Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Livingston, Frederick S. and Richard Mooney. Androgens and Isolation From Adult Tutors Differentially Affect the Development of Songbird Neurons Critical to Vocal Plasticity. J. Neurophysiol. 85: 34-42, 2001. Song learning in oscine birds occurs during a juvenile sensitive period. One idea is that this sensitive period is regulated by changes in the electrophysiological properties of neurons in the telencephalic song nucleus lateral magnocellular nucleus of the anterior neostriatum (LMAN), a structure critical for song development but not adult singing. A corollary of this idea is that manipulations affecting the pace and quality of song learning will concomitantly affect the development of LMAN's electrophysiological properties. Manipulations known to affect song development include treating juvenile male zebra finches with exogenous androgens, which results in abnormally truncated adult songs, and isolation of the juvenile from adult tutors and their songs, which extends the sensitive period for song learning. Previously, we showed that synaptic transmission in LMAN changes over normal song development and that these changes are accelerated or retarded, respectively, by androgen treatment and isolation from an adult tutor. The intrinsic properties of LMAN neurons afford another potential target for regulation by steroid hormones and experience of adult tutors. Indeed previous studies showed that the capacity for LMAN neurons to fire action potentials in bursts, due to a low-threshold calcium spike, and the width of single action potentials in LMAN, wane over development. Here we analyzed these and other intrinsic electrophysiological features of LMAN neurons over normal development, then tested whether either early androgen treatment or isolating juveniles from adult tutors affected the timing of these changes. The present study shows that androgen but not isolation treatment alters the developmental time at which LMAN neurons progress from the bursting to nonbursting phenotype. In addition, other intrinsic properties, including the half-height spike width and the magnitude of the spike afterhyperpolarization (AHP), were found to change markedly over development but only changes to the AHP were androgen sensitive. Interestingly of all of the synaptic and intrinsic electrophysiological properties in LMAN studied to date, only the half-height spike width continues to change in the late juvenile stages of song learning. Furthermore raising juveniles in isolation from an adult tutor transiently delays the maturation of this property. The present results underscore that beyond their effects on LMAN's synaptic properties, both androgens and adult tutor experience are potent and selective regulators of the intrinsic properties of LMAN neurons.
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INTRODUCTION |
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The sensitive period for the acquisition of learned song in oscine songbirds comprises three distinct phases. First, during sensory acquisition, young male zebra finches (Taeniopygia guttata) memorize the song of an adult male tutor. Next, during sensorimotor learning, the plastic song of juveniles is matched to the memorized song model. Song learning ends with crystallization, when the song becomes highly stereotyped. A major goal is to understand the neuronal properties that change during development to limit sensitive periods for song learning.
One advantage of using songbirds to study the neuronal
regulation of sensitive periods for learning is that the timing and quality of song learning can be experimentally manipulated and used to
separate general developmental neuronal changes from those that might
be specific to song learning. Young zebra finches implanted with
testosterone have shorter songs and a reduced number of syllables as
adults (Korsia and Bottjer 1991), while similar
treatment will cause white-crowned sparrows to prematurely and
abnormally crystallize their song (Whaling et al. 1995
).
In contrast, zebra finches raised in auditory and visual isolation from
an adult tutor have an extended period of sensory acquisition, past the
normal endpoint of PHD 65 (Aamodt et al. 1995
;
Eales 1985
; Jones et al. 1996
;
Livingston et al. 2000
; Morrison and Nottebohm
1993
). Here we examine whether these two behavioral
manipulations, which are known to alter the pace of song learning, also
alter the development of electrophysiological properties in brain
regions critical to song learning. Establishing this correlation would
strengthen the link between candidate electrophysiological changes and
sensitive-period regulation.
Although many brain areas are important to singing and thus
potentially important to song learning, one brain region that may be
especially important to regulating sensitive periods for song learning
is the lateral magnocellular nucleus of the anterior neostriatum
(LMAN). Lesion studies reveal that this telencephalic nucleus is
essential for juvenile song development but not for adult singing
(Bottjer et al. 1984; Scharff and Nottebohm
1991
). LMAN is likely to affect vocal plasticity via
its monosynaptic connections with the robust nucleus of the
archistriatum (RA), a vocal motor area critical to song production
throughout life. Beyond affecting juvenile vocal quality, LMAN also is
likely to be important to the memorization of tutor songs, because
blocking N-methyl-D-aspartate (NMDA) receptors
in LMAN during tutoring can interfere with song development
(Basham et al. 1996
). One hypothesis is that the
electrophysiological properties of LMAN projection neurons change over
song development, gradually altering the capacity of LMAN to influence
either song memorization or song quality (Livingston and Mooney
1997
). A corollary of this idea is that manipulations affecting
the pace of song learning should also alter the developmental time
course of any changes in LMAN's electrical properties. Specifically, if
LMAN is a crucial site for sensitive-period regulation, then exogenous
androgens, which disrupt song learning and cause premature song
crystallization, should also hasten electrophysiological changes in
LMAN that are important for limiting sensitive periods (White et
al. 1999
). Similarly, raising birds in visual and auditory
isolation from adult tutors, which extends sensitive periods for song
learning, should delay these electrophysiological changes
(Livingston et al. 2000
). Searching for these
correlations can help identify electrophysiological mechanisms
governing sensitive periods for song learning.
Indeed, there are major developmental changes in the
electrophysiological properties of LMAN neurons. At synapses between thalamic axons and LMAN neurons, the decay times of NMDA
receptor-mediated currents become faster over development
(Livingston and Mooney 1997), and the time course of
these changes can be accelerated by exogenous testosterone
(White et al. 1999
) or transiently delayed by isolation
(Livingston et al. 2000
). However, it is not known whether other electrophysiological properties of LMAN neurons, including their intrinsic properties, are also sensitive to these types
of manipulations. Normal developmental changes in these intrinsic
properties include the disappearance of a low-threshold Ca2+ spike (LTS): when injected with positive
currents, fledgling LMAN projection neurons [~25 posthatch day
(PHD)] fire in part with bursts of action potentials, due to the LTS,
yet adult neurons only fire regular spike trains (Livingston and
Mooney 1997
). This juvenile bursting could be important for
sensory acquisition perhaps by enhancing transmitter release
(Lisman 1997
) or by otherwise altering the transfer
function of LMAN neurons. In addition, the spike width of LMAN neurons
changes between juvenile and adult times (Bottjer et al.
1998
). Acute changes in action potential width can have
profound effects on neurotransmitter release (Sabatini and
Regehr 1997
), while persistent changes in spike width underlie certain forms of behavioral sensitization in Aplysia
(Siegelbaum et al. 1982
). Thus changes in either
synaptic or intrinsic electrophysiological properties can influence
neuronal and behavioral plasticity, making them both attractive
candidates for sensitive period regulation. However, while androgens
and isolation from adult tutors can alter the development of synaptic
properties in LMAN neurons (Livingston et al. 2000
;
White et al. 1999
), it remains unclear whether these manipulations also modulate the development of their intrinsic properties. Here we examine this issue and provide evidence that exogenous androgens and the quality of the juvenile bird's early auditory and visual experience of an adult tutor influence when LMAN
neurons' intrinsic properties change during development.
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METHODS |
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These experiments use behavioral and electrophysiological
techniques that have been extensively described in previous published studies (Livingston and Mooney 1997; Livingston
et al. 2000
; Mooney 1992
; White et al.
1999
). Therefore only a brief description of these techniques
is provided here.
Subjects
Brain slices were made from 48 male zebra finches, in
accordance with a protocol approved by the Duke University
Institutional Animal Care and Use Committee. The four age groups
studied here were fledglings (~25 PHD), when young male zebra finches
are near the onset of sensory acquisition; juveniles (~45 PHD),
during the early stages of sensorimotor learning and prior to the end of sensory acquisition; late juveniles (~65 PHD), when sensory acquisition ends in birds with prior exposure to tutors but before the
end of sensorimotor learning; and adults (~110 PHD), when song has
become crystallized (see Table 1 for the
numbers of cells and animals used for each group). Some of the data
from recordings of fledgling neurons have been used in a previous study (Livingston and Mooney 1997); otherwise, data have not
been previously reported. Finches were raised in our breeding colony on
a 14-h light:10-h dark cycle.
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Isolation protocol and androgen manipulations
At 25 PHD, fledglings were removed from breeding cages and
housed alone in small stainless steel cages (22 × 22 × 25 cm) until either 45 or 65 PHD; siblings could only hear and not see one another during this isolation period and were completely deprived of
auditory, visual, or other forms of contact with an adult tutor. We
have previously documented that this treatment extends sensory acquisition beyond 65 PHD and also transiently depresses serum testosterone levels ~45 PHD (Livingston et al. 2000).
Serum androgen levels in other birds were augmented using silastic
implants placed subcutaneously in the chest region; implants were made
at either 15 or 35 PHD, and birds were killed for brain slice
recordings ~10 days later (25 PHD, fledgling; 45 PHD, juvenile
see
Table 1). The implants contained ~50 µg dihydrotestosterone (a
nonaromatizable form of testosterone; similar implants of testosterone
significantly elevate serum androgen titers for 7-10 days post
implant; see White et al. 1999
).
Brain slices
Briefly, sagittal brain slices that included LMAN were cut at 400 µm and transferred to a holding chamber (room temperature); intracellular recordings were made using an interface-type chamber (30°C; Medical Systems). Artificial cerebrospinal fluid (ACSF) consisted of (in mM) 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose; equilibrated with 95% O2-5% CO2. Equiosmolar sucrose was substituted for NaCl during the tissue preparation stage.
Electrophysiological recordings
Sharp intracellular recordings were made with borosilicate glass
pipettes (Sutter Instrument) pulled to a final resistance of 80-200
M when filled with 2 M potassium acetate. Intracellular potentials
were amplified with an Axoclamp 2B amplifier (Axon Instruments) in
bridge mode, low-pass filtered at 1-3 kHz, and digitized at 10 kHz.
Data acquisition and analysis
Data acquisition and analysis for intracellular recordings were
performed using a National Instruments data acquisition board (AT-MIO-16E2), controlled by custom Labview software developed by F. Livingston and R. Neummann. Suprathreshold responses from each neuron
were measured in response to two different depolarizing current pulses
(1-s duration, +400, and +600 pA), injected from the resting potential.
Previous work has shown that prior hyperpolarization of the membrane
potential can either induce or enhance bursting (Livingston and
Mooney 1997). Thus we also elicited suprathreshold responses
beginning from a hyperpolarized state (tonic hyperpolarizing current,
400 pA; depolarizing pulses were 1-s duration, net +400 and +600 pA),
and also included them in the analysis. In total, four spike trains
were collected and analyzed from each neuron (i.e., responses to 1-s
duration, +400-, +600-pA current pulses in both normal resting and
hyperpolarized states). A software threshold-event detector was used to
measure instantaneous spike rates throughout the 1-s period of positive
current injection and the coefficient of variation in the instantaneous
rates was calculated as the standard deviation in the instantaneous
firing rate divided by the mean firing rate. Spike widths were measured at one half the spike amplitude, which was determined as the point midway between the sharp positive inflection in membrane potential immediately prior to the action potential and the spike peak; the
amplitude of the spike afterhyperpolarization (AHP) was the voltage
difference between the inflection point and the membrane potential
minimum following the spike peak. Input resistance measurements were
calculated by measuring the steady-state voltage caused by injecting
small (
200 pA) hyperpolarizing current pulses. Two-way ANOVAs (group
vs. current injection) were used to assess statistical significance,
unless otherwise noted. In all cases, the minimum significance level
was set at P < 0.05 using two-tailed comparisons. Averages are reported with the standard error of the mean (±SE).
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RESULTS |
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Developmental changes in burst firing
Intracellular sharp-electrode recordings previously revealed that
the regularity of action potential firing in LMAN projection neurons in
response to depolarizing current injection could be quite varied (Fig.
1) (also see Livingston and Mooney
1997). We originally classified neurons as either
"bursting" or "nonbursting" based solely on visual inspection
of spike trains, which could be insufficient to describe more subtle
differences that may occur over development. Therefore we wanted to use
a quantitative measure that did not rely on visual
inspection of the traces to assess whether a neuron fired in a bursting
or nonbursting mode.
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We quantified the variety of DC-evoked action potential trains by measuring the coefficient of variation of the instantaneous spike frequencies (IF-CV), which estimates how much the instantaneous frequencies vary around the mean value for a given spike train. One characteristic of fledgling LMAN neurons is that they often exhibited both very high and low instantaneous spike frequencies in a single spike train, due to the LTS, thus yielding a very high IF-CV. In contrast, neurons that varied little in their instantaneous spike frequencies throughout the train, or simply underwent a gradual monotonic decay of instantaneous firing frequency, yielded low IF-CVs. Individual current-clamp records of spike trains and plots of instantaneous firing frequencies obtained from three different fledgling neurons illustrate differences in firing behavior and IF-CV (Fig. 1). The neuron in trace 1 quickly alternated between high and low instantaneous firing frequencies, while the neuron from which trace 2 was collected exhibited a less abrupt decline from high to low instantaneous firing frequencies; both neurons had relatively high IF-CVs (Fig. 2A, top). The other fledgling neuron, from which trace 3 was collected, declined gradually in its instantaneous firing frequency over the duration of the depolarizing current injection and had a low IF-CV (Fig. 2A, top).
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Using this quantitative measure, we found that the distribution of fledgling IF-CVs is bimodal (Fig. 2), with a distinct notch at 0.4 separating the two peaks (note - - - in Fig. 2). We used this value as a cutoff, classifying a cell as bursting only if at least one of its DC-evoked action potential trains yielded an IF-CV > 0.4. The three fledgling traces shown in Fig. 1 illustrate the method: traces 1 (IF-CV = 1.23) and 2 (IF-CV = 0.85) are from different parts of the region representing bursting neurons, while trace 3 (IF-CV = 0.12) is an example from the nonbursting or regular spiking region. Ultimately, statistical significance was assessed using two-way ANOVAs on the IF-CV (group vs. level of current injected; see METHODS).
This quantitative measure allowed us to determine that the number of bursting cells in a given age group declined over development from 90 to 0% (i.e., 90% of fledgling neurons displayed at least 1 spike train with an IF-CV > 0.4, while no adult neurons had an IF-CV > 0.4; Table 1; Fig. 2). As shown in Fig. 2A, this cutoff value was never exceeded by cells in the late juvenile and adult groups even though their IF-CVs were determined both at their native resting potentials and in a tonically hyperpolarized state. This tonic hyperpolarization of membrane potential, used to relieve deinactivation of any latent LTS, ensured that slight group to group differences in resting potential (see Table 1) did not inadvertently bias our estimates of bursting (Fig. 2B). Most (90%; 26/29 cells) fledgling neurons displayed an IF-CV 0.4, while only 30% (6/20 cells) of juvenile neurons showed this behavior (mean IF-CV, fledgling: 0.58 ± 0.03 vs. juvenile: 0.27 ± 0.03, P < 0.0001). Late juvenile LMAN neurons did not display any bursting, and had a mean IF-CV less than that of juveniles (0.27 ± 0.03 vs. 0.16 ± 0.01, P < 0.0002) and equivalent to that of adults (0.16 ± 0.01 vs. 0.15 ± 0.01, P = 0.22). Thus to summarize, there are major developmental changes in the way LMAN projection neurons fire action potentials trains in response to depolarizing currents, progressing from a bursting to a nonbursting phenotype (Fig. 2C).
Effects of dihydrotestosterone and isolation on bursting
To directly test whether the developmental decline in bursting behavior contributed to sensitive periods for song learning, we examined whether factors that are known to alter the pace of song learning also alter the progression from bursting to nonbursting firing patterns. Androgens are one such factor that can perturb song development. To test whether it concomitantly alters the intrinsic properties of LMAN neurons, either fledgling or juvenile male zebra finches were implanted with dihydrotestosterone ~10 days prior to intracellular recording (see METHODS). Androgen treatment reduced bursting in both fledgling and juvenile LMAN neurons (Table 1; Fig. 3) compared with age-matched controls. At the fledgling age, dihydrotestosterone lowered the number of bursting neurons from 90 to 68% (26/29 vs. 30/44 cells), with the mean IF-CV lying between those of fledglings and juveniles (mean IF-CV, fledgling: 0.58 ± 0.03 vs. juvenile: 0.41 ± 0.03, P < 0.0001). In juveniles, dihydrotestosterone lowered the number of bursting cells from 30 to 11% (6/20 vs. 2/18 cells; mean IF-CV: 0.27 ± 0.03 vs. 0.16 ± 0.01, P < 0.001), and decreased the mean IF-CV to a value equivalent to those of both late juvenile and adult neurons.
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Auditory and visual isolation from and adult tutor is another factor that alters the pace of song development: birds raised as isolates retain their ability to learn song until after 65 PHD, a time when control birds do not copy new songs. We tested for delayed developmental changes in the firing properties from juvenile finches that were isolated from tutor song beginning at ~25 PHD. In contrast to the effects of androgens on bursting in both fledgling and juvenile neurons, isolation did not affect the developmental progression from bursting to nonbursting phenotypes. Isolation of juveniles from tutor song had no effect on the number of their LMAN neurons displaying bursting properties [Table 1; 30% (6/20) juvenile cells vs. 28% (7/25) isolate juvenile cells; mean IF-CV 0.27 ± 0.03 vs. 0.28 ± 0.03; Fig. 3]. In contrast, brain slices from late juvenile isolates did contain a small number of bursting cells (2/19 cells or 11%), suggestive of slightly delayed neuronal maturation, although the mean IF-CVs for the late juvenile isolates and their age-matched controls were equivalent (0.16 ± 0.01 vs. 0.16 ± 0.02; note the widened data distribution revealed by the 2-fold increase in the SE in Fig. 3B). In summary, dihydrotestosterone markedly accelerated the transition from the bursting to the nonbursting state, while isolation had little or no effect on the developmental time at which this property disappeared.
Spike widths and AHPs
We also examined the half-height width of single action potentials
(the half-height spike width) and the amplitude of spike AHPs of LMAN
neurons, two features that could be regulated independently of their
bursting properties. We first measured how these features change across
development and then examined the effects that androgens or raising
juvenile birds in isolation from adult tutors had on the developmental
changes of these two features. Aside from describing how features of
spike shape may relate to sensitive periods for learning, these
analyses can reveal the specificity of the actions of androgens and
isolation on the electrophysiological properties of LMAN neurons
(Livingston et al. 2000; White et al.
1999
).
The shape of action potentials changed dramatically over development;
at the fledgling age action potentials have both a slower rise and a
slower decay than seen in adults, making them much wider (Fig.
4; Table 1). The spike width previously
has been examined in LMAN from juvenile to adult times (Bottjer
et al. 1998). Here we have both confirmed and extended this
analysis to the fledgling age, a time when zebra finches can memorize
song but prior to the time they start to sing. The present study
reveals dramatic differences in shape and half-height spike width over development and illuminates how androgens and isolation affect these
developmental changes.
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Differences in the half-height spike width were seen throughout
development (Table 1; Fig. 4; P < 0.0001). The largest
change was between the fledgling and juvenile periods (1.07 ± 0.03 vs. 0.62 ± 0.01 ms, P < 0.0001), but
changes also occurred among juveniles, late juveniles, and adults
(0.62 ± 0.01 vs. 0.56 ± 0.01 vs. 0.48 ± 0.01 ms; all
comparisons P < 0.0001); these later measurements are
similar to those in Bottjer et al. (1998).
We also measured spike AHPs, the amplitude of which increased over development (P < 0.0001), specifically among fledglings, juveniles, and late juveniles (Fig. 5; 18.4 ± 0.5 vs. 22.2 ± 0.5 vs. 24.3 ± 0.6 mV; P < 0.0001 and P < 0.01, respectively). Representative examples of fledgling and adult AHPs reflective of their respective group means are provided in Fig. 5. In contrast to the changes in half-height spike width, these changes in AHPs were complete by the late juvenile period with no difference between late juveniles and adults. To determine whether a change in driving force or input resistance could underlie the change in AHP amplitude, we measured these features over development (Table 1). This analysis revealed that the resting membrane potential but not the input resistance changed slightly but significantly in the positive direction between fledgling and later time points, suggesting that early decreases in the AHP might reflect in part a diminution in driving force over development.
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Although the half-height spike width and AHPs both changed over development, androgens and isolation had distinct effects on these parameters (Table 1; Figs. 4 and 5). The half-spike width was not affected by androgen treatment at any age, but isolation did slightly increase this measurement in juveniles (Fig. 4; 0.62 ± 0.01 vs. 0.70 ± 0.01 ms, P < 0.0003); there was a similar but non-significant trend in late juveniles (0.56 ± 0.01 vs. 0.61 ± 0.03 ms, P = 0.1635). In contrast, dihydrotestosterone did increase the AHPs of fledglings (Fig. 5; 18.4 ± 0.5 vs. 20.6 ± 0.4 mV, P < 0.0001) and juveniles (22.2 ± 0.5 vs. 25.1 ± 0.6 mV, P < 0.0001), but this feature was entirely unaffected by isolation. Although resting membrane potentials and input resistances were not significantly affected by either dihydrotestosterone or isolation, the slightly more positive mean membrane potential we saw in DHT-treated fledges may account for the change in AHP amplitude (Table 1). In summary, while dihydrotestosterone did not affect the half-height spike width, isolation did have small but significant effects. In contrast, dihydrotestosterone did affect the development of AHPs, but isolation did not.
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DISCUSSION |
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Along with prior studies examining the androgen-sensitivity of
NMDA receptor-mediated currents in LMAN, the present studies show that
androgens are a potent regulator of a major subset of electrophysiological properties of LMAN neurons. Androgens have been
previously shown to regulate the decay times of NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) and the ratio of the
overall glutamate receptor-mediated EPSC to the AMPA receptor-mediated EPSC (White et al. 1999). Beyond these synaptic effects,
the present study shows that androgens act ether directly or indirectly
to also modulate specific intrinsic electrophysiological properties of
LMAN neurons, including the progression from burst to nonburst action
potential firing and the magnitude of AHPs. Furthermore androgens do
not merely act as a nonspecific maturational factor since exogenous
androgen treatment failed to alter the development of half-height spike
widths, a property that changes markedly over normal development.
The present results underscore the potent effect of sex steroids
on the electrophysiology of LMAN, a nucleus critical to the
development of song, a steroid-sensitive learned vocal behavior.
In contrast to the potent effects of exogenous androgens, raising
juveniles birds in auditory and visual isolation from adult tutors had
little or no effect on the progression from burst to nonburst firing or
on the magnitude of spike AHPs, suggesting that these parameters can
develop without exposure to an adult tutor. The two bursting cells we
saw in late juvenile isolates indicate a very slight retention of this
neuronal feature in this group, possibly indicative of slightly slower
neuronal maturation. Ultimately, because isolates older than 65 PHD can
learn song even though their LMAN neurons largely lack the capacity for
burst firing, this feature does not seem to be essential for song
learning. Furthermore because juvenile isolates have abnormally low
testosterone levels (Livingston et al. 2000), it is
unlikely that high testosterone levels are essential for either the
disappearance of the burst firing or changes in the magnitude of the
AHP even though androgens can affect these features. Therefore other
factors must be involved to explain the continued development in these
properties that are seen in juvenile isolates with depressed androgen
levels. One key factor could be the amount of singing, which is known to affect auditory and/or vocal-related activity in LMAN (Doupe and Konishi 1991
; Hessler and Doupe 1999
). In
this view, where singing-related activity is the proximal cue driving
changes in LMAN, the systemic androgen treatment used here could act
primarily through other androgen-sensitive song or nonsong areas to
augment singing rather than acting directly via LMAN neuronal androgen receptors to affect changes in excitability (see White et al. 1999
for a broader treatment of possible mechanisms of steroid action).
A major question is the functional role of burst firing in LMAN
neurons. Bursting could alter the transfer function of LMAN because it
induces a highly nonlinear function into a neuron's current-voltage
conversion, allowing increased spiking due to a given amount of current
input compared with neurons that do not have this property. This
amplification could enable LMAN neurons to fire more action potentials
in response to the weak excitatory synaptic inputs that may predominate
during early juvenile development. In addition, because the bursting is
enhanced and sometimes even induced by prior hyperpolarization of the
membrane (data not shown) (but see Livingston and Mooney
1997), this amplification could act in concert with inhibitory
inputs as a gate for information throughput. Indeed
GABAA receptor-mediated inhibition generated within LMAN, driven by local interneurons, could serve to prime excitatory throughput by enhancing the LTS (Boettiger and Doupe 1998
; Livingston and Mooney 1997
). In other
brain regions, such as the mammalian lateral geniculate nucleus (LGN),
the voltage-dependence of the LTS allows LGN relay neurons to fire in
either a "relay" or a "burst" mode (McCormick et al.
1995
; Steriade and Llinas 1988
).
Neuromodulators, including norepinephrine, can modulate inhibitory
input onto LGN relay cells and thus determine their firing mode
(McCormick 1992a
,b
). In LMAN, there are several
candidate modulators, including catecholamines (Ball
1994
; Bottjer 1993
; Soha et al.
1996
), which could act similarly to influence the firing mode
of juvenile LMAN neurons, ultimately determining their ability to
influence vocal change.
One feature of bursting that makes it an attractive candidate for
enhancing neuronal plasticity is the high instantaneous frequencies
achieved in the beginning of spike trains. These high firing rates
could enhance the probability of synaptic release (Lisman
1997; Miles and Wong 1986
) at sites where LMAN
axons form synapses, including locally within LMAN (Boettiger
and Doupe 1998
) and in basal ganglia homologue area X and
nucleus RA (Vates and Nottebohm 1995
). Although the
relative lack of bursting in isolate neurons suggests this feature is
not essential for extended song learning, it may nonetheless be
important to early song learning, especially if the probability of
transmitter release is diminished at these earlier ages. Specifically,
developmental increases in the probability of release could obviate the
need for bursting in late juveniles, although this feature may still be
needed earlier in development, when individual synapses may be less
potent. In essence, bursting could boost initially weak afferent
synapses from the thalamus as well as enhance the ability of LMAN to
excite its postsynaptic targets.
The present results confirm and extend a prior study
(Bottjer et al. 1998) by showing that there are major
developmental changes in the spike width of LMAN neurons from fledgling
through adult ages. One noteworthy feature of the developmental
decrease in spike width is that it is the only electrophysiological
feature of LMAN neurons identified to date, including synaptic
properties (White et al. 1999
), that continues to change
after 65 PHD. Although small relative to changes in spike width
occurring earlier in development, these late changes do correlate with
the period of song crystallization. Interestingly this feature was
unaffected by exogenous dihydrotestosterone, suggesting that the
isolation effects may stem directly from deprivation from the adult
tutor and its song, rather than the abnormally depressed testosterone levels seen in isolates at 45 PHD (Livingston et al.
2000
). The role of the spike width changes in LMAN is unclear,
but spike width changes underlie behavioral sensitization in
Aplysia, where presentation of noxious stimuli can induce
sensitization, which requires changes in neurotransmitter release at
specific synapses. Sensitization is due to a reduction of a
K+ current, resulting in action potential
broadening, and increased Ca2+ entry into the
presynaptic terminal, ultimately enhancing synaptic transmission
(Siegelbaum et al. 1982
). While isolation did induce a
significant widening of the action potential in LMAN neurons, it is a
small difference of 80 µs (~10% widening). However, at the
cerebellar granule cell to Purkinje cell synapse, increases in
neurotransmitter release due to a modest 23% widening of the action
potential can double the magnitude of postsynaptic currents (Sabatini and Regehr 1997
), suggesting that the subtle
changes seen here could also exert profound effects on synaptic
strength. Ultimately resolving the significance of different spike
durations in LMAN neurons will require an understanding of their
consequences for downstream events, including the resultant
postsynaptic potentials they may evoke.
Both synaptic and intrinsic electrophysiological properties can be
important in regulating neuronal plasticity. These and earlier studies
(Livingston and Mooney 1997; Livingston et al. 2000
; White et al. 1999
) show that both sex
steroids and exposure to an adult tutor, which strongly affect the pace
and quality of song learning, impact both the intrinsic and synaptic
properties of LMAN neurons. Although these results suggest that
neuronal maturation in LMAN may be influenced by both of these factors, the dissociation of the timing of these changes relative to late learning in isolates points away from them as major neuronal regulators of sensitive periods for song learning. Future studies can address whether changing single factors within one or even many song nuclei is
sufficient to regulate the closure of sensitive periods for song
learning and whether the mechanisms underlying extended learning in
isolates are similar or distinct from those that enable song learning
earlier in life.
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ACKNOWLEDGMENTS |
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This research was supported by National Institutes of Health (NIH) National Research Service Award F31 MH-11872 to F. S. Livingston, and by NIH Grant R01 DC-02524 and McKnight, Klingenstein, 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 (E-mail: mooney{at}neuro.duke.edu).
Received 30 June 2000; accepted in final form 16 September 2000.
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REFERENCES |
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