sulcus; PS,

principal sulcus. Bars = 100 mm (A,B); 1/zm (C); 10 pm (D).

Page 5: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

present across all areas of the adult prefrontal cortex. In general, the density of labeled cells was greatest along the dorsomedial aspect of the frontal lobe,

57

through area 9. The density of labeled cells decreased ventrolaterally, through both banks of the principal sulcus (area 46) and declined further through area 12,

A T ~ ~" ~ ' ,,.ab.' ~ .e' • •

,.. ,,. °;

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~®~i~ ....

I '~,I, ~ m1'~ P+o

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Fig. 4. Bright- and darkfield photomicrographs from sections through area 9 of an El50 fetus (A) and through area 46 from an E155 fetus (B,C). There are no overt changes at the later fetal stages in comparison with E135. A typical grain cluster overlying a neuron is shown at higher power

in C. Glial cells (g) are unlabeled. Bars = 100 p.m (A,B): 10 ~ m (C).

Page 6: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

58

and through areas 11, 24 and 25 on the orbital, and ventromedial surfaces (Fig. 2).

In all areas examined, the laminar distribution of labeled cells was similar. The greatest concentrations of hybridized cells were seen as two distinct bands: a superficial band coextensive with most of layer III, and a deep band, coextensive with layer VI especially its superficial half to two-thirds (Fig. 2A,B). More sparsely scattered, labeled cells were present in layers II, IV and V. No labeled cells were present in layer I or in the subcortical white matter. Autoradiographic grains appeared situated over neurons, which were distin- guished by their larger, paler nuclei in the Nissl stain. Ribonuclease treatment makes staining of the cyto- plasm weak or absent (Fig. 2C,D).

In layer Ill labeled grain clusters measured, on average 20-25 ~.m in diameter and lay over large, pale-stained nuclei, 12-15 ~m in diameter (Fig. 2C,D). The grain clusters commonly showed linear extensions, 5-10/~m long, suggesting the extension of the BDNF mRNA into proximal dendrites (Fig. 2C).

In layer VI, grain clusters, 10-15 ~m in diameter overlay medium sized, pale nuclei, 8-10 ~m in diame- ter with no evidence of labeled processes.

Cytoarchitecture of developing monkey prefrontal cortex The 6 fundamental cortical layers could be dis-

cerned at each fetal age examined (Figs. 3 and 4). However, in the two youngest cases (El10 and El21), differences in laminar and cellular architecture that enable the borders between cortical areas to be distin- guished at later ages and in adults were not apparent. At ElI0 and El21, layers ll-VI were cell dense and composed of small, darkly stained, densely packed so- mata, with little or no regional variation in laminar pattern evident (Fig. 3A). A thin, dense cortical plate was still evident at the junction of layers l and II (Fig. 3A). By El31, cell-packing density overall decreased, spinal size increased, and areal differences similar to those found in the adult were apparent (Fig. 3B). The cortical plate had disappeared by these ages. The de- velopment of prefrontal cortical architecture was es- sentially similar to that described previously 75.

Distribution of BDNF mRNA in developing monkey pre- frontal cortex

No autoradiographically labeled cells were present at El 10 in any area of the prefrontal cortex (Fig. 3A). By El21, sparsely distributed, lightly labeled cells were present in layers Ill and VI throughout all regions of the prefrontal cortex. At this stage, there were no regional variations in the density of hybridized cells. By El31 and E135 (Fig. 3), labeled cells were present in

III

IV

V

VI

EIIO E135 E155 ADULT

lit

IV

VI

III

IV

V

VI

I

II

• II

V

V

rI

Fig. 5. Schematic summary diagram of developmental changes in the distribution of cells (dots) showing BDNF probe hybridization in

area 9 from El 10 through adulthood.

all prefrontal areas examined, although the overall density of labeling was not as great as in the adult. A regional variation in density similar to that described in the adult had become evident (Fig. 3C). The greatest concentration of hybridized cells was situated dorsome- dially and extended laterally, through the principal solcus. The density of labeled cells declined ventrolat- erally, and on the orbital surface (Fig. 3C).

The laminar distribution of labeled cells at E131-- E135 was similar to that described in the adult, and was similar throughout all areas examined (Fig. 3B). The greatest number of hybridized cells was present in the deeper half of layer III and in layer VI, with fewer cells present in layers I1, IV and V (Fig. 3B). No labeled cells were found in layer I or in the underlying white matter. At these stages, clusters of autoradio. graphic grains could easily be related to underlying, palely-stained large nuclei of ~.~uror~s (!=ig. 3B). Grain density over individual cells was approximately the same as in the adult.

At the latest fetal stages examined (E150-E155), the overall density of labeled cells was greater than at younger stages but less tha~ in the adult (Flg,i. 2A, 3B, 4A and 5). Areal and lamin,tr distribution of labeled cells did not change and nc~'.roglial cells were not labeled at any stage (Fig. 4C). A schematic summary of the distribution of labeled cells throughout fetal stages and adulthood is shown in Fig. 5.

DISCUSSION

The data presented show that neurons in the adult primate prefrontal cortex express BDNF mRNA, and

Page 7: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

59

that the distinctive areal and laminar distribution of hybridized cells characteristic of the adult is estab- lished prenatally, during the latter third of gestation. The observations are consistent with previous reports of BDNF mRNA localization in neurons throughout the adult rat central nervous system m3'~5.s6 including the hippocampal formation and neocortex 3s. The develop- mental time course of BDNF mRNA expression in monkey prefrontal cortex and its maintained expres- sion by mature cortical neurons in adulthood suggests that this neurotrophic factor may play a role not only in the formation of prefrontal cortical connectivity, but also in its maintenance and thus, in the integrity of higher cerebral function throughout life.

The widespread distribution of BDNF mRNA expressing cells across areas of monkey prefrontal cor- tex as well as throughout other cortical and subcor- tical areas in monkeys (unpublished observations), rats 13,15.3s,68'86, pigs s6 and mice 3° suggests that BDNF- responsive neurons exist throughout the central ner- vous system. Whether the neurotrophin is expressed only in certain sets of neurons is not yet clear but the widespread distributiov implies that a wide variety of neurons can potentially respond to it, unlike those responsive to NGF, which seem limited to cholinergic neurons in the central nervous system. BDNF, like the other members of the neurotrophin family, NGF and NT3, are thought to be mainly target-derived factors vg'sa, so it is possible that BDNF produced in the cortical neurons is required by the parent cells of one or more of the various sets of afferent connections that target the prefrontal cortex. These include the parent cells of thalamocortical, ipsilateral cortico-cortical and callo3al projections, as well as cholinergic cells of the basal forebrain and cells in various brainstem sites, including dopamine cells of the substantia nigra/

• ventral tegmental area, serotonin cells of the dorsal raphe nuclei, and noradrenergic cells of the locus coeruleus 21,54,55,63,92. There is some evidence that cer- tain of these neurons respond to BDNF. BDNF has been shown, for example, to promote the survival of basal forebrain cholinergic neurons and of ventral mes- encephalic dopaminergic neurons in vitro 1'1°'24'37.

Thalamocortical, callosal and corticocortical affer- ents to the monkey prefrontal cortex appear to com- mence their major ingrowth into the cortex at the beginning of the period of gestation examined in the present study 18'20'52'75. In our material, BDNF mRNA was not detected at E l l0 but was evident by El21, so there appears to be a temporal correlation between the ingrowth of afferent axons and first detectable expres- sion of BDNF mRNA. This raises the possibilities that activity conveyed by the ingrowing axons triggers the

initial expression of BDNF in the prefrontal cortex as functional circuitry is established and/or that the onset of BDNF expression facilitates target finding by the afferent fibers. It has been shown that neuronal depo- larization resulting from activation of non-NMDA re- ceptors increases levels of BDNF mRNA in cultured hippocampal neurons 96, and that epileptiform activity is a powerful regulator of BDNF and NGF mRNA expression in rat hippocampus and neocortex ~6'3s. It is thought that thalamocortical, callosai and corticocorti- cai neurons utilize excitatory amino acids as their neu- rotransmitter(s) 5'~4'5°'a~. It also appears likely that pre- frontal cortical neurons :possess non-NMDA receptors at the stages of fetal development examined, since mRNAs encoding the subunits which include the kainate subclass are expressed by neurons in fetal rat cortex at developmental stages which are compara- tively much earlier than those examined in the monkey 65,67.

Although the laminar distribution of the majority of cells showing probe hybridization to BDNF mRNA appears more restricted than that of the terminal rami- fications of each of the major sets of afferent axons tT,zz.4U'41'45, only cell somata are normally visual- ized by in situ hybridization histochemistry. The major- ity of cells in layers III and VI, the major layers of probe hybridization, are pyramidal or modified pyrami- dal types with extensive dendritic ramifications in other layers including those in which thalamocortical, cortic- ocortical and callosal and other subcortical afferents end43, sT.

It is possible that cortical cells express BDNF mRNA at stages prior to E l l0 and then show non-expression for a period, since our material did not cover stages prior to E110. However, a study of the temporal distri- bution of BDNF, NGF and NT3 mRNA expression in developing rats showed that BDNF mRNA expression only rises substantially at latter stages of cortical devel- opment and into adulthood tS'Ss. BDNF mRNA expres- sion is low at earlier periods when neurogenesis and migration are the dominant events, periods that in monkeys would precede E110 7°. At these earlier stages, NT3 expression, by contrast, is high. Although the pattern of probe hybridization in the adults is regarded as the endpoint of the developmental sequence de- scribed, the use of different concentrations and types of fixatives in the fetuses and adults raises the possibil- ity that comparisons in hybridization patterns between adults and fetuses may be in part compromised. How- ever, the developmental changes in hybridization pat- terns mostly occurred in the fetuses, which were all perfused and processed in an identical manner. More- over, an adult-like distribution was largely present by

Page 8: Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

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latter fetal stages, so it is unlikely that the use of different fixatives in fetuses and adults seriously com- promises the interpretation of the changing develop- mental patterns.

BDNF and other members of the neurotrophin fam- ily exert their effects upon responsive cells through specific receptors (reviewed in ref. 83). The compo- nents of these receptors for each of the factors have not been completely characterized. Multiple receptors for NGF have been identified in cross-linking studies 32'6°. The low molecular weight NGF receptor binds equally with low affinity to NGF and BDNF 72. The trk family of proto-oncogenes appears to encode specific receptors for members of the neurotrophin family 48'5~. Recent evidence suggests that the NGF high affinity receptor is a complex composed of the NGF low molecular weight receptor and the t r k A

tyrosine kinase 25. A similar high affinity receptor for BDNF has been proposed containing the low molecu- lar weight NGF receptor and trk B 8°'s2. Additional, as yet unidentified, receptors for each of the neu- rotrophins may also exist. It is not known whether cells that give rise to afferents to the prefrontal cortex express the appropriate receptors and are thereby re- sponsive to BDNF. Transient immunoreactivity for the low-molecular weight NGF receptor has been de- scribed in the subplate of fetal cats and in ferrets 2, and has also been localized, in part, to profiles resembling growing axons in the subplate region of tile fetal moll- key visual cortex at stages prior to those examined in the present study c'). This may be an indication of the expression of neurotrophin receptors by cells of origin of afferent axons. By analogy with the peripheral ner- vous system, neurotrophin receptor expression in affer- ent neurons would be expected to follow neurotrophin expression by target cells. In the skin of the maxillary process, for example, NGF synthesis commences with the arrival of afferent axons derived from cells of the trigeminal ganglion, which only express NGF receptors after their axons reach the target t2.

The availability of BDNF or other neurotrophins as axons grow into their target layers in the cortex may stem the wave of naturally occurring cell death in the thalamus, cortex or other sites of origin of afferent fibers in a manner akin to that reported for BDNF upon rat dorsal root and retinal ganglion cells in vitro 42'57 or nodose ganglion cells in vivo 29. Neurons in the dorsal lateral geniculate nucleus of the fetal mon- key thalamus are eliminated in large numbers prior to the establishment of thalamocortical connectivity, but the cell population achieves a stable, adult-like number coincident with the period of ingrowth of geniculocorti- cal axons into target layers 9°. This is the period compa-

rable to that during which BDNF expression rises in the prefrontal cortex.

Significant changes in the distribution and morphol- ogy of populations of neurons identified by neurotrans- mitter or neuropeptide immunoreactivity occur within the developing monkey cortex during the period of gestation examined 33-36'95. It is also known that changes in afferent activity arriving in the adult monkey visual cortex regulates levels of a number of neuroactive molecules 6'26'27'44. NGF is known to promote dendritic arborization in cultured sympathetic ganglion cells TM, and when applied to basal forebrain cholinergic neu- rons, increases mRNA levels for choline acetyltrans- ferase and for NGF receptor 23'2s'64's9. NGF also in- creases levels of preprotachykinin and other neuropep- tid~ precursors when applied to cultured sensory or sympathetic ganglia cells 4'~'56'66. BDNF in the cortex could, therefore, also be acting as a local trophic agent in the developmental regulation of neurotransmitter and neuropeptide expression and their maintenance under activity-dependent conditions throughout the life of the individual.

The continued expression of BDNF mRNA by pre- frontal cortical neurons into adulthood suggests that populations of responsiv~ cells may require trophic factors for survival or maintenance of function throughout the lifetime of the animal. The integrity of the afferent systems to prefrontal cortex is essential for the maintenance of normal cognitive function 4'7'62. It is possible that a disruption in the availability, uptake or processing of BDNF by these potentially responsive cell types is associated with cognitive deficits or de- mentias which accompany neurodegenerative disor- ders such as AIzheimer's disease or disorders such as schizophrenia in which certain of the chemically defined afferent systems appear to be compro- misedg,~2,~,9,as.

Acknowledgements. Supported by Grants NS21377, NS24747 and MH4418 from the National Institutes of Health, and National Insti- tute of Metal Health, United States Public Health Service.

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