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Endocrinology Vol. 144, No. 11 5022-5029
Copyright © 2003 by The Endocrine Society

Estrogen Enhances Retrograde Transport of Brain-Derived Neurotrophic Factor in the Rodent Forebrain

Melinda K. Jezierski and Farida Sohrabji

Department of Human Anatomy and Medical Neurobiology, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114

Address all correspondence and requests for reprints to: F. Sohrabji, Department of Human Anatomy and Medical Neurobiology, Texas A&M University System Health Science Center, 228 Reynolds Medical Building, College Station, Texas 77843. E-mail: f-sohrabji{at}tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neurotrophins and their receptors activate signaling molecules to regulate neural function in development and adulthood. Neurons in the septum-diagonal band complex (or basal forebrain) derive neurotrophins through retrograde transport of these peptides from their forebrain targets. The present study tests the hypothesis that the gonadal hormone estrogen enhances retrograde transport of the neurotrophin brain-derived neurotrophic factor (BDNF). Estrogen increases BDNF expression in the horizontal limb of the diagonal band of Broca (hlDBB) and its forebrain target the olfactory bulb. In the present study, rhodamine-labeled (Rho-) BDNF injected into the olfactory bulb was rapidly transferred to neurons in the hlDBB. Significantly greater numbers of hlDBB neurons were retrogradely labeled with Rho-BDNF in animals pretreated with estrogen, compared with placebo-replaced controls. Anti-tyrosine kinase (trk) B antibodies injected into the olfactory bulb attenuated retrograde transport of Rho-BDNF in a dose-dependent manner, suggesting that estrogen may enhance BDNF transport in this circuit through regulation of its trk receptor. Anti-trkB antibodies also reduced cAMP response element binding protein phosphorylation in the hlDBB and combined injections of anti-trkA and trkB in the olfactory bulb reduced estrogen-induced increases in basal forebrain choline acetyltransferase. These studies support the hypothesis that estrogen facilitates neurotrophin transport in forebrain circuits.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEUROTROPHINS NGF (nerve growth factor) and BDNF (brain-derived neurotrophic factor) promote survival and/or differentiation of basal forebrain neurons in vitro (1) and prevent atrophy of axotomized cholinergic neurons in the adult basal forebrain (2). Neurotrophin receptors, which include p75 (3) and the tyrosine kinase receptors (trks; Refs.4, 5, 6), are intrinsic to basal forebrain neurons (7, 8) and trk receptors have been implicated in retrograde transport of growth factors. Subpopulations of basal forebrain neurons use ligand-specific tyrosine kinase receptors to transport neurotrophins from efferent sources (9, 10). The binding of neurotrophins to their trk receptor at the terminal results in the formation of a signaling endosome that propogates a retrograde signal (11 ; for reviews see Refs. 12 and 13). Hence, compounds that regulate trk receptors, such as the steroid hormone estrogen (14, 15, 16, 17), are likely to affect growth factor transport and signaling in the forebrain.

In the basal forebrain, estrogen receptors are colocalized to neurotrophin-sensitive neurons (18, 19), and estrogen increases expression of neurotrophins and their trk receptors in its telencephalic targets in the cerebral cortex, olfactory bulb and hippocampus (20, 21, 22). Our recent studies of the septobulbar circuit in young adult females suggest that estrogen may influence retrograde transport of growth factors, specifically BDNF. Within the basal forebrain, cells located in the horizontal limb of the diagonal band (hlDBB) project to the granule and periglomerular cell layers of the olfactory bulb, which synthesize BDNF and NGF, respectively (23). TrkB (24) and p75 (25) proteins are also expressed in terminals within the granule cell layer where hlDBB afferents are located and exogenous BDNF injected into the bulb is readily transported to the hlDBB (26). Both olfactory bulb and hlDBB neurons express estrogen receptors (15, 27, 28, 29) and estrogen replacement to ovariectomized females increases BDNF expression in both the olfactory bulb and the hlDBB, compared with placebo-replaced ovariectomized females. Moreover, estrogen also increases the expression of trkB, the cognate receptor for BDNF, in the olfactory bulb (15).

The present study shows that exogenous BDNF applied to the olfactory bulb is rapidly transported to the hlDBB, and estrogen treatment increases the numbers of hlDBB neurons that accumulate this peptide compared with placebo-replaced controls. Coinjections of anti-trkB antibodies into the olfactory bulb decrease BDNF transport to the hlDBB in a dose-dependent manner and rapidly inhibits cAMP response element binding protein (CREB) phosphorylation in the hlDBB. Finally, concurrent injections of trkA and trkB antibodies decrease choline acetyltransferase (ChAT) expression in the hlDBB.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal procedures
All animal procedures were reviewed and approved by the Institutional Animal Care Committee, and performed at an Association for Assessment and Accreditation of Laboratory Animal Care approved facility. Adult Sprague Dawley rats (200 g, Harlan Labs, Indianapolis, IN) were ovariectomized and immediately replaced with either a sc placebo pellet (PLC) or a 28-d time-release 17ß-estradiol pellet (E2; 0.5 mg; Innovative Research, 28-d release). Animals were injected with tracers and/or antibodies into the olfactory bulb, and were later anesthetized and perfused for histological analysis or decapitated to harvest tissue for protein analysis. Olfactory bulb and horizontal limb were rapidly dissected by the same individual (M.K.J.) to ensure consistency in microdissections. Trunk blood was collected at the time of tissue harvest for estimation of estradiol content by RIA (Diagnostics Systems Laboratories, Webster, TX) as before (15, 30). Protein extractions were performed as described previously (15, 30).

Stereotaxic surgeries
All surgeries were performed as described before (30, 31). Animals were anesthetized (ketamine, 200 mg/kg; xylazine, 10 mg/kg) and placed in a stereotaxic apparatus. Two small craniotomies were made at the following coordinates: 7.9 mm anterior to bregma and 1.0 mm lateral to the sagittal suture. A Hamilton syringe needle was briefly lowered to 3.4 mm ventral to the dura and immediately raised 0.2 µm to create a trough. Solutions were injected at a rate of 0.2 µl/20 sec. After injection, needle was raised slowly, craniotomies were covered with gel-foam, and skin flaps sutured with wound clips. To determine the size of the projection pathway, estrogen-replaced (n = 5) and placebo-replaced (n = 5) animals were injected with fluorogold (Flg) (2% wt/vol in water; Fluorochrome, Denver, CO) into the olfactory bulbs and animals were killed 6 d post surgery, by anesthetic overdose. To determine neurotrophin transport, estrogen (n = 6) and placebo-replaced (n = 6) animals were injected with 30 µg (10 µg/µl) of Rho-BDNF in PBS (see preparation below) unilaterally into the olfactory bulb. To determine receptor involvement in Rho-BDNF transport two sets of animals were prepared. In the first set, a group of intact animals was injected with 20 µg unlabeled BDNF in the olfactory bulbs before 20 µg of Rho-BDNF (n = 2) and horizontal limb sections were compared with those from rats injected with 20 µg of Rho-BDNF alone (n = 2). In the second set, animals were stereotaxically injected sequentially with anti-trkB polyclonal antibody [Transduction Laboratories (Lexington, KY); 0 µg (n = 2), 0.5 µg, 0.75 µg or 1 µg, n = 1 at each dose], fluorescein isothiocyanate-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, 1.5 µg) and Rho-BDNF (20 µg). Control animals were injected with PBS (n = 2) or anti-trkA pAB [4 µg; n = 2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)] followed by fluorescein isothiocyanate-conjugated secondary and Rho-BDNF.

Histological analysis
In all cases, animals were later overdosed with anesthetic and perfused with PBS and 4% paraformaldehyde. Brains were postfixed for 2 h in 4% paraformaldehyde, sucrose-loaded (overnight, 4 C) and later sectioned through the bulb and basal forebrain at 20 µm. Slides were inspected for labeled cells under fluorescent illumination. For all quantification, only animals with comparable injection sizes were analyzed (mean extent of injection site: PLC: 3100 ± 378 µm, E2: 2950 ± 246 µm). Hence the final sample for Flg-injected animals was n = 4 for each treatment (estrogen, placebo) and for Rho-BDNF injected animals it was n = 5 for each treatment. Every alternate section was saved through the basal forebrain. Slides were coded and an experimenter blind to treatment condition sequentially analyzed coded slides. Sections were sampled every 120 µm throughout the rostro-caudal extent of the basal forebrain and fluorescent-labeled cells were counted using an automated, image analysis software (BioQuant, R&M Biometrics, Nashville, TN). Because the hlDBB does not have distinct boundaries, it is not possible to obtain reliable volume estimates, hence it was not appropriate to use stereological cell counting techniques. Instead, the following strategy was employed. We first determined that the rostro-caudal extent of the hlDBB, as estimated by the number of sections on which Rho-BDNF or Flg-labeled cells were seen, was no different in estrogen and placebo-replaced animals. Secondly, to reduce the possibility that differences in retrograde-labeled cells was due to differences in cell density, we counted all Rho-BDNF-labeled or Flg cells in the hlDBB on a given section, as opposed to counting cells within a frame. Cells were clearly distinguished from cut axons by the presence of a nucleus or processes around the soma. The numbers reported here are total cell counts from each animal.

Phosphorylated CREB (pCREB) and ChAT analysis
To determine the contribution of trk receptors on pCREB and ChAT expression, estrogen-replaced animals were injected with anti-trkB (n = 3), anti-trkA (n = 3), or combined anti-trkA +anti-trkB antibodies (n = 3), followed by secondary as above (but no Rho-BDNF) and killed 6 h later. Controls were prepared for each antibody conditions (n = 3 in each case) and they received injections of PBS followed by secondary antibody as above. After rapid decapitation, brains were removed and microdissected regions were frozen at -80 C. Protein was extracted as before for use in Western blot analysis. Because of the short survival time, antipamezol (1 mg/kg) was administered sc to reverse effects of xylazine. The drug itself has no effect on protein expression. Western blots were performed as described before (15). Total protein from tissue lysates (50 µg) were size fractionated on a 12% gel. Membranes were sequentially incubated with primary antibodies [1:1000 pCREB-specific antibody (Upstate Biotechnology, Lake Placid, NY); 1:200 anti-ChAT (Chemicon, Temecula, CA)] followed by a biotinylated secondary [1:1000 biotinylated antirabbit for pCREB (Jackson ImmunoResearch Laboratories, West Grove, PA); 1:500 biotinylated antigoat for ChAT] and streptavidin-horseradish peroxidase [1/1000 (Amersham, Piscataway, NJ)]. An enzyme-catalyzed chemiluminescent reagent [Renaissance; NEN Life Science Products (Beverly, MA)] allowed signals to be detected on x-ray film and bands were quantified by densitometric analysis [Bio-Rad (Hercules, CA) Gel Doc 1000; version 1.4.1]. Protein loading was controlled in several ways. First, protein content of each sample was determined by bicinchoninic assay and equal amounts of protein were loaded onto the gel. Secondly, membranes were stripped and reprobed with a loading control. Because estrogen and the neurotrophins regulate many housekeeping proteins, rat IgG, which we have used previously as a reliable marker for protein loading (15, 32), was used here. In the case of CREB, stripping and reprobing for total CREB was not uniformly successful, especially in brain tissue analysis. In view of the small amounts of protein obtainable from the hlDBB and the heterogeneity of cells in this region, we found it was more accurate to use an unrelated protein as a loading control, as others have done (33). Samples were subjected to Western analysis at least twice for confirmatory data. Group differences were analyzed by Student’s t test or one-way ANOVA (P < 0.05).

Rhodamine labeling of proteins
Human recombinant BDNF [500 µg; molecular weight = 13,511; gift of Amgen (Thousand Oaks, CA)] or lysozyme (molecular weight = 14,300; Sigma, St. Louis, MO) was incubated with 50 µg of N-hydroxy succinimide-Rhodamine (molecular weight = 527; Pierce, Rockford, IL) for 2 h at 4 C. Samples were filter centrifuged at 5000 x g for 85 min at 4 C [Centricon filter YM10; Millipore (Amicon), Bedford, MA]. The extinction coefficient of BDNF and the concentration of purified conjugate (Rho-BDNF = 10 µg/µl; Rho-Lysozyme = 15 µg/µl) were determined using Beer’s Law. Efficiency of labeling was estimated at 75%, i.e. the filtered reaction product contained 0.75 moles Rho-BDNF/mol BDNF.

Rho-BDNF viability
To test viability of the labeled protein, two cell lines were used, PC12 cells that express trkA but not trkB and a conditionally immortalized olfactory bulb cell line (TCBOB; Ma, L., and F. Sohrabji, in preparation) that expresses trkB. TCBOB cells were prepared essentially similar to the procedure described for immortalized cortical cells (34). Putative bulb cells were harvested from embryonic d 12 Sprague Dawley rat pup brains under sterile conditions and transfected with the simian virus 40 large T antigen virus under the control of a temperature sensitive promoter (34). All experiments were performed with confluent cells maintained at the permissive temperature (39 C) for 3 d. PC12 cells (gift of D. Dohrman, Texas A&M University System Health Science Center) were maintained at 37 C and stimulated with 50 ng/ml NGF (80% confluent; Sigma) for 4 d.

Internalization studies
TCBOB and PC12 cells were incubated with media containing PBS (control), 10 µg Rho-Lysozyme, 10 µg unlabeled BDNF (Amgen), or 10 µg Rho-BDNF for 20 min followed by a 5-min PBS rinse. Cells were then fixed with a 4% paraformaldehyde (12 min). Fixed cells were incubated with DNA dye [Hoechst H33258, Polysciences Inc. (Warrington, PA), 10 µg/ml] for 10 min, rinsed, and coverslipped using 50% glycerol. Both Hoechst labeling and rhodamine labeling were visualized by fluorescence microscopy. To determine whether Rho-BDNF would stimulate CREB phosphorylation, olfactory bulb cells were exposed for 20 min to either PBS, unlabeled BDNF (200 ng/ml media), or Rho-BDNF (200 ng/ml media), and PC12 cells were exposed to PBS or the peptides at 1 mg/ml media for 20 min. Western blots were performed as before.

Antibody-inhibition of Rho-BDNF incorporation
Before incubating cells with Rho-BDNF (10 µg; 20 min), cells were treated with PBS (control), 1 µg anti-trkA pAB or anti-trkB pAB (10 min, Transduction Laboratories) followed by biotin-conjugated secondary antibody (1.5 µg; 10 min, Vector Laboratories). Because anti-trkB antibodies target only a portion of the ligand-binding/recognition domain, the secondary antibody was used to create a neutralizing effect, thus reducing binding for subsequent transport of the peptide. Cells were processed as above and visualized for Rho-BDNF incorporation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ovariectomized animals that received an estrogen pellet (E2) had an average plasma estradiol level of 78.5 (±5.87) pg/ml, similar to that seen at morning of proestrus in the intact cycling rat. Previous studies indicate that this level of hormone exposure resulted in increased trkA and trkB expression and decreased p75 expression (15). Placebo-replaced animals (PLC) had low (12.7 ± 3.03 pg/ml) levels of estrogen.

Flg injected into the olfactory bulbs (Fig. 1AGo) was primarily transported to neurons in the hlDBB (Fig. 1BGo), although a few Flg-labeled cells were seen in the medial septum/vertical limbs of the diagonal band. There was no difference in the numbers of Flg-labeled cells in the hlDBB of placebo-replaced (Fig. 1DGo) or estrogen-replaced animals (Fig. 1EGo). Rho-BDNF injected into the olfactory bulb allowed for specific identification of basal forebrain neurons that retrogradely transported the peptide. Rho-BDNF-labeled fibers were first detected at 30 min (data not shown) and strongly fluorescing cell bodies were first seen at 2 h. As with Flg labeling, Rho-BDNF-labeled cells were primarily seen in the hlDBB. Many more Flg-labeled neurons were detected in the hlDBB than Rho-BDNF-labeled neurons, although the dose of Flg and Rho-BDNF and their postinjection survival times were necessarily different. It is more than likely, however, that BDNF transporting-cells comprise only a portion of the projecting neurons. No labeled cells were detected in the hlDBB when animals were injected with the N-hydroxy succinimide-rhodamine compound alone at 2 or 8 h (data not shown). Rho-BDNF-labeled cells were seen in the hlDBB of both estrogen (E2)-replaced and placebo (PLC)-replaced rats; however, estrogen-treated animals exhibited significantly more Rho-BDNF-labeled cells (Fig. 1Go, F and G) at 2 h post injection.



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  		FIG. 1. Retrograde transport in the septobulbar circuit. Flg injected into the olfactory bulbs (A; needle tract indicated by arrow) is retrogradely transported to the basal forebrain. The majority of retrograde label is confined to cells of the horizontal limb of the diagonal band of Broca, shown in (B) and in Nissl stain in (C). Bar, 685 µm (in B) and 171 µm (in C). Estrogen (D) and placebo-replaced (E) animals have similar numbers of Flg-labeled neurons in the hlDBB (D). Rho-BDNF injected into the olfactory bulb is rapidly transported to the hlDBB; however, the number of hlDBB neurons labeled with Rho-BDNF is significantly greater in estrogen-treated animals (F) as compared with controls (G) (P < 0.05). Bar, 137 µm. vlDBB, Vertical limb of diagonal band; LV, lateral ventricle; AC, anterior commisure; ON, optic nerve.

 
Several tests ensured that rhodamine labeling of BDNF did not alter the biological effectiveness of the peptide. Rho-BDNF was rapidly internalized into cultured olfactory bulb cells (Fig. 2AGo), whereas these cells did not internalize another similar sized Rho-labeled protein, lysozyme (Fig. 2BGo). Furthermore, prior exposure to anti-trkB antibodies (Fig. 2CGo) attenuated Rho-BDNF uptake, whereas anti-trkA antibodies did not affect Rho-BDNF uptake (Fig. 2DGo), indicating receptor-specific internalization of the labeled peptide. PC12 cells that have trkA receptors but lack trkB receptors (35) failed to internalize Rho-BDNF (Fig. 2Go, E and F). Finally, both Rho-BDNF and unlabeled BDNF were equally effective at stimulating pCREB in cultured olfactory bulb cells (Fig. 3Go, G and H), but had no effect on PC12 cells.



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  		FIG. 2. Immortalized olfactory bulb cells internalized rhodamine-labeled BDNF (Rho-BDNF; (A) but not rhodamine-labeled lysozyme (Rho-Lys) (B), a similar-sized peptide. Nuclei are visualized with a DNA dye (green-blue). Pretreatment with anti-trkB antibody limited Rho-BDNF incorporation in olfactory bulb cells (C) in comparison to (A) pretreatment with PBS; however, pretreatment with anti-trkA (D) did not affect BDNF internalization. PC12 cells, which lack trkB receptors, internalized virtually no Rho-BDNF (E) or Rho-Lys (F). Whereas Rho-BDNF had no effect on CREB phosphorylation in PC12 cells, both unlabeled BDNF and Rho-BDNF equally stimulated CREB phosphorylation (43 kDa) in trk-B-positive olfactory bulb cells compared with PBS controls using an antibody specific for the phosphorylated form of CREB (pCREB) in a Western blot assay (G). Densitometric analysis indicated a nearly 2-fold increase in CREB phosphorylation by BDNF and Rho-BDNF (H). Bar, 50 µm.

 


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  		FIG. 3. Rho-BDNF retrograde transport in the septobulbar circuit is dependent on the trkB receptor. Rho-BDNF-labeled cells in the hlDBB visualized 2 h after olfactory bulb injection of Rho-BDNF (A). Inset shows fluorescently labeled cells at high magnification. Prior injection with unlabeled BDNF markedly decreased the number of Rho-BDNF-labeled cells compared with Rho-BDNF alone (B). Bar, 50 µm (for A and B). When compared with PBS-controls (C), very few labeled cells were seen in animals injected with 1 µg anti-trkB (D) whereas 1 µg anti-trkA (E) did not affect the number of Rho-BDNF-labeled cells. Bar, 100 µm (for D–F). Increasing concentrations of anti-trkB antibody before Rho-BDNF injection linearly decreased the number of Rho-BDNF-labeled cells (f; r = 0.9827, P < 0.05, n = 5).

 
Forebrain transport of Rho-BDNF also appeared to be receptor-mediated because coinjection with an equal concentration of unlabeled BDNF reduced the number of Rho-labeled cells by 50% (Fig. 3Go, A and B). Furthermore, the number of Rho-BDNF accumulating cells in the hlDBB decreased linearly with increasing concentrations of anti-trkB antibody (r = -0.98; df = 3; P < 0.01) (Fig. 3FGo) injected into the olfactory bulb. At the highest concentration of anti-trkB (Fig. 3DGo), the number of cells retrogradely labeled with Rho-BDNF decreased to one fifth of controls (Fig. 3EGo), whereas similar doses of anti-trkA (Fig. 3EGo) did not affect the number of Rho-labeled neurons in the hlDBB, supporting the hypothesis that BDNF transport was trkB dependent.

Antibody neutralization treatment in vivo impaired a functional signaling component in the hlDBB neurons. Anti-trkB treatment reduced endogenous CREB phosphorylation 6 h later (Fig. 4AGo) but did not affect expression of ChAT (Fig. 4BGo), the acetylcholine-synthesizing enzyme. Anti-trkA antibodies also had no effect on ChAT expression (Fig. 4BGo); however, coadministration of anti-trkA and -trkB antibodies in the olfactory bulb significantly reduced ChAT expression in the hlDBB of estrogen-replaced animals. ChAT expression in the estrogen-treated animals that received trkA/trkB antibodies was reduced toward the levels seen in estrogen-deprived controls (Fig. 4CGo), suggesting that estrogens effects on ChAT expression may be mediated by the neurotrophin receptors.



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  		FIG. 4. Blocking Trk receptors affects signaling in the septobulbar circuit. A, Anti-trkB (4 µg) antibodies injected into the olfactory bulbs of estrogen-treated rats (E2 Anti-trkB) significantly attenuated phosphorylated CREB expression in the hlDBB (pCREB; 45 kD) compared with controls injected with PBS (E2 Sham; P < 0.05). Protein samples from the same animals were used for both gels and normalized for protein loading using an unrelated peptide. Anti trkB and anti-trkA antibodies did not affect ChAT expression in the hlDBB of estrogen-treated animals (B). However, combined injections of anti-trkA and anti-trkB antibodies significantly reduced ChAT expression in the hlDBB of estrogen-treated animals, such that ChAT expression in this group was indistinguishable from placebo-replaced animals (C). Histogram depicts mean optical densities of ChAT-immunoreactive bands in estrogen and placebo replaced animals, and estrogen animals injected with anti-trkA/anti-trkB antibodies (* and **, P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data support the hypothesis that estrogen stimulates BDNF translocation from the olfactory bulb to the hlDBB via a trkB-dependent mechanism. Additionally, inhibition of the neurotrophin system in the efferent limb of the septobulbar circuit affects CREB signaling and ChAT expression in the basal forebrain. This study provides the first direct evidence that estrogen may enhance retrograde trafficking of neurotrophins, a mechanism that may contribute to the neuroprotective roles described for the hormone (30, 36, 37).

The BDNF gene contains an estrogen response element-like motif capable of binding estrogen receptors (22), and estrogen treatment increases both BDNF mRNA (22) and protein (20) in the female rat olfactory bulbs. The estrogen receptor transduces the actions of estrogen and currently two types of estrogen receptors are identified; the classical estrogen receptor now called ER-{alpha} (38) and the more recently cloned ER-ß (39). Transcripts for both ER-{alpha} and ER-ß have been localized to the olfactory bulb (28, 40) and ER-{alpha} protein is present in olfactory bulb lysates (15). However the present study does not distinguish the specific receptor that mediates estrogen actions on BDNF transport in the olfactory bulb-hlDBB circuit, or whether estrogens actions are nongenomic, via hormone action on other signaling molecules.

Two types of transmembrane receptors bind BDNF: p75, which binds all known neurotrophins, and trkB, a member of the receptor tyrosine kinase family. Both classes of neurotrophin receptors are differentially regulated by estrogen (16, 17, 41, 42). Several lines of evidence support the hypothesis that increased retrograde transport of BDNF in estrogen-replaced animals may be due to hormonal regulation of trkB. Both p75 and trkB have been shown to mediate BDNF transport in the peripheral nervous system (10, 43), although trkB is believed more crucial for its transport in central circuits (43). In the septobulbar circuit, where estrogen promotes retrograde transport of BDNF, estrogen only increases the expression of one BDNF receptor, namely trkB. Transport of exogenous BDNF in this circuit was successfully attenuated by prior injections of trkB antibodies into the olfactory bulb, further implicating this receptor in the transport of bulbar BDNF. It may be argued that increased BDNF transport may be related to estrogen-dependent changes in the size of the projection pathway between the hlDBB and the olfactory bulb. Specifically, estrogen may increase the numbers of bulb projecting neurons in the hlDBB. However, the Flg studies show that the number of neurons that project to the olfactory bulb is not altered by estrogen, indicating that estrogen favors BDNF-specific transport in this circuit. An alternative hypothesis to explain the present data are that an increase in BDNF-concentrating cells in the hlDBB may result from hormonal regulation of general transport mechanisms. In fact estrogen has been shown to promote retrograde transport of viruses in the forebrain (44) presumably by increasing axonal transport. Regulation of the motor protein dynein, which associates with trk and is thought to propel retrograde transport of trk-neurotrophin complexes (45, 46), may also provide another mechanism by which estrogen may modulate retrograde transport. Although estrogen regulation of dynein expression is currently not known, preliminary data comparing estrogen and placebo replaced animals on a DNA microarray suggests that estrogen does not regulate mRNA for light, intermediate or heavy chain dynein (Lewis, D. K., and F. Sohrabji, unpublished observations).

Previous work from this laboratory has shown that estrogen increases bulbar trkB (15). This increased bulbar trkB theoretically represents an increase in receptors located on cells intrinsic to the olfactory bulb, as well as on terminals from afferent nuclei such as the horizontal limb. Although our analysis did not distinguish between trkB regulation on intrinsic olfactory bulb fibers vs. afferent fibers, both bulbar (22) and basal forebrain (19) neurons are sensitive to estrogen, increasing the likelihood that the elevation in bulbar trkB may be due in part to receptors located on hlDBB afferents.

The functional significance of retrograde neurotrophin transport in the septobulbar circuit is underscored by the observation that CREB phosphorylation in the hlDBB is rapidly reduced when anti-trkB antibodies are injected into the olfactory bulb. Studies on NGF-trkA, have shown that neurotrophins activate several signaling molecules. Signaling endosomes, containing trk receptors on the vesicle membrane and neurotrophins in the lumen, are also associated with signaling molecules such as ERKs and PI3K (11). Recent studies also suggest that retrograde trk activation stimulates specific signaling pathways distinct from those activated at the soma, in particular CREB activation, which discriminates between local and retrograde availability of neurotrophin. CREB activation is dependent on internalization of NGF at terminals (47) and requires a novel MAPK, ERK-5 (48). Transport of the ligand-trkB receptor complex from terminals to the cell body is also critical for activating pCREB (33). Estrogen has been shown to regulate CREB phosphorylation in hypothalamic nuclei (49, 50), suggesting a mechanism by which estrogen may exert rapid cellular actions and regulate genes that lack an estrogen response element. In other forebrain nuclei, estrogens ability to regulate CREB activation is dependent on the brain region, the duration of estrogen treatment (chronic vs. acute) and may regulate CRE-DNA binding without phosphorylating CREB (51). Estrogen regulation of CREB activation may transduce hormonal action on cell morphology as in cultured hippocampal cells, where estrogen-related increases in spine density are linked to CREB activation (52). Estrogen and the neurotrophins exert similar growth promoting actions and although the acute effects of estrogen appear to be independent of the trks (53), long-term estrogen treatment may affect specific signaling pathways by regulating constitutive expression of the trks.

The pathway between the hlDBB and the olfactory bulb contains cholinergic neurons (54), whose early survival and adult function can be modulated by neurotrophins. ChAT, the acetylcholine-synthesizing enzyme, contains a cAMP response element in its 5' region. The present data showed that whereas anti-trkB injections attenuated CREB activation, it failed to affect ChAT expression in the basal forebrain. TrkA antibodies alone also did not reduce ChAT expression, although combined injections of trkB and trkA-specific antibodies for significantly reduced expression of this enzyme. Because both NGF and BDNF have been shown to regulate ChAT expression (55), this experiment suggests that, in the olfactory bulb-hlDBB pathway, the combined loss of NGF and BDNF signaling is more effective than the loss of either neurotrophin system individually. Interestingly, whereas ChAT expression is enhanced in estrogen-replaced rats, compared with placebo-treated controls, antibody treatment in estrogen-treated animals reduces ChAT. These observations suggest that estrogen actions on the cholinergic system may in part be mediated by hormonal regulation of the neurotrophin systems, as has been shown for neuron accumulation in the hormone-sensitive song nuclei of canaries (56).

Rapid neurotrophin trafficking may significantly impact on a cell’s ability to function in learning and memory events. In addition to their well-established roles in developmental differentiation and maintenance of adult neurons, these peptides may also mediate synaptic plasticity (57). BDNF enhances hippocampal synaptic efficacy and long-term potentiation (58). Furthermore, hippocampal long-term potentiation induction is impaired in the conditional trkB knockout mouse (59) and the BDNF knockout mouse (60) and in the latter, this impairment can be restored with exogenous BDNF application (61). Other evidence indicates that depolarization and elevated cAMP lead to rapid translocation of trkB to the plasma membrane (62) and that exogenous BDNF enhances synaptic hippocampal transmission via activation of trk receptors (63). Adequate BDNF occupation of newly mobilized receptors may not only enhance activity-dependent memory consolidation but maintain neuronal usage, and thus synaptic stability.

Chronic decreases in trophic availability in the forebrain may contribute to the progression of neurodegenerative diseases. In a mouse model of Down’s syndrome, defective neurotrophin transport was associated with degeneration of basal forebrain cholinergic neurons, which was reversed by exogenous NGF (64). Striking losses in trk receptors noted in the brains of AD patients, postmortem, supports the idea that deficits in transport/accumulation of trophic peptides may contribute to the etiology of the disease (10, 65, 66). If decreased trophic factor availability contributes to the neurodegenerative process, then hormones such as estrogen, that enhance trkB synthesis and transport of BDNF, will be a beneficial complement to the therapeutic process.


   Acknowledgments
 
We thank Amgen, Inc. for recombinant BDNF; Dr. D. Dohrman [Texas A&M University System (TAMUS) Health Science Center (HSC), College Station, TX] for PC12 cells; Dr. R. Miranda (TAMUS HSC) and Dr. A. Sinai (Yale University, New Haven, CT) for valuable discussions; Drs. W.-J. Chen (TAMUS HSC) and M. Zoran (Texas A&M University) for critically evaluating the manuscript and Sangeeta Neela, Leia Brundige, Angela Buchanan, Olga Marroquin, and Melissa Scarborough for technical assistance.


   Footnotes
 
This work was supported by National Institute of Neurological Disorders and Stroke/NIH Grant NS36297 and National Institute on Aging/NIH Grant AG19515.

Abbreviations: BDNF, Brain-derived neurotrophic factor; ChAT, choline acetyltransferase; CREB, cAMP response element binding protein; E2, 17ß-estradiol; Flg, fluorogold; hlDBB, horizontal limb of the diagonal band of Broca; NGF, nerve growth factor; pCREB, phosphorylated CREB; PLC, placebo; Rho, rhodamine; trk, tyrosine kinase.

Received June 9, 2003.

Accepted for publication July 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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