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The Journal of Neuroscience, July 15, 2000, 20(14):5420-5436
Different Subthreshold Mechanisms Underlie Song Selectivity in
Identified HVc Neurons of the Zebra Finch
Richard
Mooney
Department of Neurobiology, Duke University Medical Center, Durham,
North Carolina 27710
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ABSTRACT |
Songbirds learn and maintain their songs via auditory experience.
Neurons in many telencephalic nuclei important to song production and
development are song selective, firing more to forward auditory playback of the bird's own song (BOS) than to reverse BOS or
conspecific songs. Elucidating circuits that generate these responses
can localize where auditory experience influences vocalization,
bridging cellular and systems analyses of song learning. Song-selective responses in many song nuclei, including the vocal premotor nucleus robustus archistriatalis (RA) and the basal ganglia homolog area X, are
thought to originate in nucleus HVc (used as a proper name), which contains interneurons and relay cells that innervate either RA or
area X. Previous studies indicated that only X-projecting neurons have
auditory responses, leaving open the source of RA's auditory input and
the degree to which song selectivity may be refined in HVc. Here,
in vivo intracellular recordings from morphologically and electrophysiologically identified HVc neurons revealed that both
relay cell types fire song-selectively. However, their firing arises
via markedly different subthreshold processes, and only X-projecting
neurons appear to be sites for auditory refinement. RA-projecting
neurons exhibited purely depolarizing subthreshold responses that were
highly song selective and that were excitatory. In contrast,
subthreshold responses of X-projecting neurons included less-selective
depolarizing and highly selective hyperpolarizing components. Within
individual birds, these BOS-evoked hyperpolarizations closely matched
interneuronal firing, suggesting that HVc interneurons make restricted
inputs onto X-projecting neurons. Because of the two relay cell types'
subthreshold differences, factors affecting their resting membrane
potentials could enable them to transmit distinct song representations
to their targets.
Key words:
HVc; in vivo intracellular; vocal learning; songbirds; song; auditory selectivity; communication; zebra finch
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INTRODUCTION |
Songbirds and humans listen to their
own vocalizations to develop and maintain learned communication sounds
(Doupe and Kuhl, 1999 ). These processes are likely to require the
integration of auditory and vocal circuitry. Indeed, in songbirds,
neurons in many brain nuclei used for song production, development, and
perception are song selective, firing more to forward auditory playback
of the bird's own song (BOS) than to reverse BOS or conspecific songs (Margoliash, 1983; Doupe and Konishi, 1991; Janata and Margoliash, 1999). Elucidating the circuitry underlying song selectivity could reveal where audition influences vocalization, thus providing a
cellular framework for vocal learning. In fact, auditory responses in
many song-related nuclei may arise from two distinct relay cell classes
in the nucleus HVc (used as a proper name), but the correspondence
between auditory responsiveness and HVc cell type is poorly understood.
This study uses in vivo intracellular recordings from identified
HVc neurons to establish this correspondence and to probe the cellular
mechanisms underlying song selectivity.
The telencephalic nucleus HVc is a major auditory-vocal interface. HVc
exhibits singing-related premotor activity (McCasland, 1987; Yu and
Margoliash, 1996), and many HVc neurons are song selective (Margoliash,
1986; Lewicki and Arthur, 1996; Volman, 1996; Theunissen and Doupe,
1998). Beyond affording a site for auditory-vocal integration, HVc is
the probable source of auditory input to other nuclei important to song
production, development, and perception. HVc is structurally
heterogeneous, containing two relay cell types and interneurons, and
functionally heterogeneous, because its two relay cell types give rise
to anatomically separate pathways implicated in either
audition-dependent vocal plasticity or vocal control (see Fig.
2) (Nottebohm et al., 1976; Fortune and Margoliash, 1995; Foster and
Bottjer, 1998). One relay cell type innervates area X, a basal ganglia
homolog within the anterior forebrain pathway (AFP). The AFP, which is
essential to juvenile and adult audition-dependent vocal plasticity
(Bottjer et al., 1984; Scharff and Nottebohm, 1991; Brainard and Doupe,
2000) and may facilitate song perception (Scharff et al., 1998) is
thought to derive auditory input from HVc (Vates et al., 1996). In
agreement with this idea, in vivo intracellular recordings
from X-projecting neurons reveal that they have song-selective auditory
responses (Lewicki, 1996). Furthermore, certain X-projecting neurons
display BOS-evoked hyperpolarizations (Lewicki, 1996), perhaps driven by HVc interneurons, that might refine these relay cells' song selectivity relative to HVc's auditory afferents (Lewicki and Arthur,
1996). However, the auditory properties of HVc interneurons and their
interactions with either relay cell type are unknown. The second relay
cell type innervates the nucleus robustus archistriatalis (RA), which
controls the vocal and respiratory neurons used for singing (Vicario,
1993; Wild, 1993). Although RA has auditory responses requiring direct
HVc input (Doupe and Konishi, 1991; Vicario and Yohay, 1993) and
RA-projecting neurons afford attractive sites for potential
auditory-vocal interaction, previous intracellular studies concluded
they were nonauditory (Katz and Gurney, 1981; Lewicki, 1996). The
difficulty in obtaining in vivo intracellular recordings
from identified HVc neurons has obscured the nature of auditory
information transmitted from HVc to other song nuclei, while impeding
insight into the mechanisms underlying song selectivity.
Here, in vivo intracellular recordings from identified HVc
neurons were used to measure their responses to BOS playback. These studies show that HVc's two relay cell populations generate similar song-selective firing via distinct subthreshold processes, which may
arise via segregated input from highly selective interneurons onto
X-projecting cells. Ultimately, factors affecting the two relay cell
types' membrane potentials may unmask previously unidentified subthreshold differences, enabling distinct auditory representations of
song to be transmitted by HVc.
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MATERIALS AND METHODS |
Subjects. Experiments were performed using 31 adult
[85-440 posthatch days (PHD)] male zebra finches (Taeniopygia
guttata) in accordance with a protocol approved by the Duke
University Institutional Animal Care and Use Committee.
Stimuli. Before the experiment, songs were recorded from a
male zebra finch placed with an adult female zebra finch in a recording chamber (Industrial Acoustics, Bronx, NY). Songs were amplified and
low-pass filtered at 10 kHz, digitized at 20 kHz (National Instruments
data acquisition board AT-MIO-16E2; Austin, TX), and stored on a hard
drive. Songs were recorded and edited with LabView software (National
Instruments; all custom software for this study was written by M. Rosen, F. Livingston, R. Neumann, and R. Balu). Edited songs included
either one or two motifs (a motif is the largest repeated unit in the
bird's song and usually comprises single and multinote syllables;
zebra finch song bouts typically consist of several motifs). Stimuli
presented always included the BOS and reverse BOS (i.e.,
song played backward) and were 1-3 sec in duration. Forward and
reverse versions of the BOS contain the same spectral energy but differ
in both their local and global temporal organization. Differential
responsiveness to forward over reverse BOS can be used to measure
neuronal sensitivity to time-varying auditory cues (see Data analysis below).
Preparatory surgery. Two days before electrophysiological
recording, birds were food and water deprived for 1 hr, anesthetized with equithesin (2 mg/kg, i.m.: 0.85 gm of chloral hydrate, 0.21 gm of
pentobarbitol, 0.42 gm of MgSO4, 2.2 ml of 100%
ethanol, and 8.6 ml of propylene glycol brought to a 20 ml final volume with dH2O), and placed in a stereotaxic device
(45° head angle; H. Adams, California Institute of Technology). The
scalp was dissected along the midline, and HVc's location was
determined using stereotaxic coordinates (approximately  0.2 mm caudal
and 2.4 mm lateral, measured from the caudal edge of the bifurcation of
the midsagittal sinus). A stainless steel post was mounted to the
rostral part of the bird's skull with dental cement, the wound was
closed with cyanoacrylate, antibiotic (Neosporin ointment) was applied,
and the bird was warmed under a heat lamp (33°C) until recovery (2-4 hr).
In vivo electrophysiology and song presentation. On the
morning of the day of the electrophysiological recording, birds were injected in the pectoral muscle with 20% urethane (75-105 µl total; Sigma, St. Louis, MO), administered in 25-35 µl doses at 30 min intervals. Birds were immobilized via the mounted post in a
sound-attenuating chamber (Industrial Acoustics) on an air table
(Technical Manufacturing Corporation, Peabody, MA); body
temperature was maintained via an electric blanket at 37°C (Harvard
Apparatus, Holliston, MA). After topical application of xylocaine
(2%), the scalp was retracted, a small craniotomy (<300 µm wide)
was made over HVc, and the dura was slit open with a fine insect pin
(Minuten, Carolina Biological Supply).
Sharp electrodes (borosilicate glass, BF100-50-10; Sutter Instrument,
Novato, CA) were pulled to yield a resistance of 100-250 M when
filled with 3 M K-acetate and 5% neurobiotin. A hydraulic microdrive (Soma Scientific, Irvine, CA) was used to lower electrodes into the nucleus (~300 µm in depth). Brief (~1 msec) capacitance overcompensation was used to "ring" the electrode to achieve entry into the cell. An AxoClamp 2B intracellular amplifier (Axon
Instruments, Foster City, CA) was used in bridge mode to record
intracellular potentials, which were low-pass filtered at 3 kHz,
digitized at 10 kHz, and stored on a personal computer hard drive. HVc
neurons were identified on-line by their firing properties in response to injected currents (see Results) and usually by spontaneous, intermittent, high-frequency bursts of action potentials and were verified histologically after the recording session. Cells were tested
with auditory stimuli if their resting potentials were negative of 50
mV. Ten to thirty iterations of each auditory stimulus, delivered at
intervals ranging from 6 to 10 sec, were presented at ~70 dB (rms;
A-weighting) through a speaker positioned 20 cm directly in front of
the bird. Peristimulus time histograms (PSTHs; 25 msec bin width) and
median-filtered averaged membrane potential traces were computed
on-line (see Data analysis) to aid in experimental decisions.
When possible, after characterization of a cell's auditory responses
to BOS playback, its intrinsic properties were studied. Hyperpolarizing
responses to negative current pulses (200 to 400 pA) were collected
to estimate input resistance, and instantaneous and mean firing rates,
as well as the latency to the first spike, were calculated in response
to positive current pulses (+200 to 1000 pA; 1 sec duration) using
custom LabView software. Three- to five-minute-long spontaneous
membrane potential records were also collected, and a software event
detector was used to estimate spontaneous firing rates. Action
potential widths were measured at the base or shoulder of the spike,
where the membrane potential described a sharp positive inflection. The
amplitudes of spike after hyperpolarizations were measured from the
spike shoulder to the trough of the hyperpolarization after the spike.
Resting membrane potential was determined by subtracting any DC offset observed after electrode withdrawal from the membrane potential recorded during the 5-10 min before the end of the recording. All
values are reported as the mean ± SEM; statistics and tests for
statistical significance (other than those described in Data analysis)
are reported in the figure legends, Table
1, and/or the Results.
Data analysis. The suprathreshold responsiveness
(Rsupra) of cells with spiking
activity was calculated by Rsupra = SFR BFR, where
SFR and
BFR are the firing rates during each
stimulus presentation and during a 1.5 sec baseline period before each stimulus presentation, respectively. To assess subthreshold
responsiveness in spiking and nonspiking cells, raw traces first were
median-filtered (each point was replaced by the median value of the
surrounding 50 points, equivalent to 5 msec at the 10 kHz sampling rate
used here). Median-filtering removed the action potential (which was typically ~1 msec in duration) and yet did not distort slower membrane potential movements [for an example, see Jagadeesh et al.
(1997), their Fig. 1]. The subthreshold depolarizing responsiveness (RVm) of these cells was
measured by RVm = Sarea Barea, where
Sarea and
Barea are the integrals of the
positive-going deviations in membrane potential either during (i.e.,
Sarea) or before (i.e.,
Barea) stimulus presentation relative
to the mode membrane potential measured during the baseline period.
That is, the total positive area during the baseline (measured from the mode) was subtracted from the total positive area during the stimulus (measured from the mode; see Fig. 9 for examples of how these subthreshold values were measured). The mode membrane potential was
calculated for the baseline data array with an automated LabView routine and was used instead of the mean or median of the baseline data
array because we observed that it gave the most reliable measure of the
central tendency of the baseline membrane potential. Similar
calculations were made for the subthreshold hyperpolarizing area, using
the negative-going deviations in membrane potential from the baseline
mode value. The net subthreshold hyperpolarization was multiplied by
1, so that a net increase in subthreshold hyperpolarization relative
to the baseline would be represented by a negative number. It was
possible for single cells to respond to a stimulus with an increase in
both negative and positive area relative to baseline. Furthermore,
strong hyperpolarizing responses to a stimulus could yield negative
values for positive and negative area (e.g., when the baseline positive
area was greater than the positive area evoked by the stimulus).
Average Rsupra or
RVm were computed for
10-30 stimulus iterations. Significance was determined with paired
t tests comparing stimulus-evoked suprathreshold,
subthreshold depolarizing or subthreshold hyperpolarizing responses to
corresponding baseline measures.
To compare suprathreshold and subthreshold responses, response
strengths were expressed as z-scores. The suprathreshold z-score (Zsupra) is given by the difference
between the average firing rate during stimulus presentation and that
during a 1.5 sec baseline period before stimulus presentation, divided
by the SD of this difference:
where  FR is the mean
firing rate during the stimulus,
 FR is the mean firing rate
during the baseline period, and the denominator is the SD of
SFR BFR. For nonspiking cells and
median-filtered spiking cells, the subthreshold z-score
(ZVm) is given by the
difference between the average area during stimulus presentation and
that during baseline, divided by the SD of this difference. The
ZVm formula is the same
as that for Zsupra with substitutions of area for FR, where
area is the mean deviation in
Vm (from the baseline mode, calculated
separately for negative or positive area, as stated above) during song
presentation and area is the
mean deviation in Vm during baseline; the
denominator is the SD of Sarea Barea.
The selectivity of a given neuron for forward over reverse BOS playback
was measured using the psychophysical metric d' (Green and
Swets, 1966), which estimates the discriminability between two stimuli.
A difference in response to these two stimuli has been used previously
as the criterion for the auditory selectivity of neurons in HVc, as
well as in other song nuclei (Solis and Doupe, 1997; Theunissen and
Doupe, 1998; Janata and Margoliash, 1999; Rosen and Mooney, 2000). The
d' value comparing the response to BOS relative to reverse
BOS is given by:
where d'supra represents
suprathreshold responsiveness and
d'Vm represents
subthreshold responsiveness.  is the mean value of
R (as described above), and
 2 is its variance. This measure of
selectivity is similar to a ratio measure but takes into account both
the mean and the variance of a cell's responses and can report
negative values. A d' value > 0.7 (or < 0.7,
reflecting a relative excitatory bias toward reverse BOS) was used as
the criterion for identifying a cell as "selective"; this
corresponded to a significance level of p = 0.036 as
measured by a paired t test comparing
RVm or
RFR values for 20 presentations of BOS
versus reverse BOS. Note that d' values for subthreshold
responses (d'Vm) were
calculated separately for positive and negative areas. Tests for
statistical significance are reported in the figure legends, except
where noted in the Results.
To quantify the relative spike timing of the different cell types
during BOS playback, the action potential PSTH of an RA-projecting neuron was correlated (Origin, Microcal Corporation) with the PSTH of
either an X-projecting neuron or interneuron recorded from within the
same bird. To examine the relationship between spike timing and
membrane polarity (i.e., interneuron spiking vs X-projecting neuronal
polarity), the cross-correlation analysis was performed using the
interneuron action potential PSTH and the median-filtered average
membrane potential of an X-projecting cell from the same bird; the
membrane potential trace was further divided into 25-msec-long average
"points" [i.e., the trace was divided into sequential segments 250 points in length (25 msec at the 10 kHz sample rate), and the average
of these 250 points was calculated to yield a single point every 25 msec, equaling the temporal resolution of the PSTH]. In each case, the
cross-correlation analysis was performed on data collected during
forward BOS playback. Each cross-correlation was performed using data
collected from a single-cell pair within a single bird, each resultant
cross-correlogram was normalized to its peak value, and the normalized
values from a given pair type (e.g., RA-projecting vs X-projecting)
were then averaged across all pairs that were obtained (i.e., across birds).
Histology. Cells were filled with neurobiotin using positive
currents (+0.5 to +1 nA; 500 msec at 1 Hz). After the recording session, birds were deeply anesthetized with equithesin and
transcardially perfused with 0.9% saline for 3 min, followed by 4%
paraformaldehyde (PFA) in 25 mM sodium phosphate buffer for
30 min. Brains were removed and post-fixed in 4% PFA with 30% sucrose
overnight, blocked sagittally, and sectioned on a freezing microtome at
60 µm. Sections either were processed using a standard HRP-DAB
reaction technique (see Kittelberger and Mooney, 1999) or occasionally
were visualized using avidin-Oregon green (Molecular Probes, Eugene,
OR; sections were incubated overnight at 4°C in a 1:500 dilution of
avidin-Oregon green and 0.4% Triton X-100 in 0.025 M PBS,
rinsed four times for 20 min each in PBS, mounted, and coverslipped).
Camera lucida drawings were made using both 10× and 63× (Zeiss; 1.3 nA) objectives. Cell bodies were traced under the 63× objective, and
their diameters were measured along the major axis of the cell body
using an eyepiece reticule. A neuron's dendritic extent also was
estimated using the eyepiece reticule and was measured from the middle
of the cell body to the tip of the longest dendrite in the same
section. Only DAB-reacted cells were used for dendritic measurements;
because of this, as well as some incomplete fills, only a subset of
labeled cells were included for this analysis.
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RESULTS |
Intracellular recordings were made in the HVc of
urethane-anesthetized adult male zebra finches to (1) establish
the relationship between an HVc neuron's intrinsic
electrophysiological properties and its morphological type, (2) use
this information to determine which HVc neuron types are auditory, (3)
examine the subthreshold events that contribute to song-selective
action potential responses in different HVc neurons, and (4) enable
within-bird comparisons between the song-evoked responses in different
HVc neuron types.
Intrinsic properties of morphologically identified HVc neurons
Previous in vitro studies indicated that different
morphological classes of HVc neurons in adult male zebra finches
possess distinct intrinsic electrophysiological properties (Dutar et
al., 1998). The existence of similar distinctions in vivo
would enable sorting of the cells to class on the basis of their
intrinsic properties alone, especially advantageous when intracellular
recordings are too brief to afford good staining (<15 min) or when
many cells are impaled and filled in a single nucleus, making an
unambiguous assignment of an electrophysiological record to a
dye-filled cell difficult. Therefore, the in vivo intrinsic
properties of HVc neurons (morphologically identified post
hoc via intracellular neurobiotin staining; see below) were first
characterized by collecting spontaneous membrane potential records and
by injecting them with hyperpolarizing and depolarizing currents (Fig.
1A; n = 16 X-projecting, 10 RA-projecting, and 11 interneurons). The three
morphological classes of HVc neurons had distinct intrinsic properties
in vivo, differing in the distributions of their
instantaneous spike frequencies in response to similar injected
currents (Fig. 1A-C), their average firing
frequencies in response to depolarizing current pulses (Fig.
1D), their spontaneous firing rates, their action
potential widths, the amplitude of their individual spike
afterhyperpolarizations (AHPs), and the onset latencies to their first
action potential during a depolarizing pulse (Table 1). Interneurons
were readily distinguished from the two relay cell types by their
extremely high-frequency, nonadapting action potential trains in
response to depolarizing pulses (Fig. 1A-C), their
markedly steeper average firing frequencies in response to these
injected currents (Fig. 1D, Table 1), their narrower
action potential widths, their larger spike AHPs, and their higher
rates of spontaneous firing (Table 1). X-projecting neurons also fired
relatively regular action potential trains when depolarized (Fig.
1A), but with moderate spike frequency adaptation
over the first 5-10 spikes (Fig. 1C), a shallower
current-to-spike-frequency relationship (Fig. 1D, Table 1), broader action potentials, and lower rates of spontaneous firing (Table 1). Finally, unlike X-projecting neurons, RA-projecting neurons exhibited highly variable (Fig. 1A,C) spike
timing during depolarizing current injection; these evoked responses
could include very high-frequency bursts (>200 Hz: Fig.
1A,B). In further contrast to both X-projecting
neurons and interneurons, RA-projecting HVc neurons displayed firing
behavior that could be described as "sluggish," as reflected in
their significantly longer and more variable latencies to first spike
onset when injected with positive current (Table 1) and the trends
these cells manifested toward both the lowest average
current-to-frequency relationship (Fig. 1D, Table 1) and the lowest mean rates of spontaneous firing (Table 1). In practice,
this sluggish behavior along with the highly irregular action potential
discharge of RA-projecting neurons in response to moderate positive
currents (approximately +500 pA) was the most useful feature for
distinguishing them from X-projecting neurons on-line, whereas the
fast-spiking behavior of interneurons enabled them to be readily
distinguished from the two relay cell classes. Input resistances, as
well as resting potentials, were not significantly different for the
three cell classes (Table 1).
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Figure 1.
The intrinsic properties of morphologically
identified HVc cell types studied using in vivo
intracellular techniques (see also Table 1).
A1-A3, Typical
membrane potential responses (top, upper, and
middle traces) of each of the
three morphologically distinct HVc neuron types to hyperpolarizing
(400 pA) and depolarizing (+600 pA) current pulses
(bottom trace; 1 sec duration) applied
via the recording electrode. Note the high-frequency firing of the
interneuron and the highly irregular firing of the RA-projecting cell.
B1-B3
Frequency histograms of instantaneous firing frequencies in response to
a +600 pA pulse (1 sec duration) for each cell type. Histograms were
generated from all interspike frequencies measured for a given cell
class (n = 16 X-projecting, 9 RA-projecting, and 11 interneurons). B4, The cumulative
distribution of these instantaneous spike frequencies for each cell class. Note that the spike frequency
distribution for RA-projecting neurons (RAp) was
significantly higher relative to that for X-projecting neurons
(Xp) and that those for interneurons
(Int) were significantly higher than that for either
relay cell type (p < 0.05, Kolmogorov-Smirnov test). C, The average instantaneous
firing frequency for each cell class plotted as a function of spike
interval number, measured for all cells in response to a +600 pA,
1-sec-long depolarizing current pulse. These plots reveal regular
spiking with moderate spike frequency adaptation over the first 5-10
intervals in X-projecting neurons, highly variable spiking of
RA-projecting neurons, and sustained high-frequency firing of
interneurons. At +600 pA, the variability of RA-projecting HVc neuron
firing was significantly greater than that for X-projecting neurons (SD
in the mean firing rate measured across all spike intervals = 65 ± 10 Hz for RA-projecting vs 24 ± 2 Hz for X-projecting
neurons; p < 0.001, two-tailed t
test). D, The mean (± SEM) firing frequency calculated
in response to a 1-sec-long depolarizing current pulse plotted as a
function of injected current amplitude (+200 to +1000 pA), along with
linear fits of these mean values weighted by the SEMs. The average
firing frequency for interneurons was significantly greater than that
for either relay cell class (two-tailed t test,
p < 0.0001); those of the two relay cell types
were not significantly different, although the slope of the best linear
fit was lowest for RA-projecting HVc neurons (24 Hz/nA, vs 42 Hz/nA for
X-projecting neurons and 172 Hz/nA for interneurons). Linear fits:
X-projecting, 42 Hz/nA, r = 0.997, p < 0.0002; RA-projecting, 24 Hz/nA,
r = 0.91, p < 0.05; and
interneuron, 172 Hz/nA, r = 0.977, p < 0.005. See Table 1 for additional comparisons
of the intrinsic properties of these HVc neuron types.
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Morphology
In vivo intracellular neurobiotin staining revealed
that the two HVc relay cell types differed morphologically from one
another, as well as from interneurons (see Figs. 2-4). Differences
among all three cell types included the presence or absence of a
projection axon, the projection axon's termination, cell body size,
dendritic extent, and the relative abundance or absence of dendritic
spines. These differences are very similar to those that have been
noted in previous morphological analyses of HVc neurons (Nixdorf et al., 1989; Fortune and Margoliash, 1995; Benton et al., 1998).
The main axons of X- and RA-projecting HVc neurons had different
trajectories and termini, projecting either rostrally from HVc to area
X or caudally to nucleus RA, whereas interneurons elaborated processes
wholly restricted to HVc (see Figs. 2, 3). The main axons of almost all
(14/16) X-projecting neurons traveled immediately in the rostral
direction after exiting HVc (Fig.
2C). These rostral
trajectories were quite varied, including dorsal paths that initially
ran along the ventricle, above the lamina hyperstriatica (LH), as well
as more ventral routes within and between the LH and the lamina
medullaris dorsalis. X-projecting axons that described the most dorsal
trajectories turned sharply in the ventral direction close to the
rostrocaudal plane of the lateral magnocellular nucleus of the anterior
neostriatum (LMAN) and then traveled near or through LMAN en route to
area X (an example of this is shown in Fig. 2C). In two
cases, X-projecting axons traveled ventrally into the archistriatum
after leaving HVc and then ran rostrally along the ventral floor of the
telencephalon toward area X. Both of these ventral-going axons followed
a more medial trajectory in their descent from HVc and were medial to RA at the point they initiated their rostral turn toward area X. In all
RA-projecting neurons (10/10 cells), the main axon traveled ballistically in a caudal and ventral direction via the tractus dorsalis archistriatalis to RA (Fig. 2C). Both relay
cell types elaborated extensive axon collaterals within HVc, indicating
that they are likely to function as local circuit neurons in HVc as well as serving as relay cells to other song nuclei (Fig.
2C). In contrast to the two relay cell types, HVc
interneurons had processes entirely restricted to HVc; usually, an
interneuron's dendrites and axons were difficult to distinguish from
one another (Figs. 3,
4).
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Figure 2.
Schematic of the song system, the connectivity of
HVc, and the morphology of HVc relay cell types revealed via in
vivo intracellular staining with neurobiotin. A,
A sagittal view of the song system, as well as the ascending auditory
pathway (shown in highly abbreviated form by the dashed
line) that provides auditory input to HVc. HVc contains
two relay cell populations: the first (shown in red)
projects to the vocal premotor nucleus RA, which innervates the
hypoglossal motor neurons and respiratory premotor neurons used for
singing, and the second (shown in blue) projects to the
basal ganglia homolog area X, giving rise to the anterior forebrain
pathway, which includes the thalamic nucleus DLM and the anterior
telencephalic nucleus LMAN, which in turn innervates RA. Besides the
two relay cell types, HVc contains local interneurons (shown as a
small, dark blue cell in
the inset). Field L, the primary telencephalic
thalamorecipient auditory area, is the likely source of auditory input
to HVc, either directly or indirectly via NIf, a sensorimotor structure
that is a major afferent to HVc (see Janata and Margoliash, 1999),
and/or via an interposed area (the "shelf") that is contiguous to
the ventral border of HVc. B, A flowchart depicting the
major functional divisions in the forebrain song control circuitry that
arise from the two relay cell populations in HVc, including the vocal
motor pathway (red) and the anterior forebrain pathway
(blue). C, Representative examples of the
HVc relay cell types that served as the focus of this study. Camera
lucida reconstructions of RA-projecting and X-projecting neurons that
were stained with neurobiotin using in vivo
intracellular techniques are shown. These reconstructions (drawn under
a 63× objective, 1.3 nA) show the cell body and dendrites in
black and the main axon and local axon collaterals in
red. The low-power (2.5× objective) drawings show the
path of the main axon of the given relay cell type traveling from the
parent cell body to the postsynaptic target (i.e., nucleus RA or area
X). Both cell types had spinous dendrites and extensive local axon
collaterals. Scale bar: high power, 30 µm; low power, 750 µm; sections are in the sagittal orientation.
DLM, medial nucleus of the dorsolateral thalamus;
HVc (used as a proper name); L, field L;
LMAN, lateral magnocellular nucleus of the anterior
neostriatum; nAM, nucleus ambiguus; NIf,
nucleus interfacialis; nRAm, nucleus retroambigualis;
nXIIts, tracheosyringeal portion of the hypoglossal
nucleus; Ov, nucleus ovoidalis; RA,
robust nucleus of the archistriatum; X, area
X.
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Figure 3.
A camera lucida reconstruction of the two types of
HVc interneurons studied here. A, The most common
interneuron encountered here had varicose processes, without a distinct
axon, and a small cell body (10/11 cells). B, This cell
is an HVc interneuron with a morphology distinct from that of the more
common type; it had smooth rather than beaded dendrites and fine axon
collaterals that were restricted to HVc. Both were fast-spiking cells
and had otherwise similar electrophysiological properties (see
Results). Scale bar, 30 µm.
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Figure 4.
Photomicrographs illustrate the morphological
differences between the different types of HVc relay cells and
interneurons, as seen under high-magnification (100×, 1.4 nA
objective) differential interference contrast optics. A,
The dendrites of X-projecting HVc neurons were thick and heavily spined
(arrow). B, The dendrites of
RA-projecting HVc neurons were thinner and more sparsely spined, and
individual spines could be long-necked (arrow).
C, The most common type of HVc interneuron encountered
here had swellings along the length of individual processes.
D, A putative axosomatic contact between a terminal
swelling (black arrow) of a process
extending within HVc from the more common interneuron type and an
unstained cell body (white arrowhead) is
shown. Scale bars: A, B, D, 30 µm; C,
45 µm.
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In agreement with previous studies indicating that X-projecting neurons
have larger cell bodies than other HVc neuron types (Paton et al.,
1985; Alvarez-Buylla et al., 1988), the cell body diameters of
X-projecting neurons stained here were larger than those of
interneurons and RA-projecting neurons (soma diameters: X-projecting,
23.0 ± 0.9 µm; n = 16 cells; interneurons,
18.7 ± 1.0 µm; n = 10; RA-projecting, 16.8 ± 0.8 µm; n = 9; Xp > Int, p < 0.005; Xp > RAp, p < 0.0001; unpaired
t test). Qualitatively, the cell bodies of RA-projecting
neurons had more rounded profiles than had those of both X-projecting
neurons and interneurons, which tended to be polyhedral or fusiform in
shape. Both relay cell types had spinous dendrites, but the dendrites
of RA-projecting neurons were more compact (Fig. 2; mean of greatest
radial dendritic extent: X-projecting, 168 ± 4 µm;
n = 12 cells; RA-projecting, 123 ± 0.9 µm;
n = 6 cells; p < 0.001, unpaired
t test) and were thinner and more sparsely spined (Figs. 2,
4). The RA-projecting neurons stained here strongly resemble the short
dendrite class of HVc neurons described in the canary HVc, which
were tentatively classified from Golgi-impregnated material as
RA-projecting on the basis of their small soma sizes (Nixdorf et al.,
1989). In contrast to the two relay cell types, all 11 HVc interneurons had aspinous processes. The processes of 10 of the 11 interneurons had
swellings, or varicosities, along their lengths (Figs. 3A, 4C). Swellings on some interneuronal processes could be seen
in close apposition to other cell bodies, suggestive of axosomatic synapses (Fig. 4D). The one remaining interneuron had
smooth dendrites, which were readily distinguished from a thinner axon
and its very fine collaterals (Fig. 3B). This cell had the
highest average firing-frequency-to-current relationship of any
interneuron measured (~390 Hz/nA), although its action potential
width, AHP height, input resistance, and resting potential fell within
the range described for the other 10 interneurons. The combination of
fast-spiking electrophysiological properties and varicose, aspinous
dendrites seen in HVc interneurons is reminiscent of inhibitory
interneurons that have been described in other song areas (Spiro et
al., 1999) and in the mammalian telencephalon (Azouz et al., 1997;
Thomson and Deuchard, 1997).
Auditory responses of identified HVc neurons
In vivo intracellular recordings were made from a total
of 172 neurons situated within and around HVc. Many of these cells were
held too briefly to characterize their auditory responses or to
identify the cell type. Sixty neurons were sorted unambiguously to type
relying on both morphological and electrophysiological criteria, and
their auditory responses were characterized using forward and reverse
playback of the BOS. These identified HVc neurons included 20 X-projecting neurons (all confirmed morphologically), 23 RA-projecting
neurons (11 confirmed morphologically), and 17 interneurons (7 confirmed morphologically).
Previous extracellular studies have shown that the BOS is a potent
auditory stimulus for many HVc neurons (Sutter and Margoliash, 1994;
Volman, 1996). Here, all identified HVc neuron types were found to
respond robustly to forward playback of the BOS, firing significantly
above the baseline rate during presentation of the stimulus (firing
rate during BOS > baseline firing rate, p < 0.05, 17/20 X-projecting neurons; 15/23 RA-projecting neurons; 14/17 interneurons). Representative responses to BOS playback are shown for
each cell type in Figure 5, which
includes individual raw current-clamp records collected during five
consecutive playbacks of the BOS. These individual traces were used to
construct both the action potential PSTHs as well as the
median-filtered, averaged membrane potential records to quantify a
neuron's subthreshold and suprathreshold responses to song playback
(see Materials and Methods).
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Figure 5.
Representative responses of identified HVc neurons
to auditory presentation (playback) of the BOS. For each cell type
(X-projecting, RA-projecting, and interneuron), the top
five traces in each panel
are the individual current-clamp records collected during five
consecutive BOS playbacks (shown as an oscillogram at the
bottom of each panel and preceded by a
1.5 sec silent period). Immediately below the
current-clamp records are the PSTHs (spikes) of spiking
activity and the median-filtered, average membrane potential generated
from the raw traces ( m; see Materials
and Methods). For both the X-projecting and RA-projecting neurons shown
here, song-evoked action potential responses were highly phasic,
consisting of short bursts of action potentials, whereas the
accompanying subthreshold changes in membrane potential were distinctly
different; spiking in the X-projecting neuron was preceded by membrane
hyperpolarization below the resting membrane potential, whereas spiking
in the RA-projecting neuron was preceded by depolarization above its
resting membrane potential. In contrast to the phasic responses of the
two relay cells, the interneuron displayed more sustained action
potential discharge throughout BOS playback (spike height in this
example was truncated because of capacitive filtering, which more
greatly attenuated action potential height in these fast-spiking cells
than in relay cells). All of these cells were morphologically
identified after the recording session using intracellular neurobiotin
staining and were collected from three different animals.
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Several features distinguished the song-evoked auditory responses of
the three HVc cell types (Fig. 5). First, the majority of BOS-evoked
action potential responses in both relay cell classes were phasic,
occurring at restricted points during a motif (see Figs. 5, 6,
8A,B, top; 10/17 X-projecting
neurons and 9/15 RA-projecting neurons that fired significantly to
forward BOS did so in a phasic manner). In contrast, almost all
interneurons exhibited BOS playback-evoked action potential responses
that were sustained throughout the stimulus, or throughout each motif
(see Figs. 5, 6; 12/14 interneurons that responded significantly to
forward BOS fired in a sustained manner to the stimulus). Second, all
RA-projecting neurons encountered here showed robust subthreshold
responses to BOS playback (i.e., the BOS-evoked depolarizing
response > spontaneous depolarizations before playback;
p < 0.05; 23/23 cells). Even the eight RA-projecting neurons that did not show elevated firing to BOS playback (including three cells that evinced neither spontaneous nor stimulus-evoked firing) still exhibited significant subthreshold depolarizing responses
to BOS. Third, the subthreshold responses of RA-projecting neurons were
always strongly depolarizing from rest (23/23 cells), in contrast to
those of X-projecting cells, which could include hyperpolarizing (7/20
cells; p < 0.05) or depolarizing (8/20 cells; p < 0.05) components (see Materials and Methods and
below). These recordings demonstrate that all three HVc cell types have
auditory responses and raise the possibility that all three types
generate BOS-evoked firing via different synaptic mechanisms.
Song selectivity
Birdsongs vary in their temporal as well as spectral organization.
Previous extracellular studies have shown that many HVc neurons are
sensitive to time-varying acoustical features of the bird's own song,
firing selectively for forward over reverse BOS playback (these two
stimuli contain the same spectral energy but differ in their temporal
organization) (Margoliash, 1986; Lewicki and Arthur, 1996; Volman,
1996; Theunissen and Doupe, 1998). Here, intracellular recordings
confirmed that all three HVc neuron types could fire selectively for
forward over reverse BOS playback [Fig. 6; d' > 0.7, 18/20
X-projecting neurons; 15/23 RA-projecting neurons; 15/17 interneurons
(one interneuron that did not display significant firing-rate elevation
to forward BOS did display significant firing-rate suppression to
reverse BOS)].
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Figure 6.
In HVc, X-projecting and RA-projecting neurons as
well as interneurons are song selective, displaying enhanced action
potential firing to forward over reverse BOS playback. For each cell
type, the PSTHs (spikes/bin) and median-filtered
averaged membrane potential traces
[m (mV)] are shown. These three cells were
collected from a single bird; responses are to 20 iterations each of
the forward and reverse BOS playback, shown as oscillograms at the
bottom of the figure. For the X-projecting neuron, slight
membrane hyperpolarization and highly phasic subthreshold
depolarizations were evoked by forward BOS playback, with only minor
subthreshold depolarizations accompanying reverse BOS playback. For the
RA-projecting neuron, both forward and reverse BOS playback elicited
subthreshold depolarizations, although only forward BOS evoked a
significantly elevated action potential response. For the interneuron,
only forward BOS playback elicited subthreshold or suprathreshold
responses. The X-projecting neuron and interneuron were identified
morphologically, whereas the RA-projecting cell was identified on the
basis of its intrinsic electrophysiological properties.
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The median-filtered average membrane potential traces in the three
cells shown in Figure 6 illustrate that distinct subthreshold membrane
potential responses accompanied their song-selective firing. For
example, the interneuron and the X-projecting cell underwent
depolarizing and hyperpolarizing movements to forward song, with little
subthreshold responsiveness to reverse BOS playback. In contrast, the
RA-projecting neuron displayed substantial subthreshold depolarizations
to reverse as well as forward BOS playback.
Relationships between subthreshold and
suprathreshold selectivity
As a first step toward identifying differences in the subthreshold
auditory responsiveness of the three HVc neuron types, their
subthreshold depolarizations and firing-rate responses to forward and
reverse BOS playback were measured. Z-scores were calculated for
depolarizing (positive) subthreshold areas (see Materials and Methods)
and firing-rate responses to forward and reverse BOS for individual
neurons of each HVc neuron class. For individual cells, either
subthreshold area or firing-rate z-scores for forward BOS responses
were plotted against those same measures of reverse BOS responses.
These comparisons revealed that subthreshold versus suprathreshold
responsiveness varied systematically according to HVc cell type (Fig.
7A). Both X-projecting neurons
and interneurons exhibited firing-rate responses biased to forward BOS,
without a similar bias in their depolarizing subthreshold responses. In contrast, RA-projecting neurons displayed firing-rate and depolarizing subthreshold responses biased to forward over reverse BOS playback (see
Fig. 7 and legend for statistical tests of significance).
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Figure 7.
Differences in song selectivity between the three
HVc cell classes are revealed by paired comparisons of subthreshold and
suprathreshold measures of response strength and selectivity
(n = 20 X-projecting, 23 RA-projecting, and 17 interneurons). A, Within-cell comparisons of the
response strengths (z-scores) to forward BOS are plotted
against those for reverse (rev) BOS, for either firing
rate (closed circles) or subthreshold depolarizations
(open squares; these are net-positive subthreshold
responses, see Materials and Methods) for each cell type. The
unity line represents equal
responsiveness to forward and reverse BOS playback; points to the
right of the diagonal are biased to
forward BOS playback. Paired t tests
(p values are displayed above each
plot) were used to determine whether responses were
biased to either forward or reverse BOS at either a subthreshold or
firing-rate level. Left, X-projecting neurons were
biased to forward over reverse BOS at a firing-rate level but not at a
subthreshold level. Middle, RA-projecting neurons were
biased to forward over reverse BOS playback both at a firing-rate level
and at a subthreshold level. Right, Interneurons were
biased to forward BOS at a firing-rate level but not at a subthreshold
level. B, For HVc neurons of a given class,
d' statistics (see Materials and Methods) were used to
compare an individual neuron's firing-rate selectivity directly with
its subthreshold selectivity for forward over reverse BOS playback. The
unity line depicts equivalent
subthreshold and suprathreshold selectivity, whereas the
horizontal and vertical
gray bars demarcate nonselective
subthreshold or suprathreshold zones, respectively. Paired
t tests revealed greater firing-rate selectivity than
subthreshold selectivity for X-projecting neurons and interneurons
(left, right) but greater subthreshold
selectivity than firing-rate selectivity in RA-projecting neurons
(middle). The two relay cell classes had similar mean
firing-rate selectivity for forward over reverse BOS playback
(d'Xp = 1.35 ± 0.26;
d'RAp = 1.34 ± 0.17;
p = 0.8, two-tailed t test), whereas
interneurons had the highest mean firing-rate selectivity of any HVc
cell type (d'Int = 3.05 ± 0.44;
p < 0.005 vs Xp and p < 0.0005 vs RAp neurons, two-tailed t test).
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The different patterns of subthreshold and suprathreshold
responsiveness of the three HVc neuron types to forward and reverse BOS
playback suggest that X-projecting neurons and interneurons produce
more selective action potential output than would be predicted from
their depolarizing subthreshold responses alone. To confirm that this
was indeed the case, subthreshold and firing-rate d' statistics (see Materials and Methods) were computed to allow direct
comparisons of subthreshold versus firing-rate selectivity within
single HVc neurons of a given class. Plotting d' values for
depolarizing subthreshold responses (area) against those for firing
rate revealed that selectivity was greater at a firing-rate level than
at a subthreshold level for X-projecting neurons and interneurons but
lower at a firing-rate than subthreshold level for RA-projecting
neurons (Fig. 7B; see figure and legend for relevant
statistics). At a population level, mean d' values for firing rate in the three HVc cell types greatly exceeded 0.7 (i.e., were selective for forward over reverse BOS playback), with the two
relay cell classes displaying almost identical absolute levels of
firing-rate selectivity (mean d', X-projecting, 1.35 ± 0.26; RA-projecting, 1.34 ± 0.17; interneuron, 3.05 ± 0.44). In almost every case, selectivity stemmed from an augmentation
of firing rate in response to forward BOS either alone or in
combination with firing-rate suppression to reverse BOS, rather than
only a suppression of firing rate in response to reverse BOS (selective cells with an absolute excitatory bias to forward BOS, 18/18
X-projecting neurons; 15/15 RA-projecting neurons; 14/15 interneurons).
In contrast, significantly elevated firing-rate responses to reverse BOS playback were rare in all song-selective HVc neurons
(p < 0.05; 0/18 X-projecting neurons; 1/15
RA-projecting neurons; 3/14 interneurons).
Membrane potential effects on BOS-evoked subthreshold and action
potential responses
BOS playback could elicit hyperpolarizing responses in some
X-projecting neurons (Figs. 5, 6) (see also Lewicki, 1996), while evoking exclusively depolarizing responses in RA-projecting neurons (Figs. 5, 6). To test whether different patterns of song-evoked inhibition and excitation distinguish song-selective responses in the
two HVc relay cell types, tonic-positive current was injected via the
recording electrode to depolarize the resting membrane potential of
individual HVc neurons during song playback (Fig. 8). These membrane potential
manipulations were used to unmask or augment potentially latent
song-evoked inhibition and to determine whether subthreshold
depolarizations that occurred at the cell's normal resting potential
were excitatory or reversed inhibitory events. BOS-evoked excitation
and inhibition were assessed by measuring both subthreshold and
firing-rate responses for the two types of relay cells at their normal
resting potentials and when the cells were tonically depolarized. To
measure subthreshold depolarizations and hyperpolarizations, positive
and negative areas between the median-filtered average membrane
potential traces and the prestimulus baseline mode potential were
estimated before and during playback, and the positive or negative
prestimulus area was then subtracted from the stimulus-evoked area of
the same sign to yield net subthreshold responses (see Materials and Methods; Fig. 9). In parallel to these
subthreshold measurements, firing rates to forward and reverse BOS
playback relative to baseline rates were calculated at the two
different membrane potentials. Manipulating membrane potential in this
way revealed different patterns of subthreshold activity accompanied by
firing-rate suppression or augmentation in the two cell types (Figs. 8,
9).
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Figure 8.
Tonic depolarization unmasked latent inhibition
evoked by forward BOS playback in an X-projecting neuron and latent
excitation to forward and reverse BOS playback in an RA-projecting
neuron. The PSTHs and median-filtered average membrane potential
traces were generated from current-clamp records
obtained during 20 iterations of forward and reverse BOS playback
(oscillograms at bottom of each
panel), presented either at the cell's
unmanipulated resting membrane potential
(control) or with tonic depolarizing current
applied via the recording electrode to positively shift the cell's
membrane potential (depolarized). A, For
an X-projecting neuron at its normal resting potential, forward BOS
playback evoked a highly phasic action potential response that was
paralleled by a similarly phasic subthreshold depolarization. In the
tonically depolarized state, forward BOS playback evoked firing-rate
suppression paralleled by membrane hyperpolarization, suggestive of a
previously latent inhibitory mechanism. Reverse BOS playback did not
evoke subthreshold or suprathreshold responses to playback at either
the control or depolarized membrane potentials. B, For
an RA-projecting neuron observed at its unmanipulated resting
potential, highly phasic action potential responses occurred only in
response to forward BOS playback, but pronounced and sustained
subthreshold depolarizations were evoked by both forward and reverse
BOS. Tonic depolarization resulted in elevated action potential
discharge to both forward and reverse BOS playback, indicating that the
subthreshold depolarizations seen at the normal resting potential were
latent excitatory events. Note that the subthreshold
traces in the depolarized state do not reveal the
BOS-evoked hyperpolarization seen in the X-projecting neuron.
Population data for these effects are shown below (see Fig.
9).
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Figure 9.
Population analyses of the effects of tonic
depolarization on subthreshold responses in the two relay cell types
indicate that X-projecting neurons display a mixture of inhibition and
excitation evoked primarily by forward BOS, whereas RA-projecting
neurons display predominantly excitatory responses to both forward and
reverse BOS. A, Top, An example of how
the positive and negative subthreshold responses to forward and reverse
BOS playback were measured is shown. Positive or negative z-scores were
generated from positive or negative integrals calculated relative to a
baseline mode value, either before or during stimulus presentation; the
prestimulus positive area was then subtracted from the BOS-evoked
positive area to generate a positive area score (lightly
shaded; values > 0 correspond to a net increase in
positivity during stimulation). A similar subtraction was performed
with the negative integrals, which were then multiplied by 1 to
generate a negative area score (darkly shaded;
values < 0 correspond to a net increase in negativity during
playback). Bottom Left, The means (± SEM) of the
subthreshold positive and negative area z-scores for X-projecting
neurons in their control and tonically depolarized states
(Vcontrol = 76.5 ± 2.1 mV vs
Vdepolarized = 57.6 ± 3.5 mV;
p < 0.001; n = 11 cells) are
shown. Depolarization altered the responses of X-projecting neurons so
that they were net negative. Positive area:
ZareaBOScontrol = 0.42 ± 0.27;
ZareaBOSdepolarized = 1.25 ± 0.35;
p < 0.01, paired t test. Negative
areas: ZareaBOScontrol = 0.41 ± 0.30;
ZareaBOSdepolarized = 1.58 ± 0.20;
p < 0.05, paired t test.
Bottom Right, Subthreshold positive and negative area
z-scores (± SEM) for RA-projecting neurons in their control and
tonically depolarized states (Vcontrol = 79.6 ± 2.8 mV vs Vdepolarized = 57.4 ± 3.2 mV;
p < 0.0001; n = 8 cells) are
shown. Mean responses were highly positive in either state. Positive
areas: ZareaBOScontrol = 3.1 ± 0.55;
ZareaBOSdepolarized = 1.87 ± 0.38;
p < 0.05, paired t test. Negative
areas: ZareaBOScontrol = 1.23 ± 0.13; ZareaBOSdepolarized = 0.11 ± 0.76; p = 0.16, paired t test.
B, Top, In X-projecting neurons, tonic
depolarization significantly depressed firing rates to forward BOS
playback, without affecting firing-rate responses to reverse BOS
playback. Mean firing rates: ZcontrolBOS = 0.97 ± 0.10 versus ZdepolarizedBOS = 0.73 ± 0.34;
p < 0.001, paired t test;
ZcontrolBOSrev = 0.09 ± 0.12 versus
ZdepolarizedBOSrev = 0.12 ± 0.19;
p = 0.24; n = 11 cells.
Bottom, In RA-projecting neurons, tonic depolarization
resulted in a significantly elevated firing-rate response to reverse
BOS playback and a trend toward elevated firing to forward BOS
playback. Mean firing rates: ZcontrolBOS = 1.01 ± 0.15; ZdepolarizedBOS = 2.34 ± 0.60;
p = 0.12, paired t test;
ZcontrolBOSrev = 0.14 ± 0.19;
ZdepolarizedBOSrev = 1.47 ± 0.29;
p < 0.01; n = 6 cells (1 of
the 7 cells was depolarized strongly enough to inactivate spiking
totally and thus is not included here; also note that only a
significant effect on reverse BOS was observed but that the mean
response to forward BOS more than doubled in the depolarized
state).
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Tonic depolarization of X-projecting neurons resulted in periods of
pronounced membrane hyperpolarization to forward BOS playback that were
paralleled by action potential suppression, suggestive of an inhibitory
synaptic mechanism (Fig. 8A). For X-projecting neurons at their normal resting potentials, forward BOS playback could
evoke either net depolarizing (4/11 cells; p < 0.05)
or net hyperpolarizing (3/11 cells; p < 0.05)
subthreshold responses relative to baseline fluctuations in membrane
potential (Fig. 9A). Tonically depolarizing these cells
(Vnormal = 76.5 ± 2.1 mV vs
Vdepolarized = 57.6 ± 3.5 mV;
p < 0.001; n = 11 cells) strongly
augmented BOS-evoked hyperpolarization, such that all 11 cells
displayed only net hyperpolarizing responses to forward BOS playback
(Fig. 9A; net hyperpolarizing, p < 0.05;
11/11 cells; net depolarizing, p < 0.05; 0/11 cells;
see Fig. 9 legend for mean z-scores for negative and positive areas).
Although highly phasic depolarizing responses to forward BOS could
still persist during tonic-positive current injection (Fig.
8A; ~2.7 sec), they failed to exceed spontaneous
baseline depolarizations in total area. Subthreshold responses of any
kind to reverse BOS were rare, and tonic depolarization did not unmask
latent inhibition (or excitation) evoked by this stimulus (Fig.
8A; population data not shown; net depolarizing,
p < 0.05; 2/11 cells at normal
Vm and 1/11 cells at positive
Vm; net hyperpolarizing, p < 0.05; 2/11 cells at normal Vm and 1/11 cells at
positive Vm).
In X-projecting neurons, the subthreshold effects of altering membrane
potential were paralleled by changes in BOS-evoked firing (Figs.
8A, 9B). At normal resting membrane
potentials, significant firing-rate increases above baseline
accompanied forward BOS playback in all 11 cells, whereas only one of
these cells displayed a significant suprathreshold response to reverse
BOS, which consisted of firing-rate suppression. During tonic
depolarization, forward BOS playback evoked either firing-rate
suppression (BOS-evoked firing rate < baseline firing rate;
p < 0.05; 5/11 cells) or no significant suprathreshold
response of any kind relative to baseline firing rates
(p > 0.05; 6/11 cells), whereas none of these
cells displayed significant firing-rate responses to reverse BOS in the
tonically depolarized state (p > 0.5; 11/11
cells). Paired comparisons of z-scores for firing rate in response to
either forward or reverse BOS playback at the two membrane potentials show that tonic depolarization selectively altered action potential responses to forward, but not reverse, BOS (Fig. 9B).
Forward BOS-evoked firing-rate responses changed from net positive
(i.e., augmentation) to net negative (i.e., suppression) relative to baseline with depolarization, whereas reverse BOS did not evoke significant suprathreshold responses in either the resting or depolarized state (mean z-scores, ZnormalBOS = 0.972 ± 0.103 vs ZdepolarizedBOS = 0.731 ± 0.336; p < 0.001, paired t
test; ZnormalBOSrev = 0.090 ± 0.118 vs
ZdepolarizedBOSrev = 0.121 ± 0.186;
p = 0.24, paired t test; n = 11 cells). Taken together, the subthreshold and suprathreshold effects
of changing membrane potential are consistent with the idea that
X-projecting neurons receive excitatory and inhibitory inputs activated
primarily by forward BOS playback but do not receive inputs of either
sign that are strongly activated by reverse BOS playback.
Unlike X-projecting neurons, membrane potential manipulations in
RA-projecting neurons suggest that they receive predominantly excitatory inputs strongly activated by both forward and reverse BOS
playback. At their normal resting membrane potentials, RA-projecting neurons exhibited exclusively depolarizing subthreshold responses to
forward as well as reverse BOS playback (Figs. 8B,
9A; net depolarizing, p < 0.05; forward
BOS, 8/8 cells; reverse BOS, 8/8 cells; net hyperpolarizing,
p < 0.05; forward BOS, 0/8 cells; reverse BOS, 0/8
cells). Unlike X-projecting neurons, tonic depolarization (Vnormal = 79.6 ± 2.8 mV vs
Vdepolarized = 57.4 ± 3.2 mV;
p < 0.0001; n = 8 cells) did not
reveal robust forward BOS-evoked inhibition (Figs.
8B, 9A; net depolarizing,
p < 0.05; forward BOS, 5/8 cells; reverse BOS, 4/8
cells; net hyperpolarizing, p < 0.05; forward BOS, 2/8
cells; reverse BOS, 0/8 cells). Of the two RA-projecting neurons that
did display song-evoked hyperpolarization, one simultaneously
maintained a net positive subthreshold response, while the other
exhibited the highest stimulus-evoked firing rate observed for any
RA-projecting neuron, inconsistent with an inhibitory input. One
possibility is that, at higher firing frequencies, spike AHPs
contributed significantly to the median-filtered traces and increased
the average membrane potential negativity.
RA-projecting neurons differed from X-projecting neurons in that tonic
depolarization almost always augmented their song-evoked firing,
especially to reverse BOS, and never resulted in firing-rate suppression to forward BOS playback (Figs. 8B,
9B). Six of seven RA-projecting neurons displayed
significant firing-rate responses to forward BOS playback at their
normal resting membrane potentials, and five of these cells exhibited
higher absolute firing rates to forward BOS in the depolarized state.
In addition, a seventh cell that did not display a significant
firing-rate response to forward BOS playback at either membrane
potential still exhibited a >15-fold increase in response strength
after depolarization (Znormal = 0.33;
p = 0.33; vs Zdepolarized = 5.38;
p = 0.07). As a group, the mean firing-rate response of
RA-projecting neurons to forward BOS playback more than doubled after
depolarization, although this effect fell short of significance (mean
z-scores, ZcontrolBOS = 1.01 ± 0.15;
ZdepolarizedBOS = 2.34 ± 0.60;
p = 0.12, paired t test; n = 6;
two cells did not spike in the depolarized state. In RA-projecting
neurons, firing-rate suppression was never observed in response to
forward BOS playback, regardless of membrane potential (0/8 cells).
Finally, in further contrast to X-projecting neurons, tonic
depolarization could unmask latent excitatory responses to reverse BOS
in RA-projecting neurons (reverse BOS-evoked firing rate > baseline; p < 0.05; 2/8 cells at the normal membrane
potential vs 4/8 cells during tonic depolarization), and the mean
firing-rate response strength to reverse BOS playback increased
>10-fold with depolarization (ZnormalBOSrev = 0.14 ± 0.19; ZdepolarizedBOSrev = 1.47 ± 0.29; p < 0.01, paired t test). The
contrasting effects of membrane polarity on song-evoked responses in X-
and RA-projecting neurons suggest that the same auditory stimuli drive
different synaptic responses in the two HVc relay cell types;
X-projecting neurons receive excitatory and inhibitory inputs activated
primarily by forward BOS playback, whereas RA-projecting neurons
receive predominantly excitatory inputs activated both by forward and reverse BOS.
The effects of membrane polarity on song selectivity
Membrane potential manipulations differentially affected
statistical measures of selectivity in the two relay cell classes, because of contrasting effects of membrane potential on firing-rate responses to forward and reverse BOS playback. Selectivity
(d') values calculated in both the normal and depolarized
conditions revealed that the mean firing-rate selectivity for the
population of X-projecting neurons, but not RA-projecting neurons,
changed as a function of membrane polarity (Fig.
10). In X-projecting neurons, manipulating membrane potential altered mean firing-rate selectivity (d'normal = 1.65 ± 0.22 vs
d'depolarized = 1.21 ± 0.52;
p < 0.005, paired t test), whereas in
RA-projecting neurons, mean firing-rate selectivity did not vary with
membrane potential (d'normal = 1.47 ± 0.21; d'depolarized = 1.54 ± 0.27;
p = 0.85, paired t test; membrane potential
values in the two states are those given in the previous section and
are in the legend for Fig. 10). On a single-cell level, membrane
potential manipulations decreased the d' values of all
X-projecting neurons. At their normal membrane potentials, 11/11
X-projecting neurons had d' values > 0.7, whereas in
the tonically depolarized state, 9/11 cells had d'
values < 0.7 (6/9 cells; d' < 0.7), and the two
other cells that maintained d' values > 0.7 still
exhibited lower d' values when depolarized. In contrast, the
six RA-projecting neurons that were (positively) selective at their
normal resting membrane potentials maintained selectivity during
depolarization (the d' values decreased for 4 cells and
increased for 2 cells, but all were > 0.7), whereas a seventh
cell that was initially nonselective exhibited selectivity during
depolarization (d'normal = 0.54;
d'depolarized = 2.1). At a population
level, these different effects on selectivity were caused by the
distinctly different effects that depolarization had on firing rate to
forward or reverse BOS playback, as noted previously (Figs. 8, 9). For
X-projecting neurons, the specific membrane potential-dependent changes
to forward but not reverse BOS responses resulted in altered
selectivity, whereas for RA-projecting neurons, the parallel changes in
forward and reverse BOS responses resulted in equivalent selectivity at
the two membrane potentials.
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Figure 10.
Altering the membrane potential at a single-cell
level affected the firing-rate selectivity of X-projecting neurons but
not that of RA-projecting neurons. Mean firing-rate selectivity
(d') for forward versus reverse BOS playback was
measured for 11 X-projecting neurons and 7 RA-projecting neurons at
their normal membrane potentials and then during tonic depolarization
with positive current (X-projecting, Vcontrol = 76.5 ± 2.1 mV vs Vdepolarized = 57.6 ± 3.5 mV; p < 0.001; n = 11 cells; RA-projecting, Vcontrol = 79.6 ± 2.8 mV
vs Vdepolarized = 57.4 ± 3.2 mV;
p < 0.0001; n = 8 cells).
Left, In X-projecting neurons, this manipulation
altered firing-rate selectivity
(d'normal = 1.65 ± 0.22 vs
d'depolarized = 1.21 ± 0.52;
p < 0.005, paired t test), because
of changes in suprathreshold responses to forward, but not reverse, BOS
playback (see Fig. 9B). Right, In
RA-projecting neurons, tonic depolarization did not alter firing-rate
selectivity (d'control = 1.47 ± 0.21; d'depolarized = 1.54 ± 0.27; p = 0.85, paired t test),
because of a parallel trend toward increased suprathreshold
responsiveness to both forward and reverse BOS (see Fig.
9B). The horizontal gray
bar is the nonselective zone.
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Hyperpolarizing subthreshold and firing-rate responses in
X-projecting neurons
In tonically depolarized X-projecting neurons, the pronounced
hyperpolarizations evoked by forward but not reverse BOS playback suggest that these cells receive highly song-selective inhibitory input. To explore this idea further, the relationship between subthreshold hyperpolarizing responses and firing-rate selectivity was
examined for X-projecting neurons. First, as performed previously for
depolarizing responses, subthreshold hyperpolarizing responses to
forward and reverse BOS were compared using z-scores (Fig. 11A). These
comparisons revealed that hyperpolarizing responses were biased toward
forward BOS (mean z-score for hyperpolarization, BOS, 0.49 ± 0.21; BOSrev, 0.16 ± 0.12; p < 0.005, paired
t test). Second, direct comparisons of subthreshold
hyperpolarizing selectivity (d' for negative area) and
firing-rate selectivity for individual X-projecting neurons were made
(Fig. 11B). Unlike the mismatch between depolarizing
subthreshold selectivity and firing-rate selectivity (see Fig. 6),
hyperpolarizing subthreshold responses were as selective as firing-rate
measures in these cells (d'negative area = 1.05 ± 0.28; d'firing rate = 1.35 ± 0.26; p = 0.46, paired t test).
Taken together with the effects of membrane potential manipulations on
forward (but not reverse) BOS-evoked hyperpolarizations, these results
are consistent with the idea that X-projecting neurons receive highly
song-selective inhibition.
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Figure 11.
X-projecting HVc neurons exhibit inhibition that
is strongly evoked by forward but not reverse BOS playback.
A, A direct comparison of z-scores for subthreshold
hyperpolarizations (net negative area, measured with respect to
spontaneous hyperpolarizations in a 1.5 sec window before the stimulus)
evoked by forward (x-axis) versus reverse
(y-axis) BOS playback is shown. Paired
t tests indicate that membrane hyperpolarization was
more strongly evoked by forward BOS playback (n = 20 cells; p value shown above
plot). B, Within-cell comparisons of
subthreshold hyperpolarizing selectivity (d' negative
area) and firing-rate selectivity (d' firing rate) for
forward over reverse BOS playback show that the two measures are
equally biased toward forward BOS playback (n = 20 cells; p value from paired t test shown
above plot). The unity
line represents equivalent subthreshold and
suprathreshold selectivity, whereas the gray
bars demarcate zones of nonselective subthreshold
(horizontal) or firing-rate
(vertical) responses. Note that these
relationships contrast with those for response strength and selectivity
measured for depolarizing subthreshold responses and firing rate in
X-projecting neurons (see Fig. 7).
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Within-bird comparisons of the timing of song-evoked responses
HVc interneurons are one possible source of the highly
song-selective inhibition seen in X-projecting neurons. To study this possibility further, sequential recordings from two or more different HVc neuron types were obtained within a single bird. Comparisons of the
timing of forward BOS-evoked action potential firing and subthreshold
responsiveness in these different cells were made by directly
superimposing PSTHs and/or membrane potential traces collected from
within birds (Fig. 12). In addition,
cross-correlation analyses were performed on cell pairs to examine
during BOS playback the relative spike timing of RA-projecting neurons
with that of either X-projecting neurons or interneurons and to compare
interneuron spike timing with X-projecting neuronal membrane polarity
(Fig. 13; Materials and Methods). These
comparisons reveal that the timing of song-evoked responses varies
systematically according to cell class, with the closest temporal
coincidence between interneuron firing and X-projecting neuronal
hyperpolarization. In six animals, recordings were obtained from at
least one interneuron and one X-projecting neuron. In every case,
song-evoked interneuron firing closely coincided with when
hyperpolarization and firing-rate suppression occurred in the
X-projecting neuron (examples in Fig. 12). Cross-correlation analysis
of interneuron firing and X-projecting neuronal membrane
hyperpolarization during playback confirmed a maximum correlation at
zero offset (Fig. 13; n = 6 pairs in 6 birds). Although
recordings obtained from both interneurons and RA-projecting neurons
revealed that song-evoked firing could occur near the same times (Fig.
12B, compare bottom, top
PSTHs), cross-correlating their spike times revealed a slight offset,
with RA-projecting neuronal firing leading that of interneurons by
~25 msec (the resolution of the analysis; see Fig. 13;
n = 4 pairs in 4 animals). Finally, recordings from the
two relay cell types in single birds showed that they fired
reciprocally during BOS playback and that the BOS-evoked changes in
membrane potential described an antiparallel motion, with the
RA-projecting neuron exhibiting a prolonged depolarization during the
same period when the X-projecting neuron was sustaining a net
hyperpolarization (Fig. 12B). Cross-correlation
analysis confirmed a large temporal offset in song-evoked firing
between X-projecting and RA-projecting neurons, with a maximum of ~75 msec (Fig. 13; n = 4 pairs in 4 animals). These
analyses indicate that, during forward BOS playback, interneuron firing
closely coincides with X-projecting neuronal hyperpolarization,
RA-projecting neuron firing and interneuron firing are separated by
short delays, and RA- and X-projecting neurons fire reciprocally at
relatively long time intervals.
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Figure 12.
Within-bird comparisons of action potential
responses and subthreshold responses in different HVc neuron types to
forward BOS playback. A, The action potential PSTHs
(top) and median-filtered averaged membrane potential
responses (middle) aligned to forward BOS playback
(oscillogram at bottom) of an X-projecting neuron and
interneuron that were recorded sequentially from a single bird. Note
that the periods of greatest firing-rate elevation in the interneuron
(marked by asterisks) coincide with maximal firing-rate
suppression and membrane hyperpolarization in the X-projecting neuron
and that the subthreshold responses of the two cell types move in a
mirror-symmetrical manner. B, The action potential PSTHs
(top, second from bottom) and median-filtered averaged
membrane potential responses (second from top) aligned
to forward BOS playback of an X-projecting neuron, an RA-projecting
neuron, and an interneuron that were recorded sequentially from a
single bird. Note that (1) BOS-evoked firing of the RA-projecting
neuron (marked by asterisks) alternates with firing of
the X-projecting neuron, (2) the subthreshold responses of the two
relay cells move in opposite directions relative to their resting
potential (i.e., depolarizing for the RA-projecting cell and
hyperpolarizing for the X-projecting cell), and (3) the song-evoked
elevation in the interneuron firing closely coincides with when the
RA-projecting cell is firing and when the period of maximal firing-rate
suppression and membrane hyperpolarization occurs in the X-projecting
neuron. PSTHs were generated using a 25 msec bin width; all responses
shown are to 20 iterations of the stimulus.
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Figure 13.
Cross-correlation analysis shows that, during BOS
playback, peak interneuron firing closely coincides with the maximum
hyperpolarization of X-projecting neurons, whereas the firing of
RA-projecting and X-projecting neurons is separated by long time
intervals, and the firing of RA-projecting neurons and interneurons is
separated by shorter intervals. The relative spike timing and/or
membrane polarity during BOS playback was quantified by generating
cross-correlograms from the action potential PSTHs of RA-projecting and
X-projecting HVc neurons (open columns),
the action potential PSTHs of RA-projecting neurons and interneurons
(shaded columns), or the interneuron
action potential PSTHs and X-projecting neuronal membrane potential
(Vm, averaged over 25 msec intervals;
joined circles). Cross-correlation values
were generated from pairwise comparisons between two cells recorded
sequentially within a single bird and then normalized to peak and
averaged across all pairs of a given type (i.e., across birds;
RA-projecting vs X-projecting, 6 pairs in 6 birds; RA-projecting
vs interneurons, 4 pairs in 4 birds; interneurons vs X-projecting
Vm, 6 pairs in 6 birds; see Materials and
Methods). Time values are expressed in the bin widths of the PSTHs
(i.e., 25 msec); note that the different scales of the two
y-axes refer to different pairwise comparisons.
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DISCUSSION |
All HVc neuron types fire selectively for forward over reverse BOS
playback. Relay cells innervating either area X or nucleus RA generate
remarkably similar patterns of selective firing via different
subthreshold mechanisms, likely because of segregated inhibitory input
from interneurons onto X-projecting cells. Ultimately, factors that
modulate the relay cells' membrane potentials may enable them to
transmit distinct song representations, providing the potential for
auditory comparisons used in song learning, maintenance, and perception.
Different morphological classes of HVc neurons examined in
vivo display distinct intrinsic electrophysiological properties, extending previous in vitro studies (Dutar et al., 1998;
Kubota and Taniguchi, 1998). One difference in vivo was that
all cell types had lower input resistances than seen in brain slices,
likely because of higher spontaneous synaptic activity in the intact brain. In vivo, as in vitro, positive currents
drove interneurons to fire "fast" action potentials, often at high
rates, while causing X-projecting neurons to fire regular action
potential trains, although with a lower current-frequency relationship
than for interneurons. Although RA-projecting neurons could fire
repetitively in vivo, their DC-evoked spiking was
characterized by an unusually prolonged and variable spike onset. Their
firing remained intermittent and highly variable even with large
currents (~1 nA), perhaps because of spontaneous synaptic activity
interacting with injected currents. If these RA-projecting neurons are
the same that become highly active during singing, then they undergo
dramatic state-dependent modulation.
All three HVc neuron types are highly song selective. Previous
intracellular studies concluded that X- but not RA-projecting neurons
had auditory responses and left the auditory properties of HVc
interneurons undetermined (Katz and Gurney, 1981; Lewicki, 1996). Here,
interneurons fired tonically and selectively to BOS, whereas both relay
cells fired more phasically. Such tonic responses, detected in previous
intracellular studies (Lewicki, 1996), were not linked with a cell
type; the present study shows they arise from HVc interneurons with
fast spikes and varicose dendrites, two features of inhibitory
interneurons in RA and in other systems (Azouz et al., 1997; Thomson
and Deuchard, 1997; Spiro et al., 1999). If these HVc interneurons are
also inhibitory, then they likely provide song-selective inhibition to
X-projecting neurons. Previous reports classifying RA-projecting
neurons as nonauditory (Katz and Gurney, 1981; Lewicki, 1996) are
surprising because here they usually fired song-selectively and they
always displayed robust song-selective subthreshold responses. Perhaps
previous studies failed to detect auditory responses because song
stimuli were not used (Katz and Gurney, 1981) or because these cells' relatively refractory behavior caused them to be disregarded.
The existence of song-responsive RA-projecting neurons, like those
described here, has been inferred by reversibly inactivating HVc, which
transiently abolishes RA's auditory responsiveness (Doupe and Konishi,
1991). Furthermore RA's auditory responses persist even when LMAN
(RA's other likely auditory afferent) is lesioned (Doupe and Konishi,
1991; Vicario and Yohay, 1993). A confound of understanding the genesis
of RA's auditory responses in control birds is that
inactivating HVc also silences X-projecting neurons, ultimately
abolishing auditory responses in LMAN. Therefore, detecting auditory
responses in RA-projecting neurons reveals a direct path from HVc to RA
by which auditory information could influence vocalization. A
parsimonious model is one in which the same RA-projecting HVc neurons
that convey elaborate premotor patterns for singing (McCasland, 1987;
Yu and Margoliash, 1996) also respond selectively to the resulting
vocalizations, providing a cellular substrate for auditory feedback.
Separate auditory and vocal projections from HVc to RA may be less
likely to exist, because here RA-projecting neurons always responded to
the BOS. In either case, HVc is the source of two song-selective
auditory pathways.
Song-evoked subthreshold responses partly reflect the firing of neurons
presynaptic to the impaled cell and thus can illuminate the origins of
song selectivity. One caveat is that signal averaging measures
net excitation and inhibition, and stronger elements may
mask weaker components. Another qualification is that excitatory and
inhibitory contributions may differ in states other than those studied
here, perhaps via voltage-dependent effects (e.g., NMDA receptor-mediated currents). Nonetheless, one striking feature of
RA-projecting neurons is that their song-evoked subthreshold responses
appear exclusively (if latently) excitatory and, although biased toward
forward song, can be quite pronounced to reverse BOS. The elevated and
sustained firing in tonically depolarized RA-projecting neurons evoked
by either stimulus (Fig. 8B, top) suggests
that these cells receive little or no song-evoked inhibition. Instead,
such sustained depolarizations indicate that cells presynaptic to
RA-projecting neurons are excitatory and fire throughout forward and
reverse BOS playback, although with a forward song bias. Most neurons
in NIf, an HVc afferent, display sustained, song-selective firing
(Janata and Margoliash, 1999), but the nature of their connections to
different HVc neurons is unknown. In addition, a minority of neurons in
the primary avian auditory telencephalon (field L) fire selectively to
BOS (Lewicki and Arthur, 1996; Janata and Margoliash, 1999), but it is
unknown whether they innervate HVc. A necessary step will be to
determine the auditory nature and synaptic sign of extrinsic inputs
onto various HVc neurons.
Unlike RA-projecting neurons, X-projecting neurons exhibited pronounced
forward BOS-evoked hyperpolarizations and lack reverse BOS responses
altogether. The contrasting song-evoked inhibition in the two relay
cell types suggests that X-projecting neurons are privileged targets of
HVc interneurons. Indeed, within birds, elevated interneuron firing
closely coincides with X-projecting neuronal hyperpolarization during
forward BOS playback. In the songbird HVc, BOS-evoked
hyperpolarizations in a subset of X-projecting neurons may be critical
to generating their supralinear sensitivity to syllable sequences
(Lewicki and Konishi, 1995; Lewicki, 1996). Because such
combination-sensitive neurons are rare [~15-25% of HVc neurons
(Margoliash and Fortune, 1992; Lewicki and Arthur, 1996)], yet
BOS-evoked hyperpolarizations were detected in all X-projecting neurons
(at least when sufficiently depolarized), song-evoked inhibition alone
may not suffice for this form of temporal sensitivity. Finally, the
absence of reverse BOS responses in X-projecting cells contrasts
directly with RA-projecting neurons, raising the possibility that the
two relay cell types receive distinct extrinsic auditory inputs and/or
form a serial or circular hierarchy in HVc.
The function of BOS-evoked inhibition on X-projecting neurons could be
to sculpt excitatory responses to BOS, generate more precise spike
timing (Mainen and Sejnowski, 1995), elevate signal-to-noise levels
(Lewicki, 1996), or facilitate excitatory responses by either priming
the postsynaptic membrane (e.g., by deinactivating voltage-gated
conductances) or synchronizing the firing of excitatory presynaptic
inputs. If X- and RA-projecting neurons receive similar song-selective
excitatory inputs, then inhibition in X-projecting neurons could
partially suppress subthreshold depolarizing responses to forward BOS,
causing them to appear nonselective. However, postsynaptic inhibition
is unlikely to account for the lack of reverse BOS responses in
X-projecting neurons, because hyperpolarizing responses to this
stimulus were not detected even when cells were held tonically
depolarized. Support for inhibitory maintenance of signal-to-noise
levels and against postsynaptic priming comes from finding that
artificially depolarizing X-projecting neurons suppresses their mean
firing below baseline during forward BOS playback, even though highly
phasic firing persists (Fig. 8A; ~2.7 sec).
Distinguishing between these various roles will require selectively
blocking inhibition and assessing any effects on BOS-evoked responses
in X-projecting neurons. Finally, these results underscore that highly
similar action potential responses can arise from distinct subthreshold
processes, even within a single brain area; both RA- and X-projecting
neurons fired phasically and with almost identical song selectivity,
yet only X-projecting neurons displayed BOS-evoked hyperpolarizations.
Chronic recordings in nonsinging, awake zebra finches show that BOS
playback evokes responses in HVc (Dave et al., 1998; but see Schmidt
and Konishi, 1998), whereas after singing, HVc's auditory responsiveness is transiently suppressed (McCasland and Konishi, 1981).
Because of HVc's functional heterogeneity, it will be necessary to
distinguish whether auditory suppression also occurs during singing and
in all HVc cell types and whether suppression varies in different
developmental or attentional states, any of which could profoundly
impact auditory-vocal interactions. One speculation is that the same
circuitry used for singing is co-opted for the auditory processing of
song. Specifically RA-projecting neurons could act via local
interneurons to drive feedforward inhibition onto X-projecting cells
both during singing and passive listening. In the singing state, vocal
premotor-coupled inhibition may define a time window for auditory
feedback, suppressing auditory responses when they fail to coincide
closely with the vocal efference. During passive listening,
auditory-evoked activity in RA-projecting neurons may again drive
feedforward inhibition onto X-projecting neurons, shaping but not
suppressing their responses. This model predicts that NIf neurons,
which are likely to provide auditory and premotor drive to HVc,
innervate RA-projecting neurons, which in turn excite interneurons
providing segregated input to X-projecting neurons.
Dual auditory representations of song emerge from HVc, potentially
facilitating comparisons important to song learning and maintenance.
The subthreshold events underlying BOS-evoked firing in the two relay
cell types enable them to transmit different auditory representations
of song, at least under certain conditions. When sufficiently
depolarized, RA-projecting neurons fire continuously to forward BOS,
and even to reverse BOS, normally a weak stimulus for HVc neurons
(Margoliash, 1986; Lewicki and Arthur, 1996; Volman, 1996; Theunissen
and Doupe, 1998). Therefore, the HVc-to-RA pathway may be able to
convey information about song quality even when notes are distorted or
sung out of sequence, as occurs in juveniles during song learning and
in adults of those species that seasonally reexpress vocal plasticity.
In contrast, inhibitory interactions may restrict X-projecting neuronal
firing to certain times during the song, perhaps after distinct note
sequences. Coincident activation of the two pathways, perhaps when
vocalizations include these specific sequences, could then reinforce
motor patterns in HVc and/or in RA, the direct and indirect target,
respectively, of RA- and X-projecting cells. Furthermore X- and not
RA-projecting neurons are likely sites for auditory refinement; varying
the membrane potential of single X-projecting neurons altered their firing-rate selectivity, and as a whole these neurons displayed a
marked increase in firing rate over subthreshold depolarizing selectivity. Because song selectivity arises via auditory experience (Volman, 1993), synapses onto X-projecting neurons may be especially sensitive to this experience. Future work can gauge the functional significance of multiple song representations, how HVc's local circuits refine song selectivity relative to HVc's extrinsic auditory afferents, and the sources of these extrinsic afferents.
| |
FOOTNOTES |
Received Feb. 7, 2000; revised April 27, 2000; accepted May 5, 2000.
This work was supported by National Institutes of Health Grant R01 DC
02524 and by grants from the McKnight, Sloan, and Klingenstein Foundations. I am grateful for helpful discussions and comments on this
manuscript provided by Dr. David Fitzpatrick, Dr. John Spiro, Merri
Rosen, and other members of the Mooney lab, as well as two anonymous
reviewers. Special thanks to Stacey James for expert histology, Jim
Adelman for data analysis, and Merri Rosen for expert computer programming.
Correspondence should be addressed to Dr. Richard Mooney, Department of
Neurobiology, Box 3209, Duke University Medical Center, Durham, NC
27710. E-mail: mooney{at}neuro.duke.edu.
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