Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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White, Stephanie A.,
Frederick S. Livingston, and
Richard Mooney.
Androgens Modulate NMDA Receptor-Mediated EPSCs in the Zebra
Finch Song System.
J. Neurophysiol. 82: 2221-2234, 1999.
Androgens potently regulate the development of learned
vocalizations of songbirds. We sought to determine whether one action of androgens is to functionally modulate the development of synaptic transmission in two brain nuclei, the lateral part of the magnocellular nucleus of the anterior neostriatum (LMAN) and the robust nucleus of
the archistriatum (RA), that are critical for song learning and
production. We focused on
N-methyl-D-aspartate-excitatory postsynaptic currents (NMDA-EPSCs), because NMDA receptor activity in
LMAN is crucial to song learning, and because the LMAN synapses onto RA
neurons are almost entirely mediated by NMDA receptors. Whole cell
recordings from in vitro brain slice preparations revealed that the
time course of NMDA-EPSCs was developmentally regulated in RA, as had
been shown previously for LMAN. Specifically, in both nuclei,
NMDA-EPSCs become faster over development. We found that this
developmental transition can be modulated by androgens, because
testosterone treatment of young animals caused NMDA-EPSCs in LMAN and
RA to become prematurely fast. These androgen-induced effects were
limited to fledgling and juvenile periods and were spatially
restricted, in that androgens did not accelerate developmental changes
in NMDA-EPSCs recorded in a nonsong area, the Wulst. To determine
whether androgens had additional effects on LMAN or RA neurons, we
examined several other physiological and morphological parameters. In
LMAN, testosterone affected
-amino-3-hydroxy-5-methyl-4-isoxazoleproprianate-EPSC (AMPA-EPSC)
decay times and the ratio of peak synaptic glutamate to AMPA
currents, as well as dendritic length and spine density but did not
alter soma size or dendritic complexity. In contrast, testosterone did
not affect any of these parameters in RA, which demonstrates that
exogenous androgens can have selective actions on different song system
neurons. These data are the first evidence for any effect of sex
steroids on synaptic transmission within the song system. Our results
support the idea that endogenous androgens limit sensitive periods for
song learning by functionally altering synaptic transmission in song nuclei.
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INTRODUCTION |
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The learned vocalizations of zebra finches develop
through the integration of auditory and vocal-motor experience during a sensitive period restricted to the first three months of life (Immelmann 1969). This sensitive period comprises
sensory acquisition [posthatch day (PHD)
20-65], when young birds memorize the song of an adult
male tutor, and sensorimotor learning (PHD 35-90), when
juvenile birds match their song to the memorized tutor song via
auditory feedback. Song crystallization (~PHD 90) marks
the closure of sensorimotor learning, when the previously acoustically variable song becomes highly stereotyped.
A well-defined neural circuit mediates singing (Fig.
1). The vocal-motor part of this circuit
controls learned song production and includes nucleus HVc (acronym now
used as a proper name, in the convention of Fortune and
Margoliash 1992), the robust nucleus of the archistriatum (RA),
and brain stem motor areas involved in the control of syringeal and
respiratory muscles (Nottebohm et al. 1976
; Wild
1993
). A second part of the circuit, known as the anterior
forebrain pathway (AFP), indirectly connects HVc to RA via area X, the
dorsolateral part of the medial thalamus (DLM), and the lateral part of
the magnocellular nucleus of the anterior neostriatum (LMAN)
(Bottjer et al. 1989
; Nottebohm et al.
1982
; Okuhata and Saito 1987
). LMAN is an
important locus for exploring the neural mechanisms that underlie song
learning because it is essential to song development, but not to adult song production (Bottjer et al. 1984
; Scharff and
Nottebohm 1991
). LMAN terminals innervate the same vocal
premotor neurons in RA that receive HVc input, thus providing a site
for the AFP to influence the vocal-motor pathway during song learning
(Canady et al. 1988
; Kubota and Saito
1991
; Mooney and Konishi 1991
).
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N-methyl-D-aspartate (NMDA) receptors, which
depend on both glutamate binding and depolarization to gate calcium
flux into neurons, are crucial to several forms of synaptic plasticity
(Bear 1996). NMDA receptors mediate synaptic
transmission at several sites within the song system, including the
DLM-LMAN and LMAN-RA synapses. In LMAN, blockade of NMDA receptors
during tutoring decreases the number of learned notes (Basham et
al. 1996b
), which suggests that any developmental changes in
NMDA receptors in LMAN could influence song learning. Indeed, NMDA
receptor density in LMAN declines over song development, as indicated
by antagonist binding, immunolabeling, and mRNA expression levels
(Aamodt et al. 1992
; Basham et al. 1996a
;
Carrillo and Doupe 1995
). Changes in synaptic
transmission also occur in LMAN, where NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) become faster over the
same period of development (Livingston and Mooney 1997
).
In other systems, similar developmental changes in NMDAEPSCs have been invoked to explain sensitive periods for synaptic reorganization (Carmignoto and Vicini 1992
); the faster NMDA-EPSCs that
emerge during development reduce postsynaptic calcium entry and could thereby diminish certain forms of calcium-dependent synaptic remodeling (Bear 1996
).
As a first step in identifying cellular mechanisms that underlie song
learning, we tested whether factors that disrupt song learning also
alter the development of NMDA-EPSCs in the song system. Testosterone is
one factor that affects song learning, because young zebra finches
treated with testosterone during early song learning have shorter songs
and a reduced number of song syllables in adulthood (Korsia and
Bottjer 1991). Further, endogenous androgen levels fluctuate
during song development (Prove 1983
), and LMAN and RA
neurons contain androgen receptors, which provide a means for androgens
to directly alter these neurons (Balthazart et al.
1992
). Given that exogenous androgens disrupt early song learning, and that NMDA-EPSCs in LMAN are crucial for sensory acquisition (Basham et al. 1996b
) and become faster
during development (Livingston and Mooney 1997
), we
investigated whether NMDA-EPSCs are androgen sensitive. In addition to
LMAN, we examined NMDA-EPSCs in RA; because it is the site of
integration of activity from HVc and LMAN (Mooney and Konishi
1991
; Nottebohm et al. 1982
), it receives inputs
from LMAN that are primarily mediated by NMDA receptors (Kubota
and Saito 1991
; Mooney and Konishi 1991
;
Stark and Perkel 1999
), and its neurons contain androgen
receptors (Balthazart et al. 1992
).
A major finding is that exogenous androgens cause NMDA-EPSCs to become faster in LMAN and RA, but only during a developmental period that correlates with sensitive periods for song learning. These results extend previous work in the song system on sex steroid modulation of song behavior and neuronal morphology, by showing that androgens modulate NMDA receptor-mediated synaptic transmission in song nuclei. In addition, androgens have differential effects on glutamatergic synapses within the song system because they also altered AMPA-EPSCs, total dendritic length, and spine density in LMAN, but not in RA. These data provide the first evidence for an effect of sex steroids on synaptic transmission within the song system and suggest a potential mechanism for limiting sensitive periods for song learning.
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METHODS |
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Subjects
Experiments were performed using brain slices made from male
zebra finches, in accordance with a protocol approved by the Duke
University Institutional Animal Care and Use Committee. Finches were
obtained from our breeding colony where they were raised on a 14-h
day:10-h night light cycle. We defined three age groups for these
studies in LMAN and RA: fledglings (PHD 21-32), juveniles (PHD 38-49), and adults (>PHD 90). Briefly,
these ages were chosen because ~PHD 20 constitutes the
onset of sensory acquisition in zebra finches, and birds between
PHD 38 and 49 in our colony are in the early
stages of sensorimotor learning (see Livingston and Mooney
1997 for further details). By 90 days, male zebra finches have
stereotyped songs. For developmental studies in the Wulst, data from
juvenile and adult time points were combined (age range, PHD
40-117; mean, PHD 73) and were referred to as being
from juveniles.
Hormonal manipulations
To increase androgen levels, birds were implanted with 2-mm
pellets made of RTV sealant (Dow Corning, Midland, MI) containing ~50
µg of either testosterone (Steraloids, Wilton, NH) or
5-dihydrotestosterone (DHT; Steraloids); control pellets contained
RTV sealant alone. The pellets were placed subcutaneously over the
pectoral muscle, and the incision was closed with cyanoacrylate
(Elmer's Products, Columbus, OH) and dressed with antibiotic ointment
(Neosporin, Warner-Lambert, Morris Plains, NJ).
To reduce steroid levels, young zebra finches (PHD 12-17) were castrated and chronically treated with 50 µg flutamide (Sigma, St. Louis, MO), an androgen receptor antagonist, delivered through RTV pellets prepared as described for the steroid treatments. For the gonadectomy, birds were anesthetized via intramuscular injection with 35 µl of Equithesin [3-5 µl/g body mass; 1.05% pentobarbitol sodium (Abbott Laboratories, Chicago, IL), 4.25% chloral hydrate (Sigma), 7% EtOH (AAPER, Shelbyville, KY), 36% propylene glycol (Sigma), 2.1% MgSO4 (Mallinkrodt, Mundelein, IL)]. A small incision was made on the lateral wall of the body cavity, between the ribs that overlie the gonads. Testes were aspirated with a custom-fabricated glass pipette and the aid of a dissecting microscope (Zeiss, Germany). The incision was closed with cyanoacrylate and dressed with antibiotic ointment. Additionally, tetracycline (1 mg/ml) was supplied in the drinking water for 48 h postsurgery. Birds were reimplanted with flutamide pellets at 10-day intervals, following castration. Absence of gonadal tissue was visually confirmed on sacrifice with the aid of a dissecting scope, but was not histologically confirmed.
Testosterone radioimmunoassay
Blood was collected following decapitation between 10:00 and
11:00 a.m. Samples were briefly stored on ice, and then centrifuged to
isolate plasma. Testosterone was measured directly by radioimmunoassay using HPLC-purified 3H-Testosterone (Dupont-New
England Nuclear, Wilmington, DE), as well as antiserum and
HPLC-purified standards (ICN Biomedicals, Costa Mesa, CA). To
facilitate the detection of low plasma levels of testosterone, all
samples (50 µl) were spiked with 100 pg of testosterone, a value that
was in the midrange of the standard curve. Standards and samples were
extracted with ethyl acetate (Mallinkrodt), reconstituted in buffer
(phosphate-buffered saline, 1g/l gelatin; Sigma), and incubated with
antibody (1:56,000) and trace (10,000 cpm/test tube) for 1 h at
4°C for determination of free testosterone. Bound and free hormone
was separated by the dextran-coated charcoal technique (see
Nieschlag and Wickings 1978). Samples were counted by
liquid scintillation spectrometry using scintillation fluid containing
toluene (Mallinkrodt) and PPO-POPOP (Research Products International,
Mount Prospect, IL). Reported values are the measured values minus the
100 pg spike. Pilot studies revealed that the testosterone implants
augmented plasma testosterone levels over a 7- to 10-day period (data
not shown).
Brain slices
The brain slice preparation procedure has been described
previously in detail (Livingston and Mooney 1997;
Mooney and Konishi 1991
). Briefly, slices (400 µm)
were cut from the same brain, in the sagittal orientation for LMAN and
the coronal orientation for RA, and maintained on an interface-type
holding chamber at room temperature. After ~2 h, slices were
transferred to a superfusion chamber (24°C) for whole cell
recordings. LMAN and RA were readily visualized under transillumination.
Electrophysiological recordings
Whole cell recordings were obtained in brain slices made from
male zebra finches at fledgling, juvenile, and adult stages. Recording
electrodes were positioned in either LMAN or RA with the aid of a
dissecting scope (×40). Recording electrodes were made from 1.5-mm
diameter borosilicate glass (VWR, West Chester, PA), pulled on a
horizontal electrode puller (P-97, Sutter Instrument, Novato, CA) and
filled with an internal solution consisting of (in mM) 3.19% (vol/vol)
50% D-gluconic acid (Sigma), 10 EGTA (Sigma), 5 MgCl2 (Sigma), 40 HEPES (Fluka, Ronkonkoma, NY),
2 Na+-ATP (Sigma), 0.3 Na+-GTP (Boehringer-Mannheim, Indianapolis, IN),
and 1 QX-314 (RBI/Sigma, Natick, MA), and the pH was adjusted to 7.25 with CsOH (50% g/ml H2O; Aldrich Chemical
Company, Milwaukee, WI). The final electrode impedances ranged from 2 to 5 M. Whole cell currents were recorded with an Axopatch 1D (for
LMAN) or an Axoclamp 2B (for RA) intracellular amplifier (Axon
Instruments, Foster City, CA), and current traces were digitized at 10 kHz after low-pass filtering at 1-5 kHz. Glutamate EPSCs were recorded
in 50 µM picrotoxin (Sigma); NMDA-EPSCs or AMPA-EPSCs were isolated
with the addition of 5 µM
1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX, RBI/Sigma), or 100 µM
D,L-2-amino-5-phosphonovaleric acid (D,L-APV,
RBI/Sigma), respectively, accomplished by bath perfusion of the drug.
NMDA-EPSCs were electrically evoked while holding the membrane
potential 20 mV more positive than the empirically determined EPSC
reversal potential to remove the voltage-dependent blockade of the NMDA
receptor by extracellular magnesium (Mayer et al. 1984;
Nowak et al. 1984
). AMPA-EPSCs from LMAN were recorded
at the same membrane potential, but in RA, cells were held 80 mV negative of the reversal potential to increase the driving force, which
was necessary to produce synaptic currents large enough for accurate
quantification. Series resistance (<20 M
for LMAN, <30 M
for
RA) was monitored throughout the recording by measuring the current
transients resulting from small (2 mV) hyperpolarizing voltage pulses.
For experiments that measured the ratio of the peak glutamate- to peak
AMPA-EPSCs, only cells where the series resistance changed <20%
during the course of drug application were used. Input resistance
measurements were calculated by measuring the steady-state current
resulting from the application of a small (2 mV) hyperpolarizing
voltage pulse. Holding potentials were not corrected for the liquid
junction potential. Electrode solutions used in recordings from a
majority of animals contained neurobiotin (0.5%; Vector Laboratories,
Burlingame, CA) in the internal solution to ensure that recorded cells
were located within the appropriate nucleus (see below for histological methods).
Bipolar stimulating electrodes were either made from individual
tungsten microelectrodes (Micro Probe, Clarksburg, MD), or were
prefabricated concentric bipolar electrodes (FHC, Brunswick, ME). For
LMAN, stimulating electrodes were placed in the thalamic fiber tract,
which contains DLM axons that innervate LMAN (Livingston and
Mooney 1997). For RA, which receives afferent input from both LMAN and HVc, we specifically activated LMAN-RA synapses by placing stimulating electrodes dorsolateral of RA in the LMAN fiber tract (see
Mooney 1992
). Synaptic responses were elicited at
0.1-0.3 Hz by applying a brief (100 µs) electrical stimulus (5-750
µA) to the thalamic axons that innervate LMAN or the LMAN axons that innervate RA (Mooney 1992
). Stimulus intensity was
adjusted to generate an evoked EPSC with consistent amplitude that was
monosynaptic, i.e., the interval between the stimulus artifact and the
onset of the synaptic current was <5 ms, and the rising and falling phases of the synaptic current appeared smooth and monotonic. In LMAN,
this approach resulted in EPSC peak amplitudes that did not vary across
age or treatment. In RA, EPSC amplitudes were lower in slices from
younger animals because lower stimulus intensities were required to
avoid recruiting polysynaptic responses. We did not test for
statistical significance of any differences in the peak amplitudes of
evoked EPSCs because they varied depending on the slice preparation,
the stimulating electrodes, and the stimulus intensity.
Data acquisition and analysis
Data acquisition and analysis for intracellular recordings were performed with a National Instruments (Austin, TX) data acquisition board (AT-MIO-16E2), controlled by custom Labview software written by F. Livingston and R. Neummann. Five to 10 individual events were collected from a single neuron and digitally filtered (low-pass, 2 kHz) with an 8-pole Bessel filter and then averaged to obtain a representative cellular EPSC. The EPSCs shown in the figures are the averages of these cellular EPSCs for all the cells in each treatment group. The peak amplitude of the currents, the 10-90% rise time of the currents from baseline to peak amplitude, the time from the peak amplitude to 1/e of the amplitude, and the relative charge (measured by integrating currents with normalized peak amplitudes) were calculated using automated Labview software written by F. Livingston and S. White. Statistical analyses were conducted using JMP IN software (SAS Institute, Cary, NC). Nonparametric statistical tests were used because the data were not assumed to be normally distributed. Mann Whitney U tests were used to assess the significance between experimental and age-matched controls, as well as between two different developmental stages. In all cases, the minimum significance level was set at P < 0.05 using two-tailed comparisons. Only significant differences are reported in the text, unless otherwise noted. Averages are reported as means ± SE.
Song analysis
Song was analyzed by placing an individual male zebra finch in a sound-proof recording chamber (Industrial Acoustics Corporation, Bronx, NY) with a female finch. Digital audio recordings were collected using a National Instruments data acquisition board (AT-MIO-16E2) and Labview software written by R. Balu and F. Livingston. After 24 h, recordings were visually scanned for the presence of song. If no song was detected, then the animal remained in the chamber for another 24 h until a minimum of 30 bouts of song were collected. In one case, no song was detected until after the female finch was removed on the fourth day. Thirty examples of song were selected and imported into Avisoft Software (SASLab Pro 3.4, Raimond Specht, Berlin) for further analysis and generation of sonograms (frequency intensity vs. time). From these 30 renditions, one of us selected two representative 7-s sweeps for presentation to each of six observers.
Observers, blind to the experimental condition of the animal, were instructed to assign a song score ranging from 1 (very abnormal) to 5 (normal, adultlike) by listening to audio playback of the song and by visually inspecting the oscillograms (voltage vs. time) and sonograms. Observers were instructed to base their song quality judgments on the stereotypy of syllables and motifs. A song syllable was defined as a continuous marking on the song spectrogram. Mature zebra finch song is composed of one or more motifs that each contain the same stereotyped sequence of song syllables. One bout of song is typically composed of several introductory notes, followed by one or more motifs. Observers ranked the song quality of one juvenile male (PHD 57) in addition to the four castrates (>PHD 200) as well as one intact adult that had been housed in the same cage with the castrated animals from when they were PHD 35.
Morphology
To obtain filled neurons that were of sufficient quality for
morphological analysis, sharp microelectrode intracellular recordings were made in LMAN and RA from brain slices (prepared as described above) that were held in an interface-type chamber (30°C, Medical Systems, Greenvale, NY). Electrodes were made from borosilicate glass
pulled to a final resistance of 100-150 M when filled with 3 M
K-acetate (Mallinkrodt) and 4-8% neurobiotin. After establishing stable intracellular recordings, depolarizing currents (+0.5-1.5 nA,
1 s in duration at 2-s intervals) were applied for 20-40 min. The
slice was fixed in 4% paraformaldehyde in 25 mM sodium phosphate buffer for at least 24 h at 4°C, resectioned on a freezing
microtome or embedded in a gelatin-albumin mixture and cut on a
vibratome at 75 µm, and visualized with avidin-HRP (diluted 1:100;
Vector Laboratories, Burlingame, CA) and 3'3'-diaminobenzidine
tetrahydrochloride (Sigma); the reaction was intensified with 1%
CoCl2 (Sigma) and 1%
NiSO4(NH4)2SO3
(Sigma). Camera lucida drawings were made with the aid of a drawing
tube attached to a Zeiss Axioskop, using a ×63 oil-immersion objective
(nA 1.3).
Total dendritic length was measured from the camera lucida
drawings of filled cells using a PlanWheel (Scalex, Carlsbad, CA). To
determine the spine frequency, the total number of dendritic spines was
counted for each individual neuron and then divided by the total
dendritic length. To measure the extent of an individual cell's
processes, a Sholl-like analysis (Sholl 1956) was
performed from the drawings. Briefly, a series of evenly spaced
concentric circles (20-µm increments) was placed over the cell
drawing, centered around the soma, and the number of dendrites that
intersected each of these circles was plotted as a function of radius
from the cell body. For areal measurements of individual RA or LMAN cell bodies, drawings were scanned into a computer, and the borders of
the soma were traced using Scion Image software (Scion Corporation, Frederick, MD) to convert pixel values into
µm2. No corrections were made for tissue
shrinkage. Mann-Whitney U tests were used to compare the
anatomic data from control and T-treated fledglings. For the Sholl
analysis, two-way ANOVAs were used to test for any effects of radius or
experimental group on the number of intersections.
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RESULTS |
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NMDA-EPSCS in LMAN and RA change during development
To track developmental changes in NMDA receptors, we isolated NMDA-EPSCs at DLM-LMAN and LMAN-RA synapses in male zebra finches by applying NBQX (to block AMPA-EPSCs) and by holding the membrane potential 20 mV positive of the reversal potential (to relieve the voltage-dependent magnesium blockade). Whole cell recordings revealed that NMDA-EPSC decay times became markedly faster in LMAN and RA between fledgling, juvenile, and adult time points (Fig. 2). These faster currents were reflected by a decrease in the time it took the current to decay to 1/e of the peak amplitude (e-fold decay time), as well as by a decrease in the relative charge that was transferred by the current (Fig. 2A), which was measured as the area underneath the normalized current trace (see METHODS). Because these measurements generally covaried, only the e-fold decay times are reported here (but one exception is noted). The intrinsic and synaptic properties for LMAN and RA neurons throughout development are shown in Table 1. Below, significance values are reported in the text only when they do not appear in the figure legends.
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Within LMAN, the e-fold decay times of NMDA-EPSCs decreased
by ~50% over development, (94 ± 7 vs. 48 ± 4 ms,
mean ± SE; Fig. 2.) Almost all (~90%) of this shift
took place between fledgling and juvenile life. However, a small
decrease in e-fold decay time was revealed between
juveniles and adults (56 ± 3 vs. 48 ± 4 ms). This small
change in NMDA-EPSCs was not apparent in the relative charge transfer
(Fig. 2A), nor seen in an earlier study that used double
exponential fits to describe the EPSCs (Livingston and Mooney
1997). Distributions of e-fold decay times
demonstrated that the range of values narrowed over development (Fig.
2B). The decrease in NMDA-EPSC e-fold
decay times that occurred between fledgling and juvenile ages was not
accompanied by changes in EPSC rise times or in the input resistances
of LMAN neurons (Table 1). These data indicate that the changes in
e-fold decay times are not part of a more general
alteration of neuronal electrotonic properties (see
DISCUSSION). In summary, these results show that developmental changes in NMDA-EPSCs within LMAN occur predominantly during early posthatch development (Livingston and Mooney
1997
), before sensorimotor learning is complete.
In RA, NMDA-EPSCs also became faster over development, as the mean e-fold decay times declined by almost two-thirds between fledgling and adult life (159 ± 10 vs. 58 ± 3 ms; Fig. 2). These developmental changes were not only greater in magnitude than those seen in LMAN, but were also more protracted: in RA, close to one-third of the total change in e-fold decay times occurred between juvenile and adult life (i.e., 91 ± 6 vs. 58 ± 3 ms). Similar to the results obtained in LMAN, the distribution of e-fold decay times in RA narrowed markedly over development (Fig. 2B). In contrast to observations made in LMAN, the rise times of NMDA-EPSCs decreased at each developmental time point, and RA neuronal input resistance decreased between fledgling and juvenile life (Table 1). In summary, NMDA-EPSCs become faster in both RA and LMAN between fledgling and adult life, but the magnitude and timing of these changes differs between the two nuclei. Additionally, other cellular changes occur within RA over development.
Androgens hasten NMDA-EPSC maturation in fledglings and juveniles
In many types of songbirds, exogenous steroids produce dramatic
effects on both song behavior and the morphology of song control nuclei
(for review see Bottjer and Johnson 1997). In zebra
finches, androgen treatment of juvenile males at early time points
(<PHD 40) perturbs song development (Korsia and
Bottjer 1991
). One hypothesis is that androgens affect synaptic
transmission in LMAN and RA, making NMDA-EPSCs faster, which would be
reflected by decreases in the e-fold decay times. If such
effects play a role in song learning, then they should occur during the
same developmental periods when androgens affect song behavior. As a
first test of these ideas, we implanted young birds (~PHD
15) with either testosterone-containing or blank pellets and
examined NMDA-EPSCs in LMAN and RA neurons 10 days later (~PHD
25).
This chronic testosterone (T)-treatment induced premature changes in NMDA-EPSCs within both LMAN and RA (Fig. 3). In LMAN, T-treated fledgling birds had NMDA-EPSCs that were faster than those of age-matched control-implanted birds, as reflected by a 35% decrease in the mean e-fold decay time (68 ± 4 vs. 104 ± 8 ms; Fig. 3A). The NMDA-EPSC decay times in T-treated fledglings were equivalent to those in normal juvenile birds (68 ± 4 vs. 56 ± 3 ms, P = 0.09). In RA, NMDA-EPSCs from T-treated fledglings were also faster than those of control-implanted fledglings, with a 26% decrease in e-fold decay times (117 ± 10 vs. 159 ± 8 ms; Fig. 3A), but were not as fast as those of juvenile birds. In both LMAN and RA, T-treatment narrowed the distribution of e-fold decay times, similar to the tightened distribution that emerges during normal maturation (Fig. 3B). In LMAN, T-treatment produced a slight decrease in neuronal input resistance relative to control-implanted fledglings, but this decrease was not observed with DHT treatment (Table 1, see below regarding DHT treatment). In RA, T-treatment made NMDA-EPSCs faster relative to control fledglings, without the concomitant decrease in input resistance observed between fledglings and juveniles. It should be noted that in subsequent experiments, age-matched controls were not implanted with blank pellets, because there was no observed difference in any of our measures between control-implanted and unimplanted fledglings in LMAN and RA (Table 1).
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Androgen receptors within LMAN and RA neurons provide a direct
mechanism to mediate the effects seen here. However, it is possible
that androgens affect NMDA-EPSC development in many types of
telencephalic neurons, even those lacking androgen receptors, via an
indirect mechanism (see DISCUSSION and Fig. 7). To test whether these androgen effects could occur in neurons devoid of androgen receptors, we recorded NMDA-EPSCs in the Wulst, an avian telencephalic visual area that lacks androgen receptors and is not
implicated in song development (Balthazart et al. 1992).
As in LMAN and RA, NMDA-EPSCs within the Wulst became faster between fledgling and juvenile time points (312 ± 19 ms,
n = 23 vs. 185 ± 27 ms, n = 15;
Fig. 3, A and B). In contrast to LMAN and RA, however, in the Wulst, T-treatment of young birds (implanted
~PHD 15, recorded 10 days later, ~PHD 25) did
not modulate NMDA-EPSCs (mean e-fold decay time in T-treated
fledglings: 306 ± 27 ms; n = 34 cells,
P = 0.90 relative to control). Thus although NMDA-EPSCs become faster in both song and nonsong areas over development, androgens do not globally influence this transition throughout the
avian telencephalon.
Steroid hormones can exert short-term effects on neuronal excitability via nongenomic pathways, as well as long-term effects that require changes in gene expression. Although we implanted testosterone (~PHD 15) and then recorded NMDA-EPSCs 10 days later, it is possible that androgens exerted their actions within a much shorter time frame. Therefore we implanted fledgling birds (~PHD 24.5) with testosterone 12 h (rather than 10 days) before recording, and then compared the EPSC e-fold decay times to those of untreated age-matched controls. Unlike the effects of longer androgen exposure, the 12-h treatment did not alter EPSC e-fold decay times in either LMAN or RA (LMAN: 12 h = 99 ± 12 ms, n = 10, control = 104 ± 8 ms, n = 29, P = 0.58; RA: 12 h = 157 ± 15 ms, n = 14, control = 159 ± 10, n = 31, P = 0.94), even though a radioimmunoassay confirmed that the implants significantly elevated serum androgen levels before recording (Fig. 3C).
Because endogenous neuronal aromatases can convert testosterone to
17-estradiol (Schlinger 1997
), we used the
nonaromatizable androgen DHT to confirm that the effects of
testosterone were androgen-specific. As with T-treatment, young birds
were implanted with DHT (~PHD 15), and recorded from 10 days later (~PHD 25). Indeed, this chronic DHT-treatment
produced faster EPSCs in both LMAN and RA as reflected in a 42 and 22%
decline, respectively, in the e-fold decay times from
control values to those of DHT-treated fledglings (LMAN: 104 ± 8 vs. 60 ± 3 ms, RA: 159 ± 10 vs. 124 ± 9 ms; Table 1).
In LMAN, the EPSC e-fold decay times in DHT-treated fledglings were indistinguishable from normal juveniles (60 ± 3 vs. 56 ± 3 ms, P = 0.26) but in RA, EPSC
e-fold decay times from DHT-implanted fledglings were still
slower than those of juveniles (124 ± 9 vs. 91 ± 6 ms,
P < 0.005). In both LMAN and RA, DHT-treatment
produced a slight decrease in EPSC rise times, an effect not seen with
T-treatment (Table 1). In LMAN, DHT did not alter the input resistance,
although testosterone did (Table 1). Except for these small
differences, DHT caused similar effects to those of testosterone, which
suggests that androgens, rather than estrogens, are responsible for the
faster NMDA-EPSCs seen here.
Song development is disrupted by exogenous testosterone administered as
late as PHD 40 (Korsia and Bottjer 1991). To
test whether changes in NMDA-EPSCs within LMAN and RA are also
sensitive to such later treatment, recordings were made from juvenile
male finches (~PHD 45) that had received DHT implants 10 days earlier (~PHD 35). DHT was chosen to ensure that any
observed effects were androgenic and not due to estrogens. In parallel
with the effects seen at earlier ages, this later treatment yielded
NMDA-EPSCs that decayed more quickly than those of age-matched controls
(Fig. 4). In LMAN, the e-fold
decay times of NMDA-EPSCs in DHT-treated juveniles were slightly (14%)
faster than in normal juveniles (48 ± 2 vs. 56 ± 3 ms; Fig.
4) and were equivalent to adult values (48 ± 4 ms;
P = 0.22 relative to juvenile-DHT birds). In RA,
DHT-treated juveniles had NMDA-EPSCs that were also faster than those
of normal juveniles (70 ± 6 vs. 91 ± 6 ms; Fig. 4) and
equivalent to those of adults (64 ± 4 ms, P = 0.10; Fig. 4); this treatment additionally resulted in higher input
resistances of RA neurons relative to controls (Table 1). In summary,
NMDA-EPSCs in both LMAN and RA are sensitive to androgen treatment
during the developmental period when these currents normally become
faster, when endogenous androgen levels fluctuate (Prove
1983
), and when exogenous androgen disrupts song learning
(Korsia and Bottjer 1991
).
|
Endogenous androgens could provide an intrinsic signal regulating the
maturation of NMDA-EPSCs in LMAN and RA, because testosterone levels
increase over the course of song development in male zebra finches
(Adkins-Regan et al. 1990; Prove 1983
).
To confirm these hormonal changes, we measured plasma testosterone
levels from a subset of the animals from which electrophysiological
recordings were made (Fig. 3C). Adult testosterone levels
(3.2 ± 0.4 ng/ml) were higher than those of juveniles (1.4 ± 0.3 ng/ml). Although mean values for fledglings (0.6 ± 0.3 ng/ml) were half of the juvenile measure, these two groups were not
significantly different (P = 0.95).
Adult NMDA-EPSCs are not altered by androgens
To test whether exogenous androgens affect NMDA-EPSCs throughout life or, instead, only during a restricted developmental period, adult birds (>PHD 90) were implanted with DHT pellets and then recorded from 10 days later. In both LMAN and RA, this adult DHT-treatment did not affect NMDA-EPSC e-fold decay times (see Fig. 4 and Table 1). This lack of effect demonstrates that NMDA-EPSCS are sensitive to exogenous androgens only during juvenile life, and that the transition to adultlike NMDA-EPSCs is the final step both for development and for our experimentally induced androgen-mediated effects. That this limit exists experimentally suggests that the androgen sensitivity of the NMDA-EPSCs seen in young animals is not merely due to pharmacological actions of the steroid. Thus in the song system, androgen treatment can modulate NMDA-EPSCs during a fledgling and juvenile sensitive period that overlaps with periods when song learning can be disrupted by exogenous androgens (Fig. 4 and Table 1).
NMDA-EPSCs development in castrates
In addition to being sensitive to exogenous androgens, normal
development of NMDA-EPSCs within LMAN and RA might be regulated by
endogenous androgens, i.e., androgen dependent. If so, in the absence
of androgens, NMDA-EPSCs should remain in the fledgling state. To test
whether developmental changes in NMDA-EPSCs were androgen dependent,
zebra finch chicks were castrated ~PHD 14 and chronically
treated with the androgen receptor antagonist flutamide (implanted
every 10 days until recordings were made). Previous work has shown that
similar treatment produces aberrant song in a majority of adults
(Bottjer and Hewer 1992). We were not able to assess any
deleterious effects of early castration and flutamide treatment on song
behavior in juveniles because, at that age, song is highly variable and
not well-developed (Immelmann 1969
). Nevertheless, we
initially tested the androgen dependency of NMDA-EPSC e-fold
decay times in slices made from juvenile animals, rather than adults,
because this is an age when the currents are androgen sensitive. We
found no differences in e-fold decay times in either LMAN or
RA between intact and castrated juveniles (LMAN: juvenile = 56 ± 3 ms, n = 20, castrate = 57 ± 5 ms, n = 16, P = 0.81; RA: juvenile = 91 ± 6 ms, n = 26, castrate = 94 ± 5 ms, n = 41, P = 0.35). To test whether
the effects of castration emerged at later time points, we also
compared e-fold decay times between castrated adults and
age-matched controls. Once again, however, no difference was observed
between intact and castrated animals (LMAN: adult = 48 ± 4 ms, n = 23, adult castrate = 54 ± 3 ms, n = 20, P = 0.09; RA: adult 58 ± 3 ms, n = 20, adult castrate = 70 ± 8 ms,
n = 16, P = 0.20).
A potential confound is that, although it is relatively easy to augment
androgen levels, it is difficult to abolish them in zebra finches
(Adkins-Regan et al. 1990; Marler et al.
1988
). Indeed, a radioimmunoassay revealed that our juvenile
and adult castrates had serum testosterone levels similar to
age-matched controls (see Fig. 3C, adult castrate data not
shown). Thus despite the absence of observable gonadal tissue,
significant levels of androgens were present in the blood of castrates,
presumably arising from extra-gonadal sources (Adkins-Regan et
al. 1990
).
Despite the lack of effect of castration on plasma testosterone levels,
flutamide levels may have been sufficient to block receptor-mediated
actions of extragonadal androgens. To determine whether our protocol of
flutamide treatment had prevented androgen signaling, we analyzed
several androgen-sensitive features of songbirds. We measured syringeal
mass (Luine et al. 1980), frequency of singing
(Arnold 1975
), and song quality (Bottjer and
Hewer 1992
) in four surviving adults from the castrate group.
Syringeal weights were normalized for body weight to derive a
syringeal-somatic index (SSI; syrinx weight/body weight × 100).
SSIs were lower in these four successful castrates compared with normal
adults (0.11 ± 0.01, n = 4, vs. 0.20 ± 0.01, n = 10, P < 0.01), and also compared with intact fledglings (0.13 ± 0.01, n = 13, P < 0.02 as compared with adult castrates). These
SSI data indicate that flutamide treatment achieved at least a partial
peripheral block of androgen action in each of the four adult castrates.
In contrast to the somatic effects, behavioral measures did not reflect
consistent effects among flutamide-treated castrates. To obtain 30 renditions of song for the behavioral analyses, three of the four
castrates required more than two recording sessions. The fourth
castrate (Blue 7) required just 2 days, as did the intact
adult and the juvenile. Song quality (1-5) of the four castrates and
the two intact males was judged by six observers blind to the
experimental group. By these evaluations, the song quality of
Blue 7 as well as that of another castrate was deemed normal
(above 4), whereas those of two castrates and of a control juvenile
were ranked as disrupted relative to the song of an untreated adult.
These behavioral data from castrates, although from a small sample
size, are similar to those of Bottjer and Hewer (1992), which indicate that castration and flutamide treatment are ineffective in altering song quality in roughly one quarter of the cases. Given the
presence of circulating androgens in our castrates (i.e., those that
were used to determine NMDA-EPSC e-fold decay times; Fig.
3), one possibility for the lack of effect of castration on NMDA-EPSCs
is that the flutamide treatment was not consistent in blocking central
androgen receptors from these residual androgens (see
DISCUSSION).
Androgen treatment alters AMPA-EPSCs in LMAN, but not in RA
Androgenic effects at LMAN and RA synapses could be specific
to NMDA-EPSCs, or might involve more general actions on synaptic transmission at these sites. To address the specificity of androgenic actions, we examined another postsynaptic component of glutamatergic transmission, the AMPA-EPSC. Previous studies have demonstrated that
synaptic transmission at DLM terminals onto LMAN neurons occurs via
both NMDA and AMPA receptors (Livingston and Mooney 1997), whereas EPSPs at the LMAN terminals onto RA neurons are mediated largely by NMDA receptors (Mooney 1992
).
Similar to the previous experiments on NMDA-EPSCs, we measured the
e-fold decay times of AMPA receptor-mediated EPSCs in
fledgling controls and fledglings that had been implanted with DHT 10 days earlier (~PHD 15). Additionally, we tested whether
the relationship between the AMPA-EPSC and the total glutamate-EPSC was
altered by DHT. To do this, we first measured the peak amplitude of the
glutamate-EPSC at 20 mV positive of the reversal potential, and then
that of the pharmacologically isolated AMPA-EPSC (in 100 µM
D,L-APV, see METHODS for details). We
calculated the ratio of these two EPSC amplitudes (i.e.,
Glupeak:AMPApeak), and
compared these values in control and DHT-implanted fledglings.
In LMAN, androgens affected the e-fold decay times of AMPA-EPSCs, reducing the mean by 30% (7.6 ± 0.6 ms vs. 5.4 ± 0.7 ms; Fig. 5, Table 2). There was also a difference in the Glupeak:AMPApeak between control and DHT-implanted fledglings (4.0 ± 0.7 vs. 2.5 ± 0.17; Table 2 and Fig. 5). In contrast, no differences were seen in either the AMPA-EPSC rise times or the input resistances (confirming our earlier findings, see Tables 1 and 2).
|
|
At the LMAN-RA synapse, following the application of
D,L-APV, only a very small current (~4 pA) remained at
+20 mV, consistent with previous observations at this synapse
(Mooney 1992; Stark and Perkel 1999
),
which showed that nearly all of the synaptic response was mediated by
NMDA receptors. Therefore we clamped the membrane potential to 80 mV
negative of the reversal potential to increase the driving force and
augment AMPA-EPSCs in RA. The peak amplitudes for these AMPA-EPSCs were
then measured at this new holding potential, and the ratio of the peak
amplitudes for glutamate (at +20 mV) and AMPA (at
80 mV)
receptor-mediated synaptic currents was calculated. In RA, neither the
e-fold decay times of AMPA-EPSCs nor the
Glupeak:AMPApeak from birds
implanted with DHT differed from those of controls (Table 2 and Fig.
5). These results reveal that, in LMAN, androgens act more generally to alter both AMPA receptor and NMDA receptor-mediated synaptic
transmission, whereas at the LMAN-RA synapse, androgen effects are
specific to the NMDA receptor-mediated component of the synaptic current.
Testosterone treatment alters neuronal morphology in LMAN, but not in RA
The sexual dimorphism of the song system (Nottebohm and
Arnold 1976) is a compelling example of how sex steroids affect
the structure of song nuclei, including neuronal morphology
(DeVoogd and Nottebohm 1981
; DeVoogd et al.
1985
; Gurney 1981
). The effects of augmenting
early testosterone on LMAN and RA volume, neuron number, and soma size
has been examined in adult male zebra finches (Schlinger and
Arnold 1991
), but no morphological analysis has been made at
the earlier time points studied here. Because changes in dendritic or
somatic morphology could exert electrotonic effects on synaptic
currents, we assessed neuronal soma size, total dendritic length,
dendritic branching complexity via a Sholl analysis (Sholl 1956
), and spine density of LMAN and RA neurons in both control and T-treated fledglings (young birds implanted with testosterone ~PHD 15, and recorded from 10 days later). Within LMAN,
T-treatment had no effect on neuronal soma size or on dendritic
complexity (Fig. 6A and
D), although there was a slight effect on total dendritic length (T-treated = 2,464 ± 122 µm, control = 2,118 ± 92 µm; Fig. 6C). Within RA, androgen
treatment had no effect on soma size, total dendritic length, or
dendritic complexity (Fig. 6). Finally, measurements of dendritic spine
densities revealed a striking contrast in the androgen sensitivity of
LMAN and RA neurons. In LMAN, T-treatment resulted in a 64% increase
in spine density relative to control animals (control = 0.20 ± 0.016, T-treated = 0.33 ± 0.08 spines per µm; Fig.
6B) but exerted no effect on spine density in RA. As with
the contrasting effects between LMAN and RA on AMPA-EPSCs, these
effects of androgens on spine density indicate a qualitative difference
in the androgen sensitivity of LMAN neurons relative to those in RA.
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DISCUSSION |
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Here we show that developmental changes in NMDAEPSCs within
the song system can be potently modulated by androgens during fledgling
and juvenile periods, and we provide the first demonstration that sex
steroids can alter synaptic transmission in the song system. Treatment
of young zebra finches with testosterone caused NMDA-EPSCs in LMAN and
RA to become faster. The nearly identical effect of treatment with DHT,
a nonaromatizable androgen, indicates that androgens rather than
estrogens mediate this effect. NMDA-EPSCs in LMAN and RA remain
sensitive to exogenous androgens from fledgling through juvenile life,
in parallel with behavioral studies that have shown that song
development in zebra finches is disrupted by testosterone implants as
late as PHD 40 (Korsia and Bottjer 1991). In
LMAN and RA, testosterone treatment of juveniles yielded NMDA-EPSC
e-fold decay times that were as fast as those in adult animals. In contrast, exogenous androgens did not alter NMDA-EPSCs in
adult animals, suggesting that there is a sensitive period for these
steroid effects.
In addition to showing that androgens can modulate NMDA-EPSCs, our data
confirm that NMDA-EPSCs become faster within LMAN during normal song
development (Livingston and Mooney 1997), and reveal a
similar developmental trend in RA. These observations add to those of
others that document developmental changes in the physiological
properties of LMAN neurons (Boettiger and Doupe 1998
;
Bottjer et al. 1998
; Livingston and Mooney
1997
). Further, In LMAN, NMDA-EPSCs become fast early in
posthatch development, primarily during sensory acquisition and early
sensorimotor learning. In RA, changes in NMDA-EPSC e-fold
decay times are more protracted, extending further into the period of
sensorimotor learning. Although developmental changes in NMDA-EPSCs
also occurred in the Wulst, there this process was not altered by
T-treatment. Thus androgens do not globally influence NMDA-EPSCs
throughout the zebra finch telencephalon.
Whether the developmental changes in NMDA-EPSCs in LMAN and RA are
androgen dependent remains unknown, because the castration did not
lower endogenous androgens, and the efficacy of the flutamide treatment
is not clear. If flutamide was completely effective in blocking
androgen signaling, then this would suggest that NMDA-EPSC development,
although sensitive to exogenous androgens, is not regulated by
endogenous androgens, or in the absence of androgen signals, alternate
mechanisms compensate for NMDA receptor development. Alternatively,
endogenous androgens could be crucial for normal NMDA-EPSC development,
but the early castration and flutamide treatment may not have blocked
androgen signaling in all birds (Adkins-Regan et al.
1990; Bottjer and Hewer 1992
). Although the low
syringeal weight of each of the four adult castrates is consistent with
a peripheral blockade or reduction of androgens (Luine et al.
1980
), behavioral measures of androgen sensitivity such as song
frequency (Arnold 1975
) and song quality were variable
between birds, similar to what was found in a previous study
(Bottjer and Hewer 1992
). These differing effects may
indicate that peripheral and central androgen signaling have different
androgen and/or flutamide sensitivities, and that central androgen
receptors were activated in a subset of the castrates. If so, then the
e-fold decays obtained following our castration protocol
were gathered from a heterogeneous population of animals, some with
continual blockade of androgen receptors, and others with persistent
androgen signaling. Given the natural variability of e-fold
decay times seen here, a contaminant population (potentially 25%, as
suggested by behavioral data) within one experimental group would
preclude our ability to detect differences in decay times between
groups. In any case, the present results show that androgen treatment can functionally regulate NMDA-EPSCs, and provide one mechanism by
which endogenous androgen signaling could alter synaptic development within the song system.
Androgen effects on NMDA-EPSCs are not well described in any system,
even though steroids have been broadly implicated in the modulation of
neuronal excitability. In electric fish, androgens and estrogens
reciprocally modulate voltage-dependent sodium channels that underlie
sexually dimorphic electric organ discharges (Dunlap et al.
1997; Ferrari et al. 1995
), and in
Xenopus, estrogens increase quantal content at laryngeal
neuromuscular junctions (Tobias and Kelley 1995
). In the
mammalian CNS, estrogens can modulate synaptic transmission through a
variety of mechanisms (Joels 1997
), including the
augmentation of NMDA receptor currents in the CA1 region of the rat
hippocampus during estrus (Woolley et al. 1997
). The
present results suggest that in the zebra finch song system, androgens can regulate the development of NMDA-EPSCs by making them decay faster.
Although changes in NMDA-EPSC time course occur over development at a
number of central synapses, the androgen sensitivity of these changes
in LMAN and RA affords a previously unidentified means for regulating
this process in the song system. In other vertebrate systems,
stimulus-driven electrical activity plays a major regulatory role: in
the visual system, dark-rearing of the young animal or TTX treatment of
its cortex prevents the normal maturation of NMDA-EPSCs in cortical
neurons (Carmignoto and Vicini 1992). In LMAN and RA,
androgens could also affect NMDA-EPSC development through
activity-dependent pathways, either by directly altering neuronal
excitability in these areas or by inducing behavioral changes that then
alter activity (such as increased singing with a concomitant increase
in auditory stimulation, or increased attention to their own song or
that of a tutor). In contrast, androgens could alter NMDA-EPSCs via
activity-independent pathways that involve direct activation of steroid
receptors in LMAN and RA neurons (Balthazart et al.
1992
). These various possibilities for the actions of androgens
are schematized in Fig. 7. The present findings do not allow us to discriminate between activity-dependent and
-independent mechanisms. However, the NMDA-EPSC e-fold decay times were not altered within 12 h after steroid implantation, even though this treatment successfully elevated serum testosterone levels. This lack of effect argues against a fast action of androgens on NMDA receptors, although acute effects (approximately minutes) of
another sex steroid, estradiol, have been seen on NMDA
receptor-mediated potentials in rat hippocampal neurons (Foy et
al. 1999
). Further, the persistence of fast NMDA-EPSCs in
steroid-implanted birds even days after their testosterone levels had
returned to normal values (Fig. 3C) suggests that
testosterone produces a lasting change in the functional properties of
LMAN and RA neurons. Finally, androgens did not affect NMDA-EPSCs
within the Wulst, an area devoid of androgen receptors
(Balthazart et al. 1992
), which indicates that local
androgen receptors may be required to mediate the effects of androgens
seen here. Together, these results are consistent with the idea that
androgens achieve their effects through slow and long-lasting
processes, such as altered gene expression.
|
In LMAN and RA, a likely mechanism for the change in NMDA-EPSC
e-fold decay times is an altered pattern of NMDA receptor
subunit expression. A major developmental transition in the mammalian brain involves an increase in the NR2A subunit relative to NR2B (reviewed in Flint et al. 1997; Scheetz and
Constantine-Paton 1994
). For example, in rat superior
colliculus, the slower NMDA-EPSCs recorded at developing synapses can
be accounted for by the single-channel properties of the NMDA receptor
(Hestrin 1992
), and the subsequent development of faster
NMDA-EPSCs is accompanied by elevated levels of NR2A subunit protein
and transcript expression (Shi et al. 1997
). As visual
experience regulates changes in NMDA receptor subunit expression in the
rat visual cortex (Quinlan et al. 1999
), androgen-induced fluctuations in activity could trigger a subunit switch in song system nuclei. We have not yet examined the subunit composition of NMDA receptors on LMAN or RA neurons, but
NR2B-containing receptors decline in LMAN over song development
(Basham et al. 1999
), and our results are consistent
with the idea that androgens promote a relative increase in the
expression of the NR2A subunit over NR2B. In addition to changes in
subunit composition, posttranslational modifications of the NMDA
receptor by phosphatases/kinases have been reported and could
contribute to the changes in decay times seen here (Lieberman
and Mody 1994
; Tong et al. 1995
).
Androgen-induced effects on NMDA-EPSCs are likely due to changes in the
receptors themselves, and not to alterations of other cellular
properties of song system neurons. Although changes in membrane
resistance can have pronounced effects on EPSC decay times
(Spruston et al. 1993), no changes in input resistances occurred within LMAN either over development or with DHT treatment. Changes in RA input resistances that do occur with development were not
reproduced by androgen treatment (Table 1). Thus in RA, androgen
treatment does not reproduce all of the developmental changes that
occur in these neurons, but instead has more selective effects on
NMDA-EPSCs. In addition, it should be noted that the morphological
complexity of the neurons studied here could introduce space-clamp
problems, which would interfere with the measurement of input
resistance from distal dendritic regions. Altered dendritic filtering,
which can arise from the remodeling of dendritic arbors, also can
affect decay times of synaptic currents (Spruston et al.
1994
). However, our Sholl analyses show that LMAN and RA
dendritic structure does not change with androgen treatment (Fig. 6).
Although in RA there is no change in dendritic length, there was a
slight increase in LMAN. It is doubtful that this small (16%) increase in length could produce a significant increase in dendritic filtering, because dendritic lengthening would be expected to increase decay times, rather than decrease them as seen here. Finally, androgens did
not increase the cell body sizes in either LMAN or RA. These observations lend further support to the idea that androgens alter NMDA-EPSCs by regulating the NMDA receptor, and not by changing other
properties of the postsynaptic membrane.
One intriguing difference between LMAN and RA is that androgens caused
a large increase in spine frequency in LMAN, but not in RA. Another
difference is that androgens caused AMPA-EPSCs to become faster, and
decreased the Glupeak:AMPApeak evoked at the
DLM-LMAN synapse, without altering these features at the LMAN-RA synapse. Thus although androgens had comparable effects on NMDA-EPSCs in LMAN and RA, androgens affected neuronal morphology and other aspects of glutamatergic synaptic transmission only in LMAN, even though neurons in both nuclei contain androgen receptors
(Balthazart et al. 1992). The selective action of
androgens within the two song nuclei studied here, coupled with the
lack of any effect of androgens on NMDA receptor-mediated EPSCs in the
Wulst, illustrate that androgens can have specific actions on different
glutamatergic synapses in the telencephalon of juvenile male zebra
finches. Future work could reveal whether there are different androgen sensitivities of AMPA receptors within RA itself, between the LMAN-RA
synapse studied here, and the HVc-RA synapse, which is primarily AMPA
receptor mediated (Mooney 1992
; Stark and Perkel 1999
).
The effect of testosterone on LMAN spine density is notable because
raising zebra finches in isolation from adult tutors, which affects
song learning, also affects spine density in LMAN, and suggests that
changes in this structural feature may be one hallmark of synaptic
change important for song learning. Further, during normal development,
DLM efferent projections to LMAN initially increase, then later retract
(Johnson and Bottjer 1992), which suggests that new
synaptic connections are being shaped at this time. Assuming that the
new spines seen here reflect functional synapses, one possibility is
that new synapses induced by testosterone express a new physiological
phenotype that is distinct from preexisting synapses. Alternatively,
androgens could induce physiological changes at both new and
preexisting synapses.
In the song system, as in other systems modified by experience, a major
question is how sensitive periods are regulated. Several central
synapses that display experience-dependent remodeling initially exhibit
slow NMDA-EPSCs that become faster as synaptic reorganization draws to
a close (Carmignoto and Vicini 1992; Hestrin 1992
; Shi et al. 1997
). Because NMDA receptor
activation at these same synapses is critical for their modification
(Bear et al. 1990
; Schnupp et al. 1995
),
these slower NMDA-EPSCs could permit sufficient postsynaptic
Ca2+ entry to enable experience-dependent plasticity
(Bliss and Collingridge 1993
). In both LMAN and RA, the
slower synaptic currents of immature animals could allow synaptic
modification important to song learning. In LMAN, where tutor
song-evoked NMDA receptor activity is crucial to sensory acquisition
(Basham et al. 1996b
), slower NMDA-EPSCs could sustain
auditory experience-dependent changes that then instruct vocal motor
learning. The more prolonged expression of slow NMDA-EPSCs in RA could
then afford a means by which early auditory experience might continue
to guide vocal motor learning, until pubertal increases in androgens
curtail plasticity and result in song crystallization (Marler et
al. 1988
).
Here we have linked androgens, which could act to synchronize song
learning and sexual maturation, with NMDA receptors, which are known to
be important for sensory acquisition. While we have studied the effects
of exogenous androgens, behavioral studies in other songbirds suggest
that endogenous androgens play an important role in controlling various
stages of song development. In swamp and song sparrows, high levels of
androgens are needed for the transition from plastic to crystallized
song (Marler et al. 1988). A more recent finding is that
juvenile white-crowned sparrows display an extended capacity for
sensory acquisition when reared in acoustical isolation, but only when
isolation is combined with a photoperiod regimen that delays the onset
of adult testosterone levels (Whaling et al. 1998
).
Perhaps this delay in the rise to adult testosterone levels allows
adult birds to copy song because NMDA-EPSC maturation in the song
system of these birds is also delayed. An important goal will be to
determine whether the functional changes in NMDA-EPSCs induced by
androgens seen here do indeed limit sensitive periods for song
learning. Further, it will be interesting to explore other neuronal
properties that are modulated by androgens that could influence song learning.
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ACKNOWLEDGMENTS |
---|
Special thanks go to J. M. Kittelberger, R. Stacy, and C. Ellis for providing anatomic data. We acknowledge all members of the Mooney lab for providing song analyses and critical discussions. In addition, H. Chisum, R. Fernald, E. Jarvis, M. Konishi, J. McNamara, and F. Schweizer provided thoughtful critiques of the manuscript. M. Booze provided skilled histology, and E. A. Zimmerman gave expert assistance with the radioimmunoassay. S. A. White and F. S. Livingston contributed equally to this work.
This research was supported by National Institutes of Health Grant 5T32 NS-07370 and an H. H. Whitney fellowship to S. A. White, National Research Service Award F31 MH11872 to F. S. Livingston, and NIH Grant R01 DC-02524 and McKnight Foundation and Sloan Foundation awards to R. Mooney.
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FOOTNOTES |
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Address for reprint requests: R. Mooney, Dept. of Neurobiology, Duke University Medical Center, Box 3209, Durham, NC 27710.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 April 1999; accepted in final form 7 June 1999.
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REFERENCES |
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