Interactions Between Nerve Growth Factor Binding and Estradiol in Early Development of the Zebra Finch Telencephalon

Interactions Between Nerve Growth Factor Binding and Estradiol in Early Development of the Zebra Finch Telencephalon Margarita L. Contreras, 1 Juli Wade 2 1 Department of Pharmacology and Toxicology, Life Science Bldg., Program in Neuroscience, Michigan State University, East Lansing, Michigan 48824 2 Departments of Psychology, and Zoology, Program in Neuroscience, Michigan State University, East Lansing, Michigan 48824 Received 18 December 1998; accepted 11 February 1999 ABSTRACT: The zebra finch telencephalon exhibits rapid and substantial development in the first few weeks after hatching. In parallel, the rate of estradiol synthesis is very high in the zebra finch forebrain, and estradiol can have potent neurotrophic effects in specific telencephalic regions, including those that control the learning and production of song. In an attempt to elucidate mechanisms regulating telencephalic development, potentially including a role for the large capacity for estrogen production, 125 I nerve growth factor (NGF) binding was measured in homogenates of telencephalon from zebra finches age 3, 15, 30, 60, and 120 days. The highest density of low- and high-affinity 125 I-NGF binding sites was observed in 3-day-old finches. Using an aromatase inhibitor, Fadrozole, to reduce estradiol levels in 1 to 4-day-old zebra finches significantly decreased both high- and low-affinity 125 I-NGF binding sites. Conversely, treating adult or 8 to 14-day-old hatchlings with estradiol increased high-affinity 125 I-NGF binding sites. These results are consistent with the hypothesis that estradiol influences the level of NGF receptors, and suggest one mechanism through which the steroid could affect brain development. The data also indicate that estradiol and NGF activity may be important for very early development of the telencephalon. 1999 John Wiley & Sons, Inc. J Neurobiol 40: 149 157, 1999 Keywords: songbird; neurotrophin; estrogen; aromatase; trka Zebra finches hatch in a very altricial state and exhibit a remarkable rate of development in the first month of life. During that time, the nervous system must change from one that supports an individual with closed eyes, extremely limited movement, and an inability to perform basic life-supporting functions, such as feeding and thermoregulation, to one that facilitates the production of complex behaviors, such as foraging and flying. Another intriguing feature of the zebra finch brain is that the telencephalon exhibits an unusually large capacity to synthesize estrogens Correspondence to: J. Wade Contract grant sponsor: NIH; contract grant number: MH55488 1999 John Wiley & Sons, Inc. CCC 0022-3034/99/020149-09 from androgens (Schlinger, 1997). Activity of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol and androstenedione to estrone, is high both during development and in adulthood (Schlinger and Arnold, 1991, 1992; Wade et al., 1994, 1995; Cam and Schlinger, 1998). Aromatase mrna is also abundant in the zebra finch telencephalon (Shen et al., 1994). Because this high rate of estradiol production has been documented during the first few weeks after hatching in homogenates of the whole brain as well as in tissue punches, subcellular fractions, and cultures from telencephalon (Vockel et al., 1990; Schlinger and Arnold, 1992; Wade et al., 1995), it seems plausible that the steroid participates in regulating development of the tissue. Consistent 149

150 Contreras and Wade with that hypothesis, exogenous estradiol treatment in hatchlings can exert neurotrophic effects in specific regions of the telencephalon that control song production (Gurney, 1981; Simpson and Vicario, 1991b; Grisham and Arnold, 1995). These brain regions are highly sexually dimorphic, being larger in volume and containing more and larger cells in males who sing than in females who do not (reviewed in Arnold et al., 1996). Evidence exists suggesting that gonadal steroids may not normally control the process of sexual differentiation (Wade and Arnold, 1996; Wade et al., 1996; Springer and Wade, 1997). However, clues about factors regulating masculine development will likely come from analysis of the mechanisms through which exogenous estradiol exerts its effects. Thus, the present series of studies was conducted to elucidate factors involved in telencephalic development in the zebra finch, including a potential mechanism through which estradiol could support neuronal migration, survival, or differentiation. Nerve growth factor (NGF) aids in the development, survival, and maintenance of peripheral sympathetic and sensory neurons and central cholinergic neurons during development in mammals (Gibbs, 1994). These latter sets of neurons are thought to be involved in learning and memory. Similarly, cholinergic cells are abundant in the zebra finch telencephalon, including regions required for the learning of courtship song (Ryan and Arnold, 1981). Most important, though, interactions between estradiol and NGF receptors have been reported in a number of systems, including PC12 cells, explants from the developing rat forebrain, and dorsal root ganglia and medial septum of adult rats (Gibbs and Pfaff, 1992; Gibbs et al., 1994; Miranda et al., 1994; Sohrabji et al., 1994a,b; Miranda et al., 1996; Gibbs, 1997). Nerve growth factor binds to two specific receptors: trka and p75 (Chao and Hempstead, 1995). Binding to trka leads to activation of intrinsic receptor tyrosine kinase activity, resulting in autophosphorylation of the receptor and tyrosine phosphorylation of key regulatory proteins. The p75 receptor binds with similar affinity each member of a family of neurotrophins that includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). The function of the p75 receptor is not well defined. It has been shown that p75 receptors increase the rate of association of NGF with the trka receptors (Mahdeo et al., 1994), resulting in increased affinity of NGF binding and increased responsiveness to NGF. However, the mechanism for this interaction is not clear. Nerve growth factor binding has not been characterized in songbirds. Therefore, to begin to address the relationship between NGF activity and estradiol in the zebra finch telencephalon as a whole, we first assessed the developmental time course of NGF binding in telencephalic homogenates of males and females (Experiment 1). Then, to specifically test whether estradiol levels influence NGF binding, zebra finches were treated either with the potent aromatase inhibitor Fadrozole (Wade et al., 1994) (Experiment 2) or estradiol (Experiment 3) to decrease or increase estrogen exposure. MATERIALS AND METHODS Treatments and Tissue Collection Zebra finches were maintained in a breeding colony at Michigan State University. They were housed in large communal aviaries containing approximately five breeding pairs and their offspring. Nest boxes were checked daily, and each hatchling was marked with a unique toe-clip pattern. The day the bird was found was considered day 1. Individuals reach sexual maturity at about 90 days of age. Birds were banded before they fledged so that they could be tracked into adulthood when necessary. At appropriate ages, birds were rapidly decapitated, and the telencephalon was immediately removed and frozen on dry ice. Tissue was frozen at 80 C until use. The sex of each individual was determined by examination of the gonads under a dissecting microscope at the time of sacrifice. In Experiment 1, the telencephalon was collected from males and females at each of the following posthatching ages for assessment of 125 I-NGF binding: days 3, 15, 30, 60, and 120. In Experiment 2, estradiol synthesis was inhibited by injecting 20 g Fadrozole in 10 L 0.75% saline into the breast muscle on each of posthatching days 1 3. Control birds received the vehicle alone. Each telencephalon was collected on day 4. This treatment reduces telencephalic aromatase activity by more than 80% in 4 to 6-day-old zebra finches (Wade et al., 1994). In Experiment 3, birds received seven daily injections into the breast muscle of estradiol benzoate (EB; Steraloids) or steroid suspending vehicle (SSV) (recipe in Wade et al., 1997) as either adults or hatchlings (days 8 14). Telencephalons were collected approximately 24 h following the last injection. Adults received 100 g EBin20 L SSV each day. Doses were adjusted in the young birds by injecting smaller volumes to provide similar exposure to estradiol per gram body weight (based on weights obtained from untreated individuals randomly selected from colony) as follows: day 8 35 g EB; day 9 55 g; day 10 65 g; days 11 14 75 g. The ages of treatment were based on the results of Experiments 1 and 2 (see below). Sample sizes for each experiment are indicated in the figure captions.

Zebra Finch NGF Binding and Estradiol 151 Assay for Telencephalic 125 I-NGF Binding Zebra finch telencephalons were homogenized in phosphate-buffered saline (PBS) with 1 mm phenylmethylsulfonyl fluoride (PMSF). The PBS consisted of 138 mm NaCl, 2.7 mm KCl, 8 mm Na 2 HPO 4, and 1.47 mm K 2 HPO 4 (ph 7.4). The homogenates were centrifuged at 20,000 g for 10 min, and the pellets were resuspended in 20 mm Tris (ph 7.4) containing 0.32 M sucrose, 1 mm ethylenediaminetetraacetic acid (EDTA) and 1 mm PMSF. The samples were then processed by serial centrifugation as follows, to obtain a plasma membrane-enriched preparation. The samples were centrifuged at 1000 g for 10 min, and the pellets were resupended and recentrifuged. The supernatants from the two centrifugation steps were combined and centrifuged for 60 min at 100,000 g. The pellets were resuspended in PBS with 20 mm Hepes for use in the binding assays. An aliquot of each sample was used for the determination of protein concentration by the method of Bradford (1976). For the binding assay, NGF purified from male mouse submaxillary glands was iodinated using lactoperoxidase as described by Angus and Contreras (1995). Mouse NGF has successfully been used in avian tissue both to identify the anatomical distribution of putative NGF receptors and to biochemically characterize NGF binding sites (Sutter et al., 1979; Bernd, 1985). The plasma membrane enriched preparations from zebra finch telencephalons were incubated with 125 I-NGF (0.01 10 nm) in PBS containing 20 mm Hepes and 0.3% bovine serum albumin, for 180 min at 30 C in microtip microcentrifuge tubes. Nonspecific binding was defined by 100-fold excess of unlabeled NGF. Following the incubation, the membranes were centrifuged at 15,000 g for 10 min, and the supernatants were aspirated. The tip of each tube with the membrane pellet was cut off and placed in a gamma counting vial. The radioactivity associated with the membranes was quantitated in a gamma counter. The values for density of receptors were corrected for the level of protein in the assay. The equilibrium dissociation constant (K d ) and density of receptors (B max ) for both the high-affinity binding sites (R H ) and low-affinity binding sites (R L ) were calculated using nonlinear regression analysis (LIGAND program). RESULTS Experiment 1 Binding characteristics were initially determined in the telencephalic homogenates obtained from 3-dayold males. Scatchard analysis indicated the presence of both high- and low-affinity binding sites (Fig. 1). In analyses of developmental changes, age-dependent decreases in B max values (Fig. 2) clearly existed. The highest level of high- and low-affinity binding sites was seen at the lowest age examined, posthatching day 3. At day 15, there was an approximate 95% and 85% decrease in B max values for high- and low-affinity binding sites, respectively. Increases in age beyond posthatching day 15 did not result in further changes in the density of 125 I-NGF binding sites (p.05, day 3 compared to all other ages). The estimate of total binding capacity (B max ) was greater in males than females only at 3 days of age and only for the lowaffinity component (p.05) (Fig. 2). No consistent changes were detected across ages in the equilibrium dissociation constants for either the high- (K H ) or low-affinity (K L ) binding sites (all p.05) (Fig. 2). However, small (approximately 1 nm) but statistically significant differences were detected in K L only, such that the binding affinity on days 15, 30, and 60 was lower in males than in females (p.05). Since large or consistent sex differences in 125 I-NGF binding Statistical Analyses In Experiment 1, the Kruskall Wallis analysis of variance on ranks test was performed to determine whether a significant effect of age existed in K d and B max values. The Student Newman Keuls test was performed to determine which ages were statistically different from each other and whether the sexes differed significantly at any age. In Experiments 2 and 3, comparisons in B max values between control and Fadrozole- or estradiol-treated individuals were made with the Mann Whitney rank sum test. Figure 1 Specific binding of 125 I-NGF to partially purified membranes from zebra finch telencephalons. The binding of 125 I-NGF (0.01 10 nm) in the presence and absence of 100-fold excess of unlabeled NGF to partially purified plasma membranes was determined. Representative data from a 3-day-old male are shown as a Scatchard plot (line) and the two binding components (dashed lines) that contribute to the curved Scatchard plot (partial F test indicates significant improvement of fit to a two-binding component model; p.05). Characteristics of both the high- and low-affinity sites (density of binding sites, B maxh and B maxl ; equilibrium dissociation constants K H and K L ) were determined by nonlinear regression analysis.

152 Contreras and Wade Figure 2 Equilibrium constants for binding of 125 I-NGF in zebra finches at different ages. The specific binding of 125 I-NGF (0.01 10 nm) to partially purified telencephalon plasma membranes was analyzed using nonlinear regression analysis to determine the density of the high- (A) and low-affinity (B) binding sites and K d values (C) for male (closed triangles) and female (closed circles) zebra finches (n 3 of each sex at each time point). *Sex difference at a specific age, p 0.05; **values for days 15 120 are all significantly different from day 3, p.05. were not observed, a mixture of male and female birds was used in subsequent binding studies investigating the effects of manipulating estradiol levels (Experiments 2 and 3). Experiments 2 and 3 The goal of these two experiments was to determine whether modifying estradiol availability would alter the density of NGF receptors. Therefore, estradiol synthesis was inhibited by administering Fadrozole to hatchlings when NGF binding was relatively high, and estradiol was administered to both young birds (8 14 days old) and adults when the density of 125 I-NGF binding sites was low. In the telencephalons of 4-day-old zebra finches which had been treated with Fadrozole on days 1 3, there was an approximate 60 75% decrease in B max values for the high- and low-affinity 125 I-NGF binding sites compared to controls (both p.05) (Fig. 3). Treatment with Fadrozole did not produce significant alterations in the K d values (data not shown). In adult birds, treatment with estradiol resulted in a 275% increase in the density of highaffinity 125 I-NGF binding sites (p.05) (Fig. 4), but no statistically significant change in the density of low-affinity 125 I-NGF binding sites. Similarly, treatment of hatchlings with estradiol resulted in a 418% increase in the density of high-affinity 125 I- NGF binding sites (p.05), without a significant change in the density of low-affinity binding sites (Fig. 4). Treatment with estradiol also did not change the K d values for binding of 125 I-NGF (data not shown).

Zebra Finch NGF Binding and Estradiol 153 Figure 3 Effect of decreased estrogen synthesis on the binding of 125 I-NGF. Birds were injected daily on posthatching days 1 3 with either 20 g Fadrozole (treated) or vehicle (control). Subsequently, the binding of 125 I-NGF was examined in telencephalons from 4-day-old zebra finches to determine the B max values for the high- (R H ) and low-affinity (R L ) binding sites. Each value represents the mean standard error of the mean for three finches (one male and two females in each treatment group). *p 0.05, different from control. cerebellum of the developing rat, NGF receptor immunoreactivity peaks on postnatal day 6 and 14, respectively, and then declines (Eckstein, 1988). In contrast, the ontogenetic changes in NGF binding sites in zebra finches appear different from at least some mammalian systems. For example, in the rat caudateputamen and basal forebrain, NGF receptors and binding sites increase in early development and then remain at relatively high levels into adulthood (Eckstein, 1988; Mobely et al., 1989). In the zebra finch system, diminishing estradiol synthesis during the period when telencephalic concentrations of the hormone are high caused a dramatic reduction in both the high- and low-affinity 125 I-NGF binding components. Conversely, treating birds with exogenous estradiol at both young and adult stages when estradiol levels are normally relatively low caused significant increases in the level of high-affin- DISCUSSION Specific 125 I-NGF binding was detected in plasma membrane enriched preparations from the telencephalons of zebra finches ranging in age from posthatching day 3 to 120. The pattern of this binding activity was consistent with the presence of both high- and low-affinity binding components, with K d values comparable to those found in both mammalian and chick tissue. In mammalian cells, the low- and highaffinity binding components have K d values of approximately 1 nm and 10 100 pm, respectively (Chao and Hempstead, 1995), and in the chick, K d values of 1.7 nm and 23 pm have been reported (Sutter et al., 1979). General developmental patterns of NGF receptor expression similar to that in the zebra finch have also been documented in chick and some mammalian systems. The highest density of binding sites in the zebra finches was detected at 3 days of age, and the level declined by 15 days posthatching to that seen in the adult. Because of differing rates of development (among other factors), it is difficult to make direct comparisons across species. However, a similar peak of NGF receptors appears to be present during early development in the chick (which is much more precocial than the zebra finch). In whole chick brain, NGF receptor mrna peaks at embryonic day 12 (sixfold increase over day 8), and by embryonic day 18 declines to levels similar to those seen in the adult (Ernfors et al., 1988). Similarly, in the thalamus and Figure 4 Effect of estradiol treatment on the binding of 125 I-NGF. Adult (top) or 8- to 14-day-old hatchling (bottom) zebra finches were treated with seven daily injections of estradiol (E2) or vehicle (control). Subsequently, the binding of 125 I-NGF in a plasma membrane enriched preparation from the telencephalons was examined to determine the B max values for the high- (R H ) and low-affinity (R L ) binding sites. Each value represents the mean standard error of the mean for three hatchling (EB: two males and one female; control: three males) or four adult (two of each sex) zebra finches. *p.05, different from control.

154 Contreras and Wade ity 125 I-NGF binding sites. These results are consistent with those observed in mammalian systems. In the adult rat basal forebrain, trka mrna decreases in some areas (including the horizontal limb of the diagonal brand of Broca) following ovariectomy, and 3 days of estradiol treatment will reverse this effect (McMillan et al., 1996). Similarly, in rat dorsal root ganglia, p75 and trka mrna are increased during proestrus when estrogen levels are relatively high, and trka mrna is up-regulated following estrogen treatment of ovariecomized animals (Sohrabji et al., 1994). In contrast to these examples of estrogendependent up-regulation of NGF receptors, some studies report region-specific (including medial septum) decreases in p75 and trka receptors following relatively long-term exposure to estradiol (Gibbs and Pfaff, 1992; Gibbs et al., 1994). In the zebra finch telencephalon, the relationship detected between 125 I-NGF binding and estradiol levels is consistent with the hypothesis that estradiol acts as a neurotrophic factor to support some aspects of early development of the telencephalon by increasing NGF binding. We do not yet know where NGF receptors are located in the zebra finch telencephalon at any age. However, a recent study has documented the distribution of both aromatase and estrogen receptor mrna in the zebra finch brain on posthatching day 5 (earlier ages were not investigated) (Jacobs, 1998). One of the few areas of overlap in those distributions in the telencephalon is the caudomedial neostriatum (NCM). In older birds, this region is an area where the immediate-early gene, zenk, is induced by playbacks of song and where basal zenk is elevated during the period of song memorization, when male zebra finches form a template of their fathers vocalizations (Clayton, 1997). Zenk is the songbird (canary) homolog of a set of genes induced by NGF, including ngfi-a, that modulates the transcription of other genes (Milbrandt, 1987; Clayton, 1997). Thus, one possibility is that the relatively high levels of estradiol produced by the developing zebra finch telencephalon (Schlinger and Arnold, 1992; Wade et al., 1995) are synthesized and act in NCM to up-regulate NGF binding and subsequently increase transcription of genes related to development of this brain region. Both male and female zebra finches require functional development of NCM, because they must form appropriate models of zebra finch song. Males need to produce it, and females use the song to choose an appropriate mate. Although posthatching day 3 (when NGF binding is relatively high) might be too early for substantial processing of song information to occur, birds are certainly exposed to song at that age. In speculating about the functional significance of estrogen NGF interactions it would be especially helpful to know the levels of estradiol exposure in developing zebra finches. However, levels detected in plasma have been extremely variable both within and between assays (Hutchison et al., 1984; Adkins-Regan et al., 1990; Schlinger and Arnold, 1992). Preliminary results from our lab suggest that, similar to NGF binding, estradiol concentrations in telencephalic homogenates decline from posthatching day 3. However, some inconsistencies similar to those detected in plasma prevent us from drawing firm conclusions. Normally, only male zebra finches sing, and in parallel the telencephalic nuclei involved in song learning and production are larger in males (Nottebohm and Arnold, 1976). Both structural and functional masculinization can be induced in females by treatment with estradiol in the first few weeks after hatching (Gurney, 1981, 1982; Pohl-Apel and Sossinka, 1984; Simpson and Vicario, 1991a,b; Grisham and Arnold, 1995). The present data demonstrating that estradiol can increase high-affinity NGF binding raise the possibility that masculinization of the song system in females following exogenous estradiol treatment involves the up-regulation of NGF receptors, especially trka. It should be emphasized, however, that the assays we performed on homogenates of the entire telencephalon cannot assess developmental changes or sex differences specific to the song control nuclei. Still, the present data are not consistent with the idea that NGF is critical for normal sexual differentiation of the neural system for song production. The morphological sex differences in the telencephalic brain regions are due to a combination of increased neuronal migration and/or survival in males and increased cell death in females. Therefore, one would predict that if NGF is important, binding should reflect a pattern supporting the sustained migration and differentiation and reduced apoptosis in males through approximately day 50, and should decrease in females beginning between days 20 and 30, when rates of cell death are increasing in the components of the motor pathway for song production, the robust nucleus of the archistriatum (RA) and high vocal center (HVC) (Bottjer et al., 1985; Konishi and Akutagawa, 1985; Kirn and DeVoogd, 1989). These ages are only rough estimates, since the song control nuclei all vary slightly in their rates of development, and at least one related telencephalic region, lman (involved in song learning), actually shrinks in size in both sexes during this period of sexual differentiation of the motor circuit for song production (Nixdorf-Bergweiler, 1996). However, the peaks in telencephalic 125 I-NGF binding occur not only before morphological differences

Zebra Finch NGF Binding and Estradiol 155 between males and females exist in the motor circuit, but even before the telencephalic song control nuclei are distinctly visible. Furthermore, the estimate of binding capacity was greater in males than females only at that very early age (day 3), and the difference was only present in the low-affinity binding component, suggesting that it is not specific to NGF. Similarly, the small sex differences in K L seen on days 15 60 are presumably not specific to NGF. The magnitude of this sex difference in binding affinity indicates that it is unlikely to be physiologically relevant, and in any case is in the direction opposite what one would predict if increased NGF binding affinity (thus a decreased K d ) were responsible for masculinizing the song system. In fact, growth factors other than NGF may be better candidates for the facilitation of at least some components of song system development. For example, local infusions in juvenile birds of BDNF, NT-3 and NT-4/5, but not NGF, rescued RA neurons from cell death induced by removing input from lman (Johnson et al., 1997). NGF is, however, capable of preventing ibotenic acid induced deficits in singing behavior in adult zebra finches (Fiore et al., 1997). The relatively high levels of NGF binding on day 3 may be important for the migration into and/or survival of neurons in particular telencephalic regions unrelated to song. In contrast, the effect could be relatively nonspecific. For example, increased action of NGF may be required to sustain the high rate of development in the brain or nervous system in general that is especially obvious within the first several days after hatching. It is also possible that the NGF binding detected in our telencephalic homogenates was not of neural origin. Evidence exists for the presence of NGF receptors in brain structures including choroid plexus, circumventricular organs, and meninges (Raivich et al., 1987; Yan and Johnson, 1988, 1989). In avian tissue specifically (developing chick), NGF receptor mrna has been detected not only in predicted neural locations, such as dorsal root ganglia, but also in muscle, skin, and lymphoid tissues (Ernfors et al., 1988). To elucidate their role in development of the zebra finch brain, the locations of NGF receptors need to be identified. The nature and specificity of their interactions with estradiol also must be studied in more detail. However, the present data document for the first time that specific NGF binding exists in the zebra finch telencephalon, and that it is particularly high in the first week after hatching. 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