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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


    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.


    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.

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    of particular neural circuits, However, to date, most studies of BDNF mRNA localization in rodents have emphasized regional distributions rather than the cell types expressing the BDNF gene, and studies in higher primates have generally been restricted to hippocam- pus 6s'69. No studies have yet examined BDNF mRNA

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

    chemistry to examine the expression and developmen- tal regulation of BDNF mRNA 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.


    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 -70C. 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 4C. 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 ix 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 60C. 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 60C.

    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 4C, 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.


    Distribution of BDNF mRNA in adult monkey prefrontal cortex

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

    labeled probe complementary to BDNF mRNA were

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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 arrow


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