help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0879
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/12/5614    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maney, D. L.
Right arrow Articles by O’Brien, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maney, D. L.
Right arrow Articles by O’Brien, S.
Endocrinology Vol. 148, No. 12 5614-5623
Copyright © 2007 by The Endocrine Society

Rapid Neuroendocrine Responses to Auditory Courtship Signals

Donna L. Maney, Christopher T. Goode, Jessica I. Lake, Henry S. Lange and Sara O’Brien

Department of Psychology (D.L.M., C.T.G., J.I.L., H.S.L.), Emory University, Atlanta, Georgia 30322; and Department of Biology (S.O.), University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: Donna Maney, Department of Psychology, Emory University, 532 Kilgo Circle, Atlanta, Georgia 30322. E-mail: dmaney{at}emory.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In many species, courtship signals enhance reproductive function in the receiver. How these social signals are processed by the brain, particularly how they induce an endocrine response, is not well understood. Songbirds provide an ideal model in which to study this phenomenon because of the large existing literature on both their auditory neurobiology and the control of their reproductive physiology by environmental cues. To date, all of the relevant studies on songbirds have involved measuring the effects of male vocalizations on ovarian function over a period of weeks, a time course that precludes detailed analysis of the neuroendocrine mechanisms operating during song perception. We played recordings of conspecific male song to laboratory-housed female white-throated sparrows and quantified the resulting rapid changes in LH as well as the induction of the immediate early gene Egr-1in the GnRH system and mediobasal hypothalamus (MBH). Hearing song for 42 min induced LH release and Egr-1 expression in the MBH, but did not alter Egr-1 expression in GnRH neurons. The time course of LH release and the pattern of Egr-1 expression together suggest that song acts as a trigger to induce GnRH release in a manner resembling photostimulation. The Egr-1 response in the MBH was qualitatively distinguishable from the responses to either photostimulation or pharmacologically induced LH release but seemed to involve overlapping neuronal populations. Song-induced Egr-1 expression in the MBH was correlated with the expression in midbrain and forebrain auditory centers, further supporting a role for the MBH in processing social information.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COURTSHIP SIGNALS FROM a potential mate can have profound effects on reproductive development. For example, exposure to the signals of opposite-sex conspecifics advances gonadal cycles or maturation in mammals (1, 2, 3), birds (reviewed in Ref. 4), amphibians (5), reptiles (6, 7), and fish (reviewed in Ref. 8). Given the obvious salience of the auditory courtship signals of male songbirds, it is not surprising that the bulk of research on the effects of social cues on gonadal development in birds has been conducted on female songbirds listening to male song. Such research confirms that song alone, presented to photostimulated females via audio playback over a period of weeks, is sufficient to enhance LH secretion, follicle growth, egg laying, or nest-building behavior in females of many species (9, 10, 11, 12, 13, 14). Although this phenomenon has been well documented, its underlying neural mechanism is not well understood. More than 40 yr ago, Lehrman (15) and Hinde (16) were among the first to propose that courtship signals cause gonadal development by influencing the regulation of gonadotropin secretion, which is achieved primarily via the release of GnRH from the brain. An investigation of the effects of courtship signals on reproductive development should focus on brain mechanisms, including interactions between sensory centers and the neuroendocrine areas that govern GnRH release.

In the context of most previous research, in which laboratory-housed females were exposed on a daily basis to male vocalizations for a number of weeks, social signals could stimulate endocrine responses via two largely independent types of mechanisms. First, such stimulation constitutes a form of environmental enrichment and as such may have significant effects on brain function via slow-acting processes such as neurogenesis, synaptogenesis, and angiogenesis (reviewed in Refs. 17 and 18). Acting via this route, song may alter neuroendocrine function slowly, perhaps by causing recruitment of new neurons into functional circuits, increases in dendritic branching, and alterations in receptor expression or function. These sorts of changes take place on a time scale of days to weeks, which is consistent with previously reported effects of song on hypothalamic-pituitary-gonadal (HPG) function in female songbirds (9, 10, 11, 12, 13, 14). Second, social cues may act rapidly on existing cells and circuits, triggering the release of reproductive hormones within minutes. Such a mechanism would require no or minimal reorganization of neuronal circuits and would mean that the neuroendocrine response to social cues is similar to the behavioral (19) and auditory (20) responses, which happen rapidly. A rapid neuroendocrine response to social cues would likely resemble the neuroendocrine response to photic cues (long days), during which large amounts of GnRH are released from the brain in a short period of time.

In this study, we tested the hypothesis that hearing male song triggers the rapid release of GnRH. We played conspecific male song to laboratory-housed female white-throated sparrows and quantified the resulting changes in plasma LH as well as the expression of immediate early genes in areas of the brain known to be involved in the regulation of GnRH release. Birds have several forms of GnRH; the form that is released from the median eminence (ME) and involved in reproductive development is known as chicken GnRH-I (cGnRH-I) (21). The cell bodies of cGnRH-I neurons, which synthesize and store the cGnRH-I to be released, are located primarily within the septo-preoptic area of the hypothalamus and send their axons ventrally to the ME where they secrete cGnRH-I into the portal vasculature. Just dorsal to the ME lies a region known as the infundibular nucleus (IN), which together with the ME and other ventral hypothalamic structures make up the mediobasal hypothalamus (MBH) (Fig. 1Go). Several lines of evidence, primarily from studies of Japanese quail (Coturnix coturnix), implicate this region in the control of cGnRH-I release. For example, MBH lesions that spare the cGnRH-I fibers passing to the ME disrupt photoinduced gonadal development (22), and electrical stimulation of the IN induces both LH secretion and gonadal growth (23, 24). Transfer from short to long days results in striking up-regulation of the immediate early gene FOS in the MBH, along with an increase in plasma LH (25). The minimum day length required for the induction of both LH secretion and FOS is identical (25), and both are also induced by pharmacological activation of cGnRH-I release (26). Neither photic nor pharmacological induction of cGnRH-I release is accompanied by immediate early gene induction in the cGnRH-I cell bodies themselves (26, 27), suggesting that regulation of cGnRH-I secretion may be tightly governed by the MBH, near the terminals rather than by the cGnRH-I cells in the septo-preoptic area (reviewed in Ref. 28).


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 1. The MBH shown in coronal section. Egr-1-IR was quantified in two regions of the MBH, the IN and the ME. The IN in this study corresponded to the area of that name described by Meddle and Follett (25 ) and Meddle et al. (26 ). Some authors have referred to an overlapping area as the IN-IH (49 ). v.III, Third ventricle.

 
If social signals enhance reproductive development in female songbirds by a mechanism similar to photostimulation, then exposure to song should elicit both a rapid rise in LH and induction of immediate early genes in the MBH. Because this response would occur as a result of auditory input, the MBH activity should correlate with activity in auditory centers. If, however, song enhances reproductive function entirely by slow-acting processes characteristic of environmental enrichment (17, 18), then song playback need not have significant short-term effects on plasma LH or MBH activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures involving animals were approved by the Emory University Institutional Animal Care and Use Committee. Female white-throated sparrows were collected in mist nets on the campus of Emory University in Atlanta, GA, during November 2005. Sex was confirmed by PCR analysis using a small blood sample (29). In this species, birds less than about 6 months old can be identified by unossified patches of skull visible through the skin on the top of the head (30, 31). All birds used in this study showed these unossified patches at the time of capture. They therefore had hatched during spring 2005 and had no breeding experience but were all adults at the time of testing. Birds were housed in the Emory animal care facility in walk-in flight cages and supplied with ad libitum food and water. The day length was kept constant at 10 h light, 14 h dark (10L:14D), which corresponds to the shortest day they would experience on their wintering grounds in Georgia. We held the birds under these conditions until February 2006 to ensure that photorefractoriness was broken (32, 33).

Experiment 1: neuroendocrine responses to auditory stimuli
Birds were photostimulated by exposure to long days (16L:8D). Five weeks later, birds were transferred to individual cages (15 x 15 x 17 in.) inside walk-in sound-attenuated booths (Industrial Acoustics, Bronx, NY). They were housed four to six birds per sound-attenuated booth until the day before stimulus presentation, a period averaging 6 d, to acclimate them to the booths. All booths were identical and on the same photoperiod (16L:8D).

Auditory stimuli
The song stimuli and presentation protocol have been previously described (34). Briefly, recordings of singing male white-throated sparrows were downloaded from the Borror Laboratory of Bioacoustics birdsong database and edited such that there was 15 sec of silence between songs. The resulting segments were spliced together to form stimulus presentations such that the song of a novel male began every 3 min. Each presentation contained the songs of 14 males and totaled 42 min in duration. For a representative song in each of the 14 recordings, the duration of the song from beginning to end and the total duration of sound contained in the song (with periods of silence subtracted; i.e. the duty cycle) were measured using Audacity 1.3.2 (Carnegie Mellon Computer Music Group, Pittsburgh, PA), and a tone sequence was generated consisting of 1-kHz tones. Within each tone sequence, tone bursts of equal duration were arranged such that the sequences matched individual songs in total duration, duty cycle, and number of onsets and offsets of sound. Tone sequences were spliced together as described above for the song stimuli, with 15 sec of silence between them.

Sound presentation and behavioral scoring.
On the afternoon before stimulus presentation, each bird’s cage was placed in an empty sound-attenuated booth equipped with microphone, speaker, and video camera. Stimulus playbacks began 1 h after lights-on the following morning, via the speaker located inside the booth. All stimuli were delivered at a peak level of 70 decibels measured at the bird’s cage. Each bird heard either male songs (n = 8), tones (n = 8), or silence (n = 8). The stimulus presentation was followed by 18 min of silence.

Video and audio recordings were made of each bird for 60 min before, during, and for 18 min after the stimulus presentation (total 120 min). These recordings were later scored for copulation solicitation events (35) and other vocalizations, including song.

Exactly 60 min after the onset of the stimulus presentation, birds were deeply anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL), and a blood sample was immediately collected from the jugular vein into a StatSampler tube (Iris, Westwood, MA). The brains were rapidly harvested and fixed as previously described (34, 35, 36). Blood samples were centrifuged to isolate the plasma, which was harvested and stored at –20 C until LH assay (see below).

Experiment 2: rapid endocrine responses to hearing song
To test whether hearing song induces detectable LH release more rapidly than 60 min after stimulus onset, we performed a separate playback study on an additional set of females. Birds were photostimulated (16L:8D) for 5 wk and transferred to individual cages where they acclimated to the sound-attenuated booths for an additional 6 d. Playbacks were conducted as described above. To maximize statistical power, only two types of stimuli were presented: song (n = 12) and silence (n = 10). Fifteen minutes after the onset of the stimulus, the playback was ended, and a blood sample was taken from a wing vein. Samples were assayed for LH as described below.

Experiment 3: neuroendocrine responses to photostimulation
To compare the pattern of immediate early gene expression induced by song with that induced by photostimulation, 10 additional females were involved in a separate study as follows. Birds housed on short days (10L:14D) were transferred from flight cages (see above) to individual cages inside sound-attenuated booths. Two days after transfer, half of the birds (n = 5) experienced a long day; the lights went off 6 h later than usual. The remaining birds (n = 5) experienced lights-out at the normal time. Twenty-four hours after the last subjective dawn, blood samples and brains were collected as described for experiment 1.

Experiment 4: neuroendocrine responses to N-methyl-D-aspartate (NMDA) treatment
To assess our ability to detect immediate early gene expression in the ME, additional birds (n = 7) were injected sc with NMDA (20 mg/kg), which causes rapid LH release along with robust induction of immediate early genes in the IN and ME in this genus (26). Another group of birds (n = 6) was injected with saline as a control. Injections were performed on birds that had been photostimulated (16L:8D) for approximately 12 wk. Previous work has shown that NMDA injection induces LH release and expression of immediate early genes in sparrows at all stages of the reproductive cycle (26, 37 ; but see Ref. 38). Sixty minutes after injection, birds were rapidly decapitated under deep isoflurane anesthesia, and brains were collected as described above. We did not measure plasma LH in these birds; however, previous studies have shown a robust effect of this dose of NMDA on LH release in sparrows of this genus (26, 37).

LH assay
LH was measured by a postprecipitation, double-antibody RIA using a homologous chicken LH RIA technique (39) that has been validated for songbirds (40) and has been used in this species (41). The assay uses highly purified chicken LH for standard curves and for radioiodination. Goat antirabbit {gamma} globulins were used as the secondary antibody. Further details of the LH assay are described by Wingfield et al. (42). All samples collected for experiments 1, 2, and 3 were run in duplicate in a single assay, thereby eliminating between-assay variation. The within-assay variation was 12.3%. The data on plasma LH levels were square-root transformed to normalize them.

Immunocytochemistry (ICC)
Brain tissue was processed for ICC as previously described (26, 34, 35). Briefly, every second 50-µm section was incubated with an antibody against Egr-1 (known in the songbird literature as ZENK; Santa Cruz Biotechnology, Santa Cruz, CA), which was subsequently labeled using a biotinylated secondary antibody and the ABC method (Vector Laboratories, Burlingame, CA). The specificity of this antibody has been established in songbirds (43). Labeling was visualized using diaminobenzidine enhanced with nickel (26, 35, 44). In experiments 1 and 4 (playback and NMDA injection), the brain sections were divided into a rostral set containing the preoptic area and a caudal set containing the ME. The rostral set underwent ICC to label Egr-1 immunoreactivity (IR) as above, followed by a second ICC to label cGnRH-I using an anti-GnRH antibody (HU60) kindly donated by H. Urbanski. This antibody recognizes both cGnRH-I and -II (45), but the population of cells we examined in this study, which is located in the septo-preoptic-infundibular region and projects to the ME, is known to contain only cGnRH-I (reviewed in Ref. 21). Sections were incubated with anti-GnRH primary 1:5000 and subsequently labeled using a protocol identical to that used to label Egr-1, except that diaminobenzidine was used without the nickel enhancement. Double-labeled cells thus appeared as a purplish-black nucleus surrounded by reddish-brown cytoplasm. Sections from experiment 3, the caudal set of sections from experiment 1, and the caudal sets of six of the birds from experiment 4 (three NMDA-treated and three saline-treated) were single-labeled for Egr-1. Within experiments, staining was performed either in one run of ICC or two to three runs with treatment groups balanced across runs. All sections were mounted onto microscope slides and coverslipped in DPX.

Quantification of Egr-1-IR
Egr-1-IR in cGnRH-I neurons.
cGnRH-I neurons of the septo-preoptic-infundibular cGnRH-I system were inspected in every second 50-µm section. This population begins as a cluster of cells ventral to the septomesencephalic tract and extends dorsocaudally, ending rostral to the paraventricular nucleus. The number of cGnRH-I neurons in this population that were also immunopositive for Egr-1 was counted and expressed as a percentage of the total number of cGnRH-I neurons counted.

Egr-1-IR in the MBH.
A Leica DFC480 camera attached to a Zeiss Axioskop microscope was used to photograph the MBH at 100-µm intervals throughout its rostrocaudal extent (26), which encompassed seven consecutive sections per brain. The x10 objective was used to capture all images, which were approximately 4.4 megabytes. The light level on the microscope was set exactly the same for each picture. To quantify Egr-1-IR in the IN, Image J software (National Institutes of Health, Bethesda, MD) was used to place the largest circle that would fit (200–275 µm diameter) just dorsal to the ME and adjacent to the third ventricle (Fig. 1Go). This selection encompassed an area defined as IN in a majority of published sources (reviewed in Ref. 46 ; see also Refs. 25 , 47 , and 48) and may have included tissue that some researchers have called the inferior hypothalamic nucleus (IH) (e.g. Ref. 49). There is no clear boundary between the IN and IH (25, 49). Egr-1-positive nuclei were counted within the circle on one side of the brain, chosen at random, in each section by a blind observer. Very pale cell nuclei were considered background and were not counted. Egr-1-positive cells were also counted throughout the extent of the ME (26). To convert these counts to cells per unit area, the total number of cells per section was divided by the area of the region counted, either the area of the circle in the IN or the area of the ME as traced.

Egr-1-IR in auditory regions.
The caudomedial nidopallium (NCM) and the avian homolog of the inferior colliculus (in birds known as nucleus mesencephalicus lateralis pars dorsalis, MLd) were identified with reference to Bailey et al. (50), Mello and Clayton (51), and Stokes et al. (52). Egr-1-IR was quantified in two consecutive sections 100 µm apart on one side of the brain, chosen at random except when one side was damaged; in these cases, the intact side was chosen. Images were acquired using the x10 (NCM) or the x4 (MLd) objective. Egr-1-positive nuclei with an OD darker than a threshold value were counted by a blind observer as previously described (34, 35). Because of variability in background staining among brains, the threshold was set manually for each image such that clusters of pixels highlighted by Image J agreed with what the observer considered to be labeled nuclei. For NCM, labeled cells were counted inside a 0.4-mm2 square placed as close as possible to the midline, adjacent to the hippocampus (34). MLd was traced in the three consecutive sections where it was largest and its borders most obvious with respect to the nucleus intercollicularis and surrounding tissue (34). Consequently, the sections of MLd we chose to analyze were very similar with respect to surrounding landmarks.

Statistical analysis
Data collected for experiment 1 (plasma hormone levels, number of GnRH-positive neurons, and number of Egr-1-positive cells in brain regions of interest) were entered into a single multivariate ANOVA with sound stimulus as a between-subjects factor. Post hoc Scheffé tests were subsequently used to make pairwise comparisons across treatment groups. In addition to being included in this analysis, plasma LH levels in experiment 1 were analyzed together with LH levels from experiment 2 in a two-way ANOVA with stimulus (song, tones, or silence) and time (15 or 60 min) as between-subjects factors. Post hoc Scheffé tests were used to test for effects of stimulus at each time point. The effects of photostimulation on plasma LH (experiment 3) and the effects of NMDA injection on Egr-1 induction in cGnRH-I neurons (experiment 4) were assessed using unpaired t tests. To describe the distribution of Egr-1 activation in the MBH in more detail, the IN and ME cell counts from experiments 1, 3, and 4 were entered into repeated-measures ANOVA with stimulus (experiment 1) or treatment (experiments 3 and 4) as between-subjects factors and section number (proceeding from rostral to caudal throughout the MBH) as the repeated measure. To assess the relative contributions of the auditory regions NCM and MLd to Egr-1 induction in the IN, we entered the average number of Egr-1-positive nuclei per unit area in each region into a stepwise multiple regression using values for the IN as dependent variables and those for NCM and MLd as independent variables. This procedure automatically identifies predictive variables using a sequence of F tests. To test whether Egr-1-IR in our regions of interest may have been affected by the behavioral response to stimuli, we used Spearman correlations to test for relations between vocal behavior and both plasma LH and Egr-1-IR in our regions of interest.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Behavior
It is well established that in ring doves, a female’s own vocalizations can induce LH release (53). To monitor vocalizations and other behaviors, we made audio and video recordings of each bird for 2 h, a period that included the stimulus presentation, before tissue collection. One bird in the silence group sang more than 100 times during the 120-min observation period and was subsequently excluded from the analysis. A second bird was excluded because the DVD recorder failed, and we therefore had no record of her behavior during the observation period. The remaining birds produced primarily vocalizations such as contact calls that were not aggressive or song-like in nature. No copulation solicitation display activity was noted; in captivity, plasma estradiol in females of this genus do not typically reach levels necessary to support this behavior (54). There was no relation between vocal behavior and LH level or between vocal behavior and Egr-1 expression in any of our brain regions of interest.

Neuroendocrine effects of hearing song
A multivariate ANOVA that included the variables quantified in experiment 1 (plasma LH, number of GnRH-positive neurons, and Egr-1 expression in cGnRH-I neurons, the IN, ME, NCM, and MLd) revealed a significant overall main effect of sound stimulus (Wilks’ {lambda} F12,28 = 5.009; P < 0.0002). Differences among stimulus groups are reported below for each variable as appropriate.

LH.
Hearing song induced a rise in LH (Fig. 2Go) that was detectable 60 min after the onset of a 42-min presentation of song playback (experiment 1) but not after 15 min of song playback (experiment 2). An ANOVA conducted only on data from experiment 1 showed a significant effect of stimulus (F2,19= 3.928; P = 0.0373). A two-way ANOVA conducted on combined data from experiments 1 and 2 revealed a significant interaction between stimulus and duration of playback (F1,32 = 7.750; P = 0.0089) and nonsignificant trends toward main effects of these factors on LH (effect of stimulus, F1,32 = 3.728, P = 0.0624; effect of stimulus duration, F1,32 = 2.823, P = 0.1027). Post hoc Scheffé tests showed that birds hearing 42 min of song had higher LH than those hearing silence (P = 0.0205). LH levels in birds that heard song were higher but statistically not different from in birds that heard tones (P = 0.0752). There was no difference in plasma LH between birds hearing tones and those hearing silence (P = 0.4759).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 2. Hearing song induced a rise in plasma LH that was detectable at 60 min after the onset of playback. Hearing 1-kHz tones did not significantly increase plasma LH over levels seen after hearing silence. Birds in the 15-min group heard only silence or song. *, Significantly different from silence, P = 0.0205.

 
Expression of Egr-1 in cGnRH-I neurons.
To test for an effect of song on new transcription activity within cGnRH-I neurons, we quantified Egr-1 in the GnRH neurons of the septo-preoptic-infundibular cGnRH-I system. The number of GnRH-positive neurons was similar in all groups (means and SEM were 289 ± 25, 306 ± 33, and 313 ± 32 in the silence, song, and tones groups, respectively; ANOVA F2,19 = 0.155; P = 0.8576). Many of the GnRH-positive cells expressed Egr-1-IR (Fig. 3AGo), but this expression was unrelated to sound stimulus (Fig. 3BGo). Birds hearing song, tones, and silence expressed similar levels of Egr-1 in the cGnRH-I neurons (P > 0.80 for all pairwise comparisons).


Figure 3
View larger version (52K):
[in this window]
[in a new window]

  		FIG. 3. A, Coexpression of Egr-1 protein and GnRH in the same cells (black arrows). GnRH is visible as a brown stain in the cytoplasm, whereas Egr-1 is represented by a purplish stain in the nucleus. Single-labeled cells immunopositive for only Egr-1 or GnRH are indicated by white arrows. B, Hearing song did not induce Egr-1-IR within cGnRH-I neurons of the septo-preoptic-infundibular system. Roughly the same percentage of cGnRH-I neurons coexpressed Egr-1-IR in all three stimulus groups.

 
Expression of Egr-1 in the MBH.
In Experiment 1, birds hearing song had significantly higher numbers of Egr-1-positive cells in the IN than did birds hearing silence (P = 0.0046) or tones (P = 0.0072; Fig. 4Go). Egr-1-IR levels in the IN were similar in birds hearing silence and those hearing tones (P = 0.7589). Song did not, however, induce Egr-1-IR within the ME (Fig. 4Go; P > 0.30 for all pairwise comparisons). Egr-1 expression in the ME was, overall, very low in all groups.


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 4. Hearing song increased Egr-1 expression in the IN but not the ME. *, Significantly different from both silence and tones, P < 0.008.

 
Comparison with other LH releasers
To better understand the responses of the MBH to the sound stimuli, we compared these responses to those induced by two stimuli known to elicit LH release and immediate early gene expression in the MBH: photostimulation (experiment 3) and injection of NMDA (experiment 4). Photo-induced LH release was confirmed in experiment 3; birds exposed to one long day had higher LH levels (0.652 ± 0.095 ng/ml) than those that remained on short days (0.186 ± 0.102 ng/ml; P = 0.0100). We plotted the numbers of Egr-1-positive cells in the IN and ME for experiments 1, 3, and 4 to show Egr-1 induction as a function of rostrocaudal level through the MBH (Figs. 5Go and 6Go) and performed repeated-measures ANOVA on these data for each experiment. As expected, there were significant main effects of treatment in the IN for all three experiments (effects of auditory stimulus type, F2,19 = 7.263, P = 0.0045; effect of day length, F1,8 = 46.708, P = 0.0001; effect of NMDA treatment, F1,4 = 42.511, P = 0.0029). In the ME, there were significant main effects of day length (F1,8 = 18.039; P = 0.0028) and NMDA treatment (F1,4 = 44.800; P = 0.0026) but not auditory stimulus type (F2,19 = 0.278; P = 0.7600). There were highly significant main effects of rostrocaudal distance through the MBH in both the IN and ME in all three experiments (IN: experiment 1, F6,114 = 5.415, P < 0.0001; experiment 3, F6,48 = 7.401, P < 0.0001; ME: experiment 1, F6,114 = 23.974, P < 0.0001; experiment 3, F6,48 = 4.372, P = 0.0013; experiment 4, F6,24 = 6.464, P = 0.0004), with the exception of a nonsignificant trend toward an effect of distance in the IN in experiment 4 (F6,24 = 0.382; P = 0.1000). We were primarily interested in whether each treatment induced a different pattern of Egr-1 expression throughout the MBH, and we therefore looked for interactions between treatment and distance in each experiment. The only significant interaction was between distance and day length in the IN in experiment 3 (F6,48 = 4.450; P = 0.0012). Otherwise, the effects of treatment did not depend on the rostrocaudal level of the MBH (IN: experiment 1, F12,114 = 1.477, P = 0.1430; experiment 4, F6,24 = 0.382, P = 0.8831; ME: experiment 1, F12,114 = 1.115, P = 0.3549; experiment 3, F6,48 = 2.028, P = 0.0801; experiment 4, F6,24 = 1.702, P = 0.1638).


Figure 5
View larger version (53K):
[in this window]
[in a new window]

  		FIG. 5. Comparison of Egr-1 induction in the IN by auditory stimuli (experiment 1), photostimulation (experiment 3), and NMDA treatment (experiment 4). A, Egr-1-IR is shown as a function of rostrocaudal level of the MBH. In most cases, the magnitude of Egr-1 responses did not depend on rostrocaudal level. The response to photostimulation, however, was greater in the caudal portion of the IN than in the rostral portion. *, Significantly different from one or more control groups, P < 0.05. B, Photomicrographs of Egr-1-IR in the IN in this study.

 

Figure 6
View larger version (48K):
[in this window]
[in a new window]

  		FIG. 6. Comparison of Egr-1 induction in the ME by auditory stimuli (experiment 1), photostimulation (experiment 3), and NMDA treatment (experiment 4). A, Egr-1-IR is shown as a function of rostrocaudal level of the MBH. Expression of Egr-1 in the ME of control animals was low in all groups; the relatively higher levels in the saline group of experiment 4 could have been related to a longer duration of photostimulation (12 vs. 6 wk in experiment 1) or to being processed in different runs of ICC. *, Significantly different from the control group, P < 0.05. B, Photomicrographs of Egr-1-IR in the ME in this study. Egr-1-IR was noted in the internal zone but not the external zone of the ME (46 ). Some staining seen outside the ME along its ventral aspect (visible only in the leftmost panel) was present in all animals and not counted.

 
NMDA did not induce Egr-1 in GnRH neurons.
As was found for experiment 1, some cGnRH-I neurons expressed Egr-1, but this expression was unaffected by treatment (P = 0.8679). This result is consistent with that reported by Meddle et al. (26) who found in the congeneric white-crowned sparrow that this dose of NMDA did not induce FOS expression in cGnRH-I neurons.

Egr-1 induction in the ME.
In the ME, Egr-1-IR was not induced by hearing song, but it was induced by photostimulation and by NMDA treatment (Fig. 6Go). Labeled cells were found primarily in the internal zone (layers I–III) rather than the external zone (layers IV and V; see Ref. 45). In all groups, the external zone including the palisade layer was virtually devoid of Egr-1-IR. Much of the stimulus-induced labeling in the internal zone did not appear to represent neuronal nuclei (e.g. in the example labeled NMDA in Fig. 6BGo) but was nonetheless strongly induced by photostimulation and by NMDA treatment.

Auditory areas
In both NCM and MLd, each type of auditory stimulus induced levels of Egr-1 expression that were significantly different from that induced by each of the other two types of stimuli (Fig. 7Go). Hearing song induced more Egr-1-IR in both regions than hearing silence (NCM, P = 0.0002; MLd, P < 0.0001) or tones (NCM, P = 0.0291; MLd, P = 0.0068). Hearing tones induced more Egr-1-IR than hearing silence (NCM, P = 0.0232; MLd, P = 0.0265). Egr-1 expression was correlated in MLd and NCM (r = 0.600, P = 0.0025) and in each of these regions was correlated with expression in the IN (MLd, r = 0.569, P = 0.0049; NCM, r = 0.503, P = 0.0159). These correlations were expected, because the expression in each of these regions was significantly affected by stimulus (Figs. 4Go and 7Go). To explore the nature of auditory influences on the IN response to sound, we assessed the relative contributions of each auditory region to activity in the IN using a stepwise regression with Egr-1 expression in the IN as the dependent variable and expression in the two auditory regions as potential explanatory independent variables. This procedure automatically constructs a model, using one or more of the independent variables, that best explains variability in the dependent variable, in this case, Egr-1 expression in the IN. According to the resulting model, Egr-1-IR in the IN was significantly predicted by that in MLd (F = 9.572; P = 0.0057). The predictive power of the model was not increased by the addition of Egr-1-IR in NCM, meaning that expression in the IN was better predicted by expression in MLd alone than by expression in NCM alone or in both MLd and NCM. Thus, song-induced transcription activity in the IN may be more closely related to the activity in MLd, an auditory center in the midbrain, than in NCM, an auditory center in the forebrain.


Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 7. Hearing song significantly increased Egr-1-IR in two regions of the auditory system, the NCM and the avian homolog of the inferior colliculus, the MLd. Hearing tones induced Egr-1-IR at a level intermediate between hearing song and hearing silence. *, Significantly different from both silence and song, P < 0.03; **, significantly different from both silence and tones, P < 0.03.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides significant new insight into the progonadal effects of conspecific courtship signals, a long-studied phenomenon described more than 40 yr ago by Lehrman (15) and Hinde (16). First, we show that the endocrine response to auditory courtship signals is rapid. All other studies on awake female birds involved measuring reproductive function after exposure to male vocalizations over periods of weeks rather than minutes (9, 10, 11, 12, 13, 14). Here, 42 min of song produced a significant rise in LH. Second, we describe the concomitant neural response in the septo-preoptic-infundibular cGnRH-I system and MBH, which govern HPG function, and show how this response may be related to the response in auditory centers. This study thus lays the groundwork for studying the brain mechanisms underlying neuroendocrine responses to sensory cues, particularly social signals, in a well-established endocrine model.

Introducing novel or complex sensory stimuli to animals in a laboratory setting has the effect of enriching the environment, which over time can have profound effects on neural development and function. Such effects include neurogenesis, increases in dendritic branching, changes in synaptic proteins, receptors, and neurotrophins, cortical reorganization, enhancements in learning and memory, angiogenesis, and even significant increases in brain size (reviewed in Refs. 17 and 18). It would not be surprising if Hinde’s female canaries listening to male song (11, 16) experienced many of these changes, which may have led directly to the enhancement of ovarian function. If such changes could completely explain the effects of social signals on the HPG axis, however, we would not expect to see rapid release of hormones and activation of the MBH in response to the first songs of the breeding season. Processes such as the creation of new blood vessels and incorporation of new neurons into functional circuits take longer than 60 min. Our data show that the mechanism underlying song-induced enhancement of reproductive function may more closely resemble the process of photostimulation, during which cGnRH-I is released rapidly into the portal vasculature upon perception of the relevant cue.

In this study, the effects of song on LH release were rapid, but not immediate. Hearing song for 15 min failed to increase LH above control levels (Fig. 2Go). We expected to see a significant effect at this time point because in female ring doves (Streptopelia risoria), presentation of male or female nest coo vocalizations increases LH within 10 min (55). In that study, vocalizations were presented to females under urethane anesthesia, which was required to withdraw blood directly from the pituitary vasculature. It is unclear whether the LH rise could have been detected as quickly using more traditional blood collection techniques. Rapid endocrine responses to social cues have also been demonstrated in free-living male song sparrows (56), which show a rise in both LH and testosterone by 10 min after the onset of a simulated territorial intrusion by a conspecific male. The endocrine effects, however, depended on the presence of a visual stimulus (a live decoy) in addition to song playback and were not elicited by auditory stimuli alone. Even 4.5 h of playback to laboratory-housed males did not significantly affect hormone levels, which together with our results suggests that the endocrine response to auditory social signals may involve processes more complex than neuronal firing alone.

A time course longer than 15 min but shorter than 60 min may suggest involvement of new gene transcription and protein synthesis, which we investigated by quantifying the expression of the immediate early gene Egr-1 (known in the songbird literature as ZENK). We found that despite significant song-induced LH release, hearing song did not increase the expression of Egr-1 in cGnRH-I neurons (Fig. 3Go). The lack of an effect of song on immediate early gene expression in the cGnRH-I system during LH release is consistent with reports of similar insensitivity during LH release in other species. Previous studies have shown that after photostimulation (25) or NMDA treatment (26), despite massive LH and presumably cGnRH-I release, cGnRH-I cell bodies do not express FOS protein. Such a result is difficult to interpret, however, because cGnRH-I neurons may never express FOS at all. We show here that they do express Egr-1 (Fig. 3Go) but that neither song nor NMDA treatment affects this expression within 60 min. Many authors have suggested that rather than induce the synthesis of new cGnRH-I, photostimulation and NMDA treatment induce cGnRH-I release by having effects at the cGnRH-I terminals (25, 26, 28, 57).

The control of cGnRH-I release from the terminals in the ME is thought to involve cells in the IN (22, 23, 24, 25, 26). Photo- and NMDA-induced cGnRH-I release are accompanied by striking activation of immediate early gene expression in this region (25, 26, 47). In this study, hearing song induced expression of Egr-1 in the IN that was significantly greater than the response to synthetic tones or silence (Fig. 4Go). Because this activation of new gene transcription occurred together with an increase in LH, we hypothesize that it is related to the release of cGnRH-I and could represent transcription common to both photoinduced and socially induced endocrine changes. Qualitative comparison of song-induced IN activation with that induced by photostimulation or NMDA treatment suggested that these responses are likely not identical. Although the Egr-1 response to song was significantly higher than the response to control stimuli, its magnitude above control levels was much lower than were the photo- or NMDA-induced responses (Fig. 5AGo). The distribution of Egr-1-IR may have also been slightly different; the response to photostimulation was greater in the caudal portion of the IN than in the rostral portion, whereas the response to song was uniform throughout the extent of the IN (Fig. 5AGo). It is possible therefore that song activates gene transcription in a unique population of IN cells.

Whereas song induced a significant Egr-1 response in the IN, there was no effect on the response in the ME (Fig. 4Go). Egr-1-IR was very low throughout the ME in experiment 1 (Fig. 6Go). We are confident in our ability to detect Egr-1 in the ME, however, because both photostimulation and NMDA treatment induced significant Egr-1 expression in this region (Fig. 6Go). The responses to the latter stimuli are consistent with reports of photo- and NMDA-induced FOS expression in the ME in quail and white-crowned sparrows (25, 26, 47). This FOS expression is colocalized with glia-specific markers and is therefore thought to represent glial activation that in turn affects cGnRH-I terminals in the ME (25, 47). The Egr-1 expression we observed in the ME, which is strongly induced by both photostimulation and NMDA treatment (Fig. 6Go), is mostly likely also glial in nature. First, in an exhaustive anatomical study of the ME in the white-crowned sparrow, a congener of the white-throated sparrow, Oksche and Farner (46) reported that neuron somata were never found even in layers of the ME that contain an occasional neuron soma in other avian species, such as mallards (Anas platyrhynchos) (58). Second, although Egr-1 induction occurs primarily in neurons (59), it has been described in glia as well (60). The Egr-1 expression we describe in the ME is more likely to represent activation of astrocytes (25) or tanycytes (48, 61) and may be related to the retraction of glial end-feet processes that, during short-day conditions, prevent contact between cGnRH-I terminals and the portal vasculature and block cGnRH-I release (57). It is possible that the lack of song-induced Egr-1 expression in the ME in this study reflects the fact that the birds were already photostimulated, and the glial end-feet therefore already retracted. Future experiments should make use of glial markers to characterize further the Egr-1-positive cells in the ME.

Because transcription activity in the MBH was brought on by an auditory stimulus, we also characterized sound-induced Egr-1-IR at two levels of the auditory system: a midbrain processing center homologous to the inferior colliculus and a forebrain center analogous to auditory cortex. We found that the level of Egr-1 expression in these regions was directly related to the presumed behavioral relevance of the stimulus (Fig. 7Go), which is consistent with previous studies in this and other species (e.g. Refs. 20 , 34 , and 35). Across treatment groups, auditory activation in both regions was correlated with expression in the IN. The correlations alone, however, do not give much information regarding possible neural connections between auditory centers and the hypothalamus, because both regions could be activated independently by song. Direct connections have not been described in songbirds but are likely to exist. In ring doves, the auditory thalamus may project directly to hypothalamic regions such as the preoptic area and the ventromedial nucleus (62), which lies just dorsal to the IN in the MBH. Playbacks of nest-coos elicit selective firing from neurons in these regions (55). A projection from the auditory thalamus to the endocrine hypothalamus may be common to many vertebrates, because it is also present in some amphibians (63).

To investigate the possibility of a similar connection in songbirds, we tested whether Egr-1 expression in the IN was better predicted by the expression in MLd, which is a direct source of input to the auditory thalamus, or by NCM, which is upstream, removed by one or more synapses, and involved in higher order forebrain processing. We hypothesized that if auditory responses in the IN originate from a direct projection from the thalamus, they would be better predicted by activity in MLd than in NCM. Our analysis supported this hypothesis. We are not suggesting, however, that the endocrine response to auditory social signals arises entirely from ascending rather than descending input. Across taxa, the auditory midbrain is known to receive descending inputs (64, 65), and forebrain activity modulates the response properties of auditory midbrain neurons (66, 67, 68). MLd most likely integrates ascending auditory input with descending input from the forebrain to impart selectivity to its targets, which include the auditory thalamus and, possibly indirectly, hypothalamic areas. Auditory-hypothalamic projections, if they exist in songbirds, could help explain song-induced enhancements of reproductive behavior and function and form the basis for testable hypotheses concerning interactions between sensory and endocrine systems.

The response to song consists of at least three components: a behavioral response characterized by copulation solicitation displays (19), an auditory response characterized by increased firing and gene transcription in auditory centers (20), and an endocrine response (9, 10, 11, 12, 13, 14). In this study, we looked at all three. Some authors have speculated that each of these components is under separate control; in other words, the neural filters that govern these responses may operate via different mechanisms (4, 9). For example, the behavioral and endocrine responses to simulated territorial intrusion in male song sparrows are dissociable (56), as are the behavioral and endocrine responses to hearing song in female canaries and white-crowned sparrows (reviewed in Ref. 4). The present study provides further evidence that auditory and endocrine responses do not depend on the behavioral response, because the former were robust despite a complete lack of the latter. This lack of display behavior was likely related to low plasma estradiol, which in captivity does not reach levels required to support this behavior (54). Our data show that performing the behavioral response does not cause the auditory and endocrine responses, which may be closely related to each other.

In addition to promoting the behavioral response to song, estradiol levels may affect the selectivity of the auditory and endocrine responses. We previously showed that although estradiol-treated females showed more Egr-1 induction in NCM and MLd in response to song than control sounds (tones), in untreated short-day females, the responses to these stimuli were indistinguishable (34). If the endocrine response is related to the auditory response, as our data suggest, then the endocrine response may not be specific to conspecific song under conditions of low estradiol. In this study, although the Egr-1 response in the IN was highly selective for song vs. tones, the LH response to song was not significantly different from the response to tones (P = 0.0752). The lack of a significant difference most likely reflects an issue of sample size but nonetheless implies that there was some response to sound in general. Nonspecific responses to sound could be related to low estradiol level or a state of incomplete or slow gonadal development. Bentley et al. (9) held female canaries on a photoperiod of 11L:13D, which stimulates slower than normal gonadal development, and exposed them to conspecific song, heterospecific song, or silence. Although the females listening to conspecific song laid more eggs and laid them sooner than those listening to heterospecific song, the difference in follicle size between the two groups was, although compelling, not statistically significant. It is important to note that the birds listening to heterospecific song in the canary study had larger ovarian follicles and laid more eggs than birds hearing silence. It would not be surprising if heterospecific song normally affects ovarian development, because the songs of sympatric species may in some cases help signal optimal breeding conditions. Future studies should explore the functional distinction between heterospecific song and other ‘control’ sounds, such as the synthetic tones used here.

Conclusions
In this study, we have shown in a female songbird that the MBH and gonadotrophs can respond within 60 min to the sound of male courtship song. The time course of the LH response and the anatomical distribution of Egr-1 induction suggest that song acts as a trigger to initiate rapid release of cGnRH-I and that the well-documented ovarian effects of hearing song may not depend on slow-acting processes that involve structural remodeling of the brain. It is important to note that our data do not rule out a role for slow processes in song-induced enhancement of ovarian function and that such processes are likely to contribute toward the long-term effects of hearing song on a day-to-day basis. Future studies should focus on the connections between auditory centers and the regions of the hypothalamus implicated in song-induced cGnRH-I release.


   Acknowledgments
 
We are indebted to Henryk Urbanski for providing the GnRH antibody, to John Wingfield for the use of his RIA facility, and to George Bentley for advice regarding the experimental design and LH assay. We also thank Laura Mayer and Madiha Raees for technical assistance, Joanna Hubbard for collecting the animals, and Rashidat Ayantungi, Tulasi Ghimirey, and Marsha Howard for expert animal care. The manuscript benefited from the suggestions of three anonymous reviewers.


   Footnotes
 
This work was supported by National Science Foundation Grants IBN-0346984 and HHMI 52003727 and the Center for Behavioral Neuroscience.

Disclosure Summary: The authors have nothing to disclose.

First Published Online September 6, 2007

Abbreviations: cGnRH-I, Chicken GnRH-I; HPG, hypothalamic-pituitary-gonadal; ICC, immunocytochemistry; IH, inferior hypothalamic nucleus; IN, infundibular nucleus; IR, immunoreactivity; 10L:14D, 10 h light, 14 h dark; MBH, mediobasal hypothalamus; ME, median eminence; MLd, nucleus mesencephalicus lateralis pars dorsalis; NCM, caudomedial nidopallium; NMDA, N-methyl-D-aspartate.

Received June 29, 2007.

Accepted for publication August 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Marsden HM, Bronson FH 1964 Estrous synchrony in mice: alteration by exposure to male urine. Science 144:1469[Abstract/Free Full Text]
  2. McComb K 1987 Roaring by red deer stags advances the date of oestrus in hinds. Nature 330:648–649[CrossRef][Medline]
  3. Whitten WK, Bronson FH, Greenstein JA 1968 Estrus-inducing pheromone of male mice: transport by movement of air. Science 161:584–585[Abstract/Free Full Text]
  4. Ball GF, Balthazart J 2002 Neuroendocrine mechanisms regulating reproductive cycles and reproductive behavior in birds. In: Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, eds. Hormones, brain and behavior. Vol 2. San Diego: Academic Press; 649–798
  5. Lea J, Dyson M, Halliday T 2001 Calling by male midwife toads stimulates females to maintain reproductive condition. Anim Behav 61:373–377[CrossRef]
  6. Lindzey J, Crews D 1988 Psychobiology of sexual behavior in a whiptail lizard, Cnemidophorus inornatus. Horm Behav 22:279–293[CrossRef][Medline]
  7. Uller T, Olsson M 2005 Continuous male presence required for fertilization in captive painted dragons, Ctenophorus pictus. J Exp Zool 303A:1115–1119
  8. Stacey N, Chojnacki A, Narayanan A, Cole T, Murphy C 2003 Hormonally derived sex pheromones in fish: exogenous cues and signals from gonad to brain. Can J Physiol Pharmacol 81:329–341[CrossRef][Medline]
  9. Bentley GE, Wingfield JC, Morton ML, Ball GF 2000 Stimulatory effects on the reproductive axis in female songbirds by conspecific and heterospecific male song. Horm Behav 37:179–189[CrossRef][Medline]
  10. Brockway BF 1965 Stimulation of ovarian development and egg-laying by male courtship vocalization in budgerigars (Melopsittacus undulates). Anim Behav 13:575–578[CrossRef][Medline]
  11. Hinde RA, Steel E 1978 The influence of daylength and male vocalizations on the estrogen-dependent behavior of female canaries and budgerigars, with discussion of data from other species. Adv Study Anim Behav 8:39–73
  12. Kroodsma DE 1976 Reproductive development in a female songbird: differential stimulation by quality of male song. Science 192:574–575[Abstract/Free Full Text]
  13. Leboucher G, Depraz V, Kreutzer M, Nagle L 1998 Male song stimulation of female reproduction in canaries: features relevant to sexual displays are not relevant to nest-building or egg-laying. Ethology 104:613–624
  14. Morton ML, Pereyra ME, Baptista LF 1985 Photoperiodically induced ovarian growth in the white-crowned sparrow (Zonotrichia leucophrys gambelii) and its augmentation by song. Com Biochem Physiol A 80:93–97[CrossRef]
  15. Lehrman DS 1965 Interaction between internal and external environments in the regulation of the reproductive cycle of the ring dove. In: Beach, FA ed. Sex and behavior. New York: John Wiley Sons; 355–380
  16. Hinde RA 1965. Interaction of internal and external factors in integration of canary reproduction. In: Beach, FA ed. Sex and behavior. New York: John Wiley Sons; 381–415
  17. Olson AK, Eadie BD, Ernst C, Christie BR 2006 Environmental enrichment and voluntary exercise massively increase neurogenesis in the adult hippocampus via dissociable pathways. Hippocampus 16:250–260[CrossRef][Medline]
  18. van Praag H, Kempermann G, Gage FH 2000 Neural consequences of environmental enrichment. Nat Rev Neurosci 1:191–198[Medline]
  19. Searcy WA, Marler P 1981 A test for responsiveness to song structure and programming in female sparrows. Science 213:926–928[Abstract/Free Full Text]
  20. Mello CV, Vicario DS, Clayton DF 1992 Song presentation induces gene expression in the songbird forebrain. Proc Natl Acad Sci USA 89:6818–6822[Abstract/Free Full Text]
  21. Sharp PJ, Dunn IC, Main GM, Sterling RJ, Talbot RT 1990 Gonadotropin releasing hormones: distribution and function. In: Wada M, Ishii S, Scanes CG, eds. Endocrinology of birds. Berlin: Springer-Verlag; 31–42
  22. Juss TS 1993 Neuroendocrine and neural change associated with the photoperiodic control of reproduction. In: Sharp PJ, ed. Avian endocrinology. Bristol, UK: Society for Endocrinology; 47–60
  23. Konishi H, Foster RG, Follett BK 1987 Evidence for a daily rhythmicity in the acute release of luteinizing hormone in response to electrical stimulation in the Japanese quail. J Comp Physiol A 161:315–319[CrossRef][Medline]
  24. Ohta M, Wada M, Homma K 1984 Induction of rapid testicular growth in Japanese quail by phasic electrical stimulation of the hypothalamic photosensitive area. J Comp Physiol A 154:583–589[CrossRef]
  25. Meddle SL, Follett BK 1997 Photoperiodically driven changes in Fos expression within the basal tuberal hypothalamus and median eminence of Japanese quail. J Neurosci 117: 8909–8918
  26. Meddle SL, Maney DL, Wingfield JC 1999 Effects of N-methyl-D-aspartate on luteinizing hormone release and Fos-like immunoreactivity in the male white-crowned sparrow (Zonotrichia leucophrys gambelii). Endocrinology 140:5922–5928[Abstract/Free Full Text]
  27. Peczely P, Kovacs KJ 2000 Photostimulation affects gonadotropin-releasing hormone immunoreactivity and activates a distinct neuron population in the hypothalamus of the mallard. Neurosci Lett 290:205–208[CrossRef][Medline]
  28. Dawson A, King VM, Bentley GE, Ball GF 2001 Photoperiodic control of seasonality in birds. J Biol Rhythms 16:365–380[Abstract]
  29. Griffiths R, Double MC, Orr K, Dawson RJ 1998 A DNA test to sex most birds. Mol Ecol 7:1071–1075[CrossRef][Medline]
  30. Mellencamp WR 1969 Skull ossification in the white-throated sparrow. EBBA News 32:109–111
  31. Wiley RH, Piper WH 1992 Timing of cranial pneumatization in white-throated sparrows. Condor 94:336–343
  32. Shank MC 1959 The natural termination of the refractory period in the slate-colored junco and in the white-throated sparrow. Auk 76:44–54
  33. Wolfson A 1958 Regulation of refractory period in the photoperiodic responses of the white-throated sparrow. J Exp Zool 139:349–379[CrossRef][Medline]
  34. Maney DL, Cho E, Goode CT 2006 Estrogen dependent selectivity of genomic responses to birdsong. Eur J Neurosci 23:1523–1529[CrossRef][Medline]
  35. Maney DL, MacDougall-Shackleton EA, MacDougall-Shackleton SA, Ball GF, Hahn TP 2003 Immediate early gene response to hearing song correlates with receptive behavior and depends on dialect in a female songbird. J Comp Physiol A 189:667–674[CrossRef][Medline]
  36. Maney DL, Erwin KL Goode CT 2005 Neuroendocrine correlates of behavioral polymorphism in white-throated sparrows. Horm Behav 48:196–206[CrossRef][Medline]
  37. Meddle SL, Bush S, Sharp PJ, Millar RP, Wingfield JC 2006 Hypothalamic pro-GnRH-GAP, GnRH-I and GnRH-II during the onset of photorefractoriness in the white-crowned sparrow (Zonotrichia leucophrys gambelii). J Neuroendocrinol 18:217–226[CrossRef][Medline]
  38. Dawson A 2005 Seasonal differences in the secretion of luteinizing hormone and prolactin in response to N-methyl-DL-aspartate in starlings (Sturnus vulgaris). J Neuroendocrinol 17:105–110[CrossRef][Medline]
  39. Follett BK, Scanes CG, Cunningham FJ 1972 A radioimmunoassay for avian luteinizing hormone. J Endocrinol 52:359–378[Abstract/Free Full Text]
  40. Dawson A, Goldsmith AR 1982 Prolactin and gonadotrophin secretion in wild starlings (Sturnus vulgaris) during the annual cycle and in relation to nesting, incubation and rearing young. Gen Comp Endocrinol 48:213–221[CrossRef][Medline]
  41. Spinney LH, Bentley GE, Hau M 2006 Endocrine correlates of alternative phenotypes in the white-throated sparrow (Zonotrichia albicollis). Horm Behav 50:62–771
  42. Wingfield JC, Hegner RE, Lewis D 1991 Circulating levels of luteinizing hormone and steroid hormones in relation to social status in the cooperatively breeding white-browed sparrow weaver, Plocepasser mahali. J Zool (Lond) 225:43–58
  43. Mello CV, Ribeiro S 1998 ZENK protein regulation by song in the brain of songbirds. J Comp Neurol 393:426–438[CrossRef][Medline]
  44. Shu S, Gong J, Fan L 1988 The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett 85:169–171[CrossRef][Medline]
  45. Urbanski HF 1992 Photoperiod modulation of luteinizing hormone secretion in orchidectomized Syrian hamsters and the influence of excitatory amino acids. Endocrinology 131:1665–1669[Abstract]
  46. Oksche A, Farner DS 1974 Neurohistological studies of the hypothalamo-hypophysial system of Zonotrichia leucophrys gambelii. Adv Anat Embryol Cell Biol 48:1–136[Medline]
  47. Meddle SL, Follett BK 1995 Photoperiodic activation of fos-like immunoreactive protein in neurones within the tuberal hypothalamus of Japanese quail. J Comp Physiol A176:79–89
  48. Sharp PJ 1972 Tanycyte and vascular patterns in the basal hypothalamus of Coturnix quail with reference to their possible neuroendocrine significance. Z Zellforsch 127:552–569[CrossRef][Medline]
  49. Yasuo S, Watanabe M, Okabayashi N, Ebihara S, Yoshimura T 2003 Circadian clock genes and photoperiodism: comprehensive analysis of clock genes expression in the mediobasal hypothalamus, the suprachiasmatic nucleus and the pineal gland of Japanese quail under various light schedules. Endocrinology 144:3742–3748[Abstract/Free Full Text]
  50. Bailey DJ, Rosebush JC, Wade J 2002 The hippocampus and caudomedial neostriatum show selective responsiveness to conspecific song in the female zebra finch. J Neurobiol 52:43–51[CrossRef][Medline]
  51. Mello CV, Clayton DF 1994 Song-induced ZENK gene expression in auditory pathways of songbird brain and its relation to the song control system. J Neurosci 14:6652–6666[Abstract]
  52. Stokes TM, Leonard CM, Nottebohm F 1974 The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates. J Comp Neurol 156:337–374[CrossRef][Medline]
  53. Cheng MF 1986 Female cooing promotes ovarian development in ring doves. Physiol Behav 37:371–374[CrossRef][Medline]
  54. Moore M 1983 Effect of female sexual displays on the endocrine physiology and behaviour of male white-crowned sparrows, Zonotrichia leucophrys. J Zool 199:137–148
  55. Cheng MF, Peng JP, Johnson P 1998 Hypothalamic neurons preferentially respond to female nest coo stimulation: demonstration of direct acoustic stimulation of luteinizing hormone release. J Neurosci 18:5477–5489[Abstract/Free Full Text]
  56. Wingfield JC, Wada M 1989 Male-male interactions increase both luteinizing hormone and testosterone in the song sparrow, Melospiza melodia: specificity, time course and possible neural pathways. J Comp Physiol A 166:189–194
  57. Yamamura T, Hirunagi K, Ebihara S, Yoshimura T 2004 Seasonal morphological changes in the neuro-glial interaction between gonadotropin-releasing hormone nerve terminals and glial endfeet in Japanese quail. Endocrinology 145:4264–4267[Abstract/Free Full Text]
  58. Calas A 1973 L’innervation monoaminergique de l’eminence mediane: etude radioautographique et pharmacologigue chez le Canard Anas platyrhynchos. Z Zellforsch 138:503–522[CrossRef][Medline]
  59. Mello CV, Clayton DF 1995 Differential induction of the ZENK gene in the avian forebrain and song control circuit after metrazole-induced depolarization. J Neurobiol 26:145–161[CrossRef][Medline]
  60. Brinton RD, Yamazaki R, Gonzalez CM, O’Neill K, Schreiber SS 1998 Vasopressin-induction of the immediate early gene, NGFI-A, in cultured hippocampal glial cells. Brain Res Mol Brain Res 57:73–85[Medline]
  61. Saldanha CJ, Silverman AJ, Silver R 2001 Direct innervation of GnRH neurons by encephalic photoreceptors in birds. J Biol Rhythms 16:39–49[Abstract]
  62. Cheng MF, Zuo M 1994 Proposed pathways for vocal self-stimulation: met-enkephalinergic projections linking the midbrain vocal nucleus, auditory-responsive thalamic regions and neurosecretory hypothalamus. J Neurobiol 25:361–379[CrossRef][Medline]
  63. Wilczynski W, Allison JD, Marler CA 1993 Pathways linking social and environmental cues to endocrine control regions of amphibian forebrains. Brain Behav Evol 42:252–264[Medline]
  64. Mello CV, Vates GE, Okuhata S, Nottebohm F 1998 Descending auditory pathways in the adult male zebra finch (Taeniopygia guttata). J Comp Neurol 395:137–160[CrossRef][Medline]
  65. Winer JA, Chernock ML, Larue DT, Cheung SW 2002 Descending projections to the inferior colliculus from the posterior thalamus and the auditory cortex in rat, cat, and monkey. Hear Res 168:181–195[CrossRef][Medline]
  66. Endepols H, Walkowiak W 2000 Integration of ascending and descending inputs in the auditory midbrain of anurans. J Comp Physiol A 186:1119–1133[CrossRef][Medline]
  67. Yan J, Ehret G 2002 Corticofugal modulation of midbrain sound processing in the house mouse. Eur J Neurosci 16:119–128[CrossRef][Medline]
  68. Yan W, Suga N 1998 Corticofugal modulation of the midbrain frequency map in the bat auditory system. Nat Neurosci 1:54–55[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. Balthazart
Activation of Luteinizing Hormone Secretion by Photoperiod and Social Stimuli: Different Paths to the Same Destination
Endocrinology, December 1, 2007; 148(12): 5611 - 5613.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available