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The Journal of Neuroscience, February 1, 2002, 22(3):946-958
The Role of Auditory Experience in the Formation of Neural
Circuits Underlying Vocal Learning in Zebra Finches
Soumya
Iyengar and
Sarah W.
Bottjer
Department of Biology, University of Southern California, Los
Angeles, California 90089-2520
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ABSTRACT |
The initial establishment of topographic mapping within developing
neural circuits is thought to be shaped by innate mechanisms and is
primarily independent of experience. Additional refinement within
topographic maps leads to precise matching between presynaptic and
postsynaptic neurons and is thought to depend on experiential factors
during specific sensitive periods in the animal's development. In male
zebra finches, axonal projections of the cortical lateral magnocellular
nucleus of the anterior neostriatum (lMAN) are critically important for vocal learning. Overall patterns of topographic organization in the majority of these circuits are adult-like throughout the sensitive period for vocal learning and remain stable
despite large-scale functional and morphological changes. However,
topographic organization within the projection from the core subregion
of lMAN (lMANcore) to the motor cortical robust nucleus of
the archistriatum (RA) is lacking at the onset of song development and emerges during the early stages of vocal learning. To
study the effects of song-related experience on patterns of axonal
connectivity within different song-control circuits, we disrupted song
learning by deafening juvenile zebra finches or exposing them to loud
white noise throughout the sensitive period for song learning.
Depriving juvenile birds of normal auditory experience delayed the
emergence of topographic specificity within the
lMANcore
RA circuit relative to age-matched controls,
whereas topographic organization within all other projections to and
from lMAN was not affected. The projection from lMANcore to
RA therefore provides an unusual example of experience-dependent
modification of large-scale patterns of brain circuitry, in the sense
that auditory deprivation influences the development of overall
topographic organization in this pathway.
Key words:
topographic organization; sensory experience; sensitive
periods; zebra finch; songbird; vocal learning.
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INTRODUCTION |
An animal's interactions with its
external world during sensitive periods of development have a profound
and lasting influence on its behavioral capacities. For example,
songbirds learn their species-typical vocal pattern after hearing it
during a restricted period of development. Although the role of sensory
experience in complex behavioral and perceptual functions is well
established, the mechanisms whereby experiences shape neural circuits
are not well understood. The development of highly precise topographic circuits has generally been thought to involve two broad classes of
mechanisms: an activity-independent phase in which the expression of
various molecules guides the initial patterning of developing axonal
connections, followed by an activity-dependent phase in which
experientially driven patterns of synaptic activity act to refine
topographic maps (Cline, 1991
; Goodman and Shatz, 1993; Roskies et al.,
1995; Tessier-Lavigne and Goodman, 1996). The initial level of
precision in the projections of presynaptic neurons onto their
postsynaptic targets during development, before any contribution from
sensory experience, is usually extremely high (Catalano et al., 1991;
Agmon et al., 1993, 1995; Crair et al., 1998; Crowley and Katz,
2000; but see Simon and O'Leary, 1992), and abnormal experience
seems to have little or no influence on the development of nascent
topographic order (O'Leary and Cowan, 1983; Udin, 1985; Weliky and
Katz, 1997; Crair et al., 1998; Crowley and Katz, 1999). This pattern
of results indicates that the initial specificity of neural circuitry
relies primarily on innate mechanisms, and that such mechanisms are
able to confer a remarkable degree of precision. In fact, although the
exact level of refinement induced by experiential activity is generally
not known, it is clear that experience may account for a rather small
increment in the grain of any particular neural map (Crair et al.,
1998). Of course, it should be stressed that even if only the final
5-10% refinement of a neural circuit were attributable to
experientially induced activity, it could nevertheless be true that
complex behavioral capabilities may rely primarily on such final levels
of refinement.
Tract-tracing studies have shown that a subset of the neural circuits
underlying song learning in zebra finches are topographically organized
(Johnson et al., 1995; Nixdorf-Bergweiler et al., 1995; Vates and
Nottebohm, 1995; Iyengar et al., 1999; Bottjer et al., 2000). That is,
broad patterns of axonal connectivity between subregions of
vocal-control nuclei are systematically organized in adult birds. We
have reported previously that topographic patterns of connectivity in
most vocal-control circuits are similar in juvenile male zebra finches
during the sensitive period for song learning and in adult birds that
have finished learning to produce a stereotyped song pattern. However,
one vocal-control pathway provides a salient exception to the general
rule that initial circuits have a high degree of precision: the axonal
projection from the cortical lateral magnocellular nucleus of the
anterior neostriatum (lMAN)core to the
motor-cortical robust nucleus of the archistriatum (RA) lacks
the level of topographic specificity seen in other song-control
projections during early vocal development (Iyengar et al., 1999; cf.
Simon and O'Leary, 1990). The refinement of this projection to match
the topographic organization seen in adult birds occurs during early
stages of vocal learning (20-35 d), when birds are learning about the
auditory and motor characteristics of their vocalizations and the
lMANcoreRA projection is necessary for the
production of normal "subsong" behavior (Iyengar et al., 1999; cf.
Marler, 1991; Margoliash, 1997; Nordeen and Nordeen, 1997). The purpose
of the present study was to begin to assess the role of experience in
topographic mapping of axonal connections to and from lMAN. We found
that depriving juvenile birds of normal auditory experience did not
influence the maintenance of topographic organization in those
song-control circuits that already matched the adult pattern, as has
been found in other systems. However, disrupting normal auditory
experience did delay the emergence of topographic specificity within
the lMANcoreRA projection. This result
provides a rare example in which abnormal experience influences the
development of initial, large-scale topographic organization in a
developing neural circuit.
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MATERIALS AND METHODS |
All birds used in this study were bred in our aviaries. The
surgical procedures used were in accordance with National Institutes of
Health guidelines and the Animal Care and Use Committee at the
University of Southern California.
Deafening. Juvenile male zebra finches (n = 10 at 12 d after hatching; n = 6 at 20 d
after hatching) were anesthetized with 0.04-0.06 ml of the barbiturate
anesthetic Equithesin. An incision was made in the skin over each ear
and the tympanic membrane covering the middle ear cavity was exposed.
The tympanic membrane as well as the columella overlying the oval
window were removed, and a pair of fine forceps was used to remove the
cochlea. Small plugs of sterile Gelfoam were placed into the inner ear
cavity and the incision was closed with Collodion. Birds were returned
to their parents in group breeding aviaries after they recovered from
surgery. Of the 10 birds deafened at 12 d after hatching, 4 were
allowed to survive until 35 d of age. The remaining 12 deafened (DF) birds were removed from the breeding aviaries and placed
in separate cages in auditory and visual contact with other adult zebra
finches when they were ~40 d of age and allowed to survive until adulthood.
White noise exposure. Previous work has shown that exposure
to white noise (WN) at high sound pressure levels (SPLs) (112-120 dB)
causes little or no damage to hair cells in zebra finches (Ryals et
al., 1999). Preliminary results from our lab have shown that white
noise played continuously for long periods of time (3 months) at
~100-115 dB disrupts normal song behavior in adult zebra finches
without damaging hair cells (Zevin et al., 2000). Therefore, we decided
to test the effects of chronic exposure to white noise on the
topographic organization of circuits to and from lMAN as a means of
disrupting normal auditory experience without directly damaging
auditory pathways (cf. Marler et al., 1973; Marler and Waser, 1977).
Eleven clutches of juvenile birds (12-22 d of age, including both
males and females) were housed with their parents in individual cages
placed in sound-attenuating chambers fitted with speakers. To mask
normal auditory input to these birds, a Quan-Tech 420 noise generator
(Tucker Electronics, Garland, TX) was used to produce white noise that
was amplified to either 100 or 116 dB through a NAD 902 multichannel
power amplifier and played continuously. One group of juvenile
male birds (n = 9 at 100 dB; n = 3 at
116 dB) was raised in white noise from 15 d until 35 d after
hatching, and a second group of juvenile male birds (n = 5 at 100 dB; n = 3 at 116 dB) was continuously
exposed to white noise from 15 d until they reached adulthood
(>90 d of age). Parents and female siblings of the second group of
birds were removed from the white-noise boxes when the juvenile male birds were 50 d of age.
Noise levels within the white-noise chambers were monitored daily with
a sound-level meter (Realistic; Radio Shack, Forth Worth, TX) to
ensure that they did not fluctuate more than ±3 dB. Speakers were
mounted on the upper part of one side of each chamber, just above the
level of the cages in which birds were housed. Sound levels were
measured just opposite the speakers, and also at the center and the
base of the cages, where birds spent the majority of their time. When
white noise at a 100 dB SPL was played through the speaker, SPLs of 94 dB and 88 dB were recorded at the center and at the base of the
chamber, respectively. Increasing the SPL of the white noise to 116 dB
at the speakers gave rise to SPLs of 100 dB at the center and 97 dB at
the base of the boxes, respectively.
Song recordings. The songs of birds from both experimental
groups that survived until adulthood were recorded when birds were >90
d of age (n = 12 DF; n = 8 WN).
The songs of six control adult birds that had experienced normal
patterns of auditory input throughout the period of song learning were
recorded, as were the songs of the fathers of all WN birds before white
noise exposure. We attempted to record the songs of all 35 d birds
(control, DF, and WN), but most birds did not sing during the time we
attempted to record their behavior (i.e., we were unsuccessful in most
cases). Furthermore, the temporal order and structure of syllables in
the vocalizations of normal 35 d birds are highly variable, making
it difficult to compare them with the songs of experimental 35 d
birds. Therefore, we did not attempt to analyze the songs of 35 d
DF and WN birds. Songs of adult birds were recorded using a TEAC
X-300 tape recorder (TEAC, Montebello, CA) and analyzed using a Kay
Elemetrics DS 5500 Sona-Graph (Pinebrook, NJ), or were recorded
digitally using a voice-activated system with an eight channel ARC88
sound card (SEK'D, Bretzfeld-Schwabbach, Germany) and software from
Avisoft (SASlab, Berlin, Germany). To ensure that WN birds did not have access to a song model, they were placed in the recording room individually and their songs were recorded for a period of 3-4 hr
every day until they sang for a total of at least 10 min over the
course of a week. At the end of each recording session, they were
returned to the white-noise boxes. A total of 10-15 song bouts from
each bird were selected randomly and printed as plots of frequency over
time (sonograms); these plots were used to analyze the sequence
of syllables and syllable morphology (see below). Songs of birds that
were recorded on tape were later transcribed into digital files for
additional analysis using Avisoft.
Song analysis. Zebra finch song is produced as discrete
syllables, which appear as continuous tracings on sonograms and are separated from other syllables by 5-10 msec gaps of silence (Zann, 1996). A group of distinct syllables sung in a highly stereotyped order
by an adult male zebra finch constitutes a song phrase or motif. Song
phrases may be repeated several times, which constitutes a bout (Price,
1979; Sossinka and Böhner, 1980; Cynx, 1990). Song bouts are
initiated by a variable number of short, simple introductory notes,
whereas individual song syllables are generally of longer duration and
more complex. Different song syllables contain elements that may be
noisy, highly frequency modulated, or consist of harmonic stacks
(Price, 1979; Zann, 1996). Although the syllables in an individual
bird's songs are repeated in a highly stereotyped manner, birds
occasionally produce motif variants in which some syllables are dropped
from their motifs (Zann, 1990; Nordeen and Nordeen, 1992).
Most birds sang readily in the absence of other zebra finches. Such
"undirected" songs are slower in tempo and consist of a smaller
number of motifs per bout of song compared with songs directed toward
other birds (Morris, 1954; Hall, 1962; Sossinka and Böhner,
1980). However, the structure of individual syllables and the sequence
of syllables remain the same in both directed and undirected songs
(Zann, 1996). Therefore, for three of five 100 dB WN adult birds that
did not sing for several recording sessions, songs of these males
directed toward a female were recorded.
To quantify the degree of stereotypy in the song phrases of control and
experimental birds, we used a computer program (The Songinator:
http://siva.usc.edu/~jdzevin/song help.html, by J. D. Zevin,
1999) to calculate measures of linearity, consistency, and stereotypy
based on those published by Scharff and Nottebohm (1991). Our measure
of whether syllables were produced in a specific linear order in a
bird's song behavior was modified from the sequence linearity score of
Scharff and Nottebohm (1991) and was calculated as follows:
linearity = (number of different syllables 
1)/number of
syllable transitions.
In their calculation of this measure, Scharff and Nottebohm used all
syllables produced by a bird as the numerator and all transitions after
each syllable as the denominator (including the transition between the
last syllable and the ending of the song phrase). However, we included
only transitions between song syllables (i.e., we did not include the
transition between the last syllable and the end of the song motif, and
therefore we used one less than the total number of syllables in the
numerator). We calculated this "internal" linearity score to
account for the following: normal birds occasionally end their songs at
different syllables, although the songs are highly linear. For example, the occurrence of each syllable in two phrases of a song ABCD#ABC# (where "#" signifies the end of a song phrase) can be accurately predicted, given the preceding syllable. Using Scharff and Nottebohm's measure of linearity, this sequence would receive a linearity score <1
(i.e., 4/5 = 0.8), although it is perfectly linear by our measure
(i.e., 3/3 = 1.0). (For additional details, see
http://siva.usc.edu/~jdzevin/song_help.html.)
A consistency score (Scharff and Nottebohm, 1991) was calculated to
measure how often specific variations of the song phrase were sung by
each bird (e.g., a bird that produces ABCD#ABC#, as above, has a linear
song, but not a consistent song). The consistency with which each bird
produced a specific sequence of syllables was calculated in the same
way as described by Scharff and Nottebohm using the following equation:
consistency = 
[T(d)/T(a)]/N. In this
calculation, the dominant (most frequent) transition for each syllable
[T(d)] was divided by all transitions for that syllable, designated T(a). The sum of
[T(d)/T(a)] for all syllables in each bird's
songs was divided by N (the total number of syllables in that bird's songs). In this measure, we counted end-stops (#) as
notes, so as to capture variability at both the beginnings and ends of
songs. A completely stereotyped song bout with four syllables in each
motif (e.g., #ABCD#ABCD#ABCD#) has five dominant transitions (AB, BC,
CD, D#, and #A), and all transitions for each syllable fall into one of
these dominant transition types (i.e., each
T(d)/T(a) equals 1), yielding an overall
consistency score of 1.0. Linearity and consistency scores for each
bird were averaged to determine an overall stereotypy score:
stereotypy = (linearity + consistency)/2.
The total number of syllables produced by each bird was also counted
and compared with those in the songs of controls to test whether
auditory deprivation produced impoverished vocal repertoires. The
stereotypy of individual syllable morphology for each bird was analyzed
by visually examining different syllables in printed sonograms and
comparing those sung in different bouts.
Dye injections into lMAN. The procedures used to make dye
injections into different subregions of lMAN as well as qualitative and
quantitative methods for analyzing the resulting anterograde and
retrograde label from these injections have been described in detail
previously (Iyengar et al., 1999). Briefly, lMAN consists of a
magnocellular core of neurons as well as a surrounding shell primarily
composed of parvocellular neurons (lMANcore and
lMANshell, respectively) (cf. Johnson et al.,
1995; Bottjer et al., 2000). All birds were anesthetized with
Equithesin and placed in a stereotaxic apparatus (see Table
1 for summary of dye injections).
Approximately 2-5 nl of the fluorescent tracers rhodamine dextran
amine (RDA; 10% solution in 0.02 M PBS)
or fluorescein dextran amine (FDA; 20% solution in 0.02 M phosphate buffered saline) (Molecular Probes, Eugene, OR) was targeted to lMANcore or
lMANshell using a Picospritzer. Dye
injections that entered both core and shell regions were included with
injections that were restricted to only lMANcore
or lMANshell for analysis in this study, because
retrograde and anterograde label resulting from these injections was
comparable in normal male zebra finches (Johnson et al., 1995; Iyengar
et al., 1999). Birds exposed to chronic white noise were removed from
their sound chambers just before dye injections and were returned to
the white-noise boxes immediately after they recovered from surgery.
After a survival time of 3 d after surgery to allow for axonal
transport of the dyes, birds were deeply anesthetized and perfused
transcardially with 0.7% saline followed by 10% buffered formalin.
Brains were removed and post-fixed in 10% buffered formalin for 5-7 d
and then cryoprotected in 25% sucrose overnight. Brains were sectioned coronally at a thickness of 50 µm, and two alternate series of sections were collected on gelatin-coated slides. One series was air-dried after sectioning, coverslipped with buffered glycerol, and
stored at 4°C. The second series was allowed to dry overnight, Nissl-stained with thionine, and coverslipped with Permount.
Analysis of topography. Injection sites within
lMANcore and the resulting retrograde label in
the medial dorsolateral nucleus of the thalamus (DLM) and anterograde
label in RA and area X were photographed using an epifluorescence
microscope fitted with rhodamine and fluorescein filters (Chroma
Technology, Brattleboro, VT) for RDA and FDA, respectively. The
location of RDA and FDA injection sites within lMAN as well as the
retrograde label within DLM produced by these injections were traced
onto Nissl-defined outlines of these nuclei obtained from camera lucida
tracings of the thionine-stained series of sections. (There was no
differential shrinkage of the tissue in fluorescent vs Nissl-stained
sections.) Whereas RDA produced intense anterograde labeling of axons
and terminal arborizations, the anterograde fluorescent label produced
by injections of FDA into lMAN was faint and difficult to photograph.
Therefore the pattern of anterograde label described in this study is
derived primarily from RDA injections.
We quantified the volume of anterogradely labeled
lMANcore arbors within both RA and area X using
methods published previously (Iyengar et al., 1999). Briefly, an image
analyzer was used to capture images of anterograde label within RA and
area X and injection sites within lMANcore. Only
those injection sites that produced well-defined, intensely labeled
terminal fields within RA and area X were selected for quantitative
analysis. Images of the Nissl-defined profiles of RA and area X from
thionine-stained alternate sections were also collected. Ten injections
in adult DF and WN birds and 17 injections in 35 d DF and WN birds
were used to analyze anterograde label in RA, whereas 9 injections in
adult DF and WN birds and 16 injections in 35 d DF and WN birds were used to analyze anterograde label over area X. Image Pro Plus
software from Media Cybernetics (Silver Spring, MD) was used to
manually outline the area of anterograde label within both nuclei as
well as the injection sites within lMANcore on
each section in which they appeared. The investigator was not aware of
the experimental treatment for each subject during tracing. Criteria
for tracing included well-labeled individual processes that formed a
terminal field of highly branched, extremely fine processes with
numerous varicosities; areas that included only unbranched axons (i.e.,
axons of passage within the nucleus) were not included. The volume of
anterograde label within RA and area X and the volume of the dye
injections within lMANcore were estimated by
adding these areas and multiplying the sum by the sampling interval
(100 µm). The total volumes of RA and area X were measured in
Nissl-stained sections in the same manner. The percentage of each
nucleus occupied by anterogradely labeled axons from
lMANcore was then calculated by dividing the
volume of labeled axonal arbors within each nucleus by the total volume
of the respective nucleus (RA or area X) in each bird. Quantitative
data obtained from juvenile and adult DF and WN birds in this study
were compared with previously published data from normal 20 d,
35 d, and adult birds (Iyengar et al., 1999) (see Results and
Tables 3 and 4).
The average size of injection sites in lMAN was roughly comparable
across different experimental groups. However, despite the fact that
larger injections tended to produce a greater amount of terminal label
(and vice versa), there was not a strong correlation between the size
of an injection site and its terminal field in RA or area X, as is
typical of any tract-tracing study (Simon and O'Leary, 1992; Feldman
and Knudsen, 1997; Iyengar et al., 1999; Scharff et al., 2000). This
lack of tight correspondence between the volume of the injection site
and the volume of anterograde label could stem not only from
differences in the amount of dye contained within each injection site
but also from the amount of dye actually incorporated and anterogradely
transported by lMAN neurons, which cannot be quantified. Furthermore,
the volume of the terminal field in each nucleus includes variations in
intensity of anterograde label. Because we wished to quantify the total proportion of RA and area X that received input from lMAN neurons, we
included all levels of anterograde label in our quantitative assessment, regardless of variations in the density of fine labeled processes. This source of variability also contributes to the lack of a
systematic relationship between the volume of the injection site and
that of the terminal field. The important point to note, however, is
that our previous work carefully documented that injections of similar
size produce a substantially greater proportion of anterograde label in
RA of 20 d birds compared with older birds, regardless of location
within lMAN (Iyengar et al., 1999). Thus, despite variability in the
volume of anterograde label after comparable injections, the methods
used here (and in our previous study) are clearly adequate to reveal
changes in overall patterns of topography as a function of age or experience.
Another important factor that could affect our measurements of
anterograde label within RA is the density of
lMANcore projection neurons in different groups
of birds in our study. Specifically, neuronal density within
lMANcore must be comparable between DF and WN
birds as well as between age-matched normal birds, so that injections
of similar volume can encompass comparable numbers of neurons in
different birds. Previous studies have reported that the total number
of lMANcore projection neurons as well as neuronal density within lMAN remains constant throughout song learning
(Bottjer et al., 1985; E. J. Nordeen and Nordeen, 1988; Burek et
al., 1991). In addition, Burek et al. (1991) showed that early
deafening does not affect neuronal density within lMAN in juvenile or
adult birds. Therefore, injection sites of comparable volume should
include similar numbers of lMANcore neurons in
hearing-deprived and normal juvenile and adult birds (Iyengar et al.,
1999).
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RESULTS |
Analysis of song in adult DF and WN birds
Controls
The songs of all normal adult males (controls and the fathers of
WN adult males) comprised four to seven syllables (Table 2), which were produced in a highly
stereotyped sequence with few motif variants. This stable song behavior
was reflected in high scores for linearity and consistency, which were
averaged to yield high overall stereotypy scores (Table 2). In
addition, individual syllables were easily identified in different
renditions of the songs sung by these birds and did not vary in
structure across different motifs (Fig.
1C).
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Figure 1.
Sonograms comparing songs of adult birds deprived
of normal auditory input during the sensitive period for song learning
and an adult control. Letters above sonograms denote
different syllables in the songs of the WN bird and his father.
A, The song of an adult bird deafened at 12 d was
composed of noisy syllables repeated several times in succession, with
little frequency modulation and poorly resolved harmonics as well as
abnormal call-like notes (C'). B, The
adult WN (116 dB) bird produced motifs consisting of a variable number
of simple introductory note-like syllables (A),
followed by one rendition of a song-like syllable
(B) and a variable number of call-like notes
(syllable C). C, His father's song
(B) is highly stereotyped and consists of
syllables A-G. Calibration, 200 msec.
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Deafened birds
Adult male zebra finches that had been deafened by bilateral
cochlear removal at 12 or 20 d produced highly disrupted songs compared with normal controls (Konishi, 1965; Nottebohm, 1968; Price
1979; Scharff and Nottebohm, 1991) (Fig. 1). Syllables in the songs of
DF birds were not counted because they were produced in such a variable
manner that it was impossible to consistently identify them across
different renditions. Compared with syllables in control adult birds'
songs, the length and structure of individual syllables produced by
deafened birds were highly variable with poorly resolved harmonics and
little or no frequency modulation, and highly abnormal call-like notes
were incorporated at the end of song phrases in some of these birds. In
addition, the songs of DF birds lacked a stable song phrase, such that
the temporal sequence of syllables tended to be variable across
different iterations of song phrases. An example of song from an adult
bird that was deafened at 12 d is shown in Figure
1A. This song comprised a string of syllables that
resembled introductory notes followed by three to five renditions of a
noisy amorphous syllable that seemed to represent variable iterations
of the same syllable (C').
White noise birds
WN birds produced abnormal songs as adults consisting of simple
syllables that resembled introductory- and call-like notes in normal
songs (Fig. 1). Careful visual inspection of sonograms indicated that
the structure of individual syllables in adult songs of WN birds was
stable across different renditions and was similar to comparable
syllables of control adults (unlike the noisy, poorly structured
syllables produced by DF birds). However, songs of WN birds included
significantly fewer syllables than did songs of adult controls (Table
2) (F(1,12) = 6.88; p = 0.022). In addition, the song phrases of WN birds were less stable
than those of controls, resulting in lower scores for linearity and consistency. Although the behavior of birds exposed to 100 dB of white
noise tended to be slightly less disrupted than those exposed to 116 dB, there were no significant differences between the two groups (data
not shown). Behavioral data from birds raised in white noise at 100 and
116 dB were therefore pooled and compared as a group against adult
controls. Adult WN birds received significantly lower scores for
linearity (F(1,12) = 7.40;
p = 0.02), consistency (F(1,12) = 5.99; p = 0.03), and stereotypy (F(1,12) = 7.61;
p = 0.02). These lowered scores were attributable to
the tendency of WN birds to repeat one or two syllables a variable
number of times within a motif, which is not typical of normal song. In addition, WN birds produced different temporal sequences of notes on
occasion and tended to include a variable number of long calls in their
songs that were not seen in songs of control adults.
As an example, the song of an adult bird reared in white noise and his
father's song (recorded before the father was placed in white noise)
are shown in Figure 1, B and C, for comparison. The WN-reared bird produced a very simple adult song consisting of
syllable A, which was repeated a variable number of times within different motifs and resembled introductory notes in normal songs. It
was frequently followed by one iteration of a song-like syllable (B)
and a variable number of call-like notes (C). On other occasions, this
bird produced song phrases consisting of syllable A only or syllable C
only, and in some song phrases included a note D after A (data not
shown). His song phrases were variable in terms of both length (i.e.,
number of notes) and sequence of notes; thus he lacked a stable song
phrase. His father's song consisted of syllables A through G, which
were sung in a very stereotyped manner. Syllables A and B of the WN
bird are somewhat similar to syllables A and B in his father's song,
respectively. In addition, the call-like note (C) in the WN adult
bird's song resembles syllable E in his father's song. However, the
WN-reared bird's song is highly variable and impoverished compared
with his father's song. In summary, birds exposed to loud white noise
during the sensitive period for song learning produce highly abnormal
song behavior consisting of few, simple syllables produced in a
variable manner when they reach adulthood.
Topographic organization within different circuits to and
from lMAN
Injections into lMANcore of all birds
produced ipsilateral retrograde label in an oval region corresponding
to the dorsolateral part of DLM (DLMDL) and
anterograde label within ipsilateral RA and area X (Figs. 3, 4).
Injections into lMANshell produced ipsilateral retrograde label in a crescent-shaped region corresponding to ventromedial DLM and anterograde label in the ipsilateral dorsal archistriatum (Ad), parolfactory lobe, and dorsolateral caudal neostriatum (a cortical region dorsal and lateral to RA and Ad) in all groups of birds studied (cf. Bottjer et al., 2000). Careful qualitative examination revealed no differences in the topography of
axonal connections to and from lMANshell or
within the DLMDLlMANcore circuit in any of the experimental groups in this study compared with
normal 35 d and adult birds from a previous study (cf. Johnson et
al., 1995; Iyengar et al., 1999). Therefore, we describe patterns of
anterograde label from lMANcore to RA and area X
only (Figs. 3, 4).
Topographic organization within the
lMANcoreRA circuit
The lMANcoreRA circuit in normal adult
male zebra finches has a topographic organization such that
ventromedial, ventrolateral, and dorsal regions of RA receive
projections from lateral, intermediate, and medial subregions of
lMANcore, respectively (Johnson et al., 1995). In
addition, injections into intermediate-lateral
lMANcore label medial and central parts of RA,
whereas injections into intermediate-medial
lMANcore label dorsal RA as well as small regions
extending along the medial and lateral borders adjacent to this dorsal
"cap" (Iyengar et al., 1999) (Fig.
2). However, our previous work has shown
that this topographic organization is lacking at the onset of song
learning (20 d), and becomes organized to match the adult pattern by
35 d after hatching, during the early part of song learning and
auditory-motor integration (see control data, Table
3). In addition, the location of
anterograde label within RA produced by dye injections into different
subregions of lMANcore at 20 d is not well
matched to the adult pattern, whereas the location of anterograde label
in RA of 35 d birds does match that seen in adult birds after
injections within corresponding subregions of
lMANcore (Iyengar et al., 1999).
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Figure 2.
Schematic depicting the overall topographic map of
the axonal connections of right lMANcore onto RA in a male
zebra finch (see Results) (cf. Johnson et al., 1995; Iyengar et al.,
1999). D, Dorsal; I, intermediate;
L, lateral; M, medial;
I+M, intermediate + medial; L+I, lateral + intermediate; M + C, medial + central;
VL, ventrolateral; VM, ventromedial;
D ± L ± M, dorsal ± lateral border ± medial border.
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Table 3.
Summary data for quantitative analysis of label over RA
after injections into lMANcore in control and experimental
birds
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Qualitative analysis
Qualitative examination of anterograde label in all adult birds
that had not experienced normal patterns of auditory input during
development revealed that topographic mapping within the lMANcoreRA projection was similar to that in
normal adult birds. Twenty-one of 21 injections into different
subregions of lMANcore in DF and WN adult birds
produced anterograde label that was similar to controls in terms of
both location within RA and size of terminal field (Table 1; see below
for quantitative analysis). For example, anterograde label stemming
from an injection into dorsal-intermediate lMANcore in an adult DF bird was restricted to
the ventrolateral part of RA (Fig. 3A). This pattern of
label is comparable with that seen in control adult and 35 d
birds, in which dye injections anywhere in the intermediate part of
lMANcore produce anterograde label within the
ventrolateral RA (Fig. 2) (cf. Iyengar et al., 1999).
In contrast, patterns of axonal connectivity between
lMANcore and RA in DF and WN birds at 35 d
were substantially less refined than those in normal 35 d or adult
birds. The majority of injections into lMANcore
of 35 d DF and WN birds (17 of 19 injections; Table 1) produced
label that encompassed a much greater proportion of RA compared with
that seen after comparable injections in normal 35 d birds. In
addition, labeled lMANcore axons in 35 d DF
and WN birds were not consistently localized within the target regions of RA that they will ultimately innervate (11 of 19 injections). For
example, an injection into the intermediate part of
lMANcore (extending across the dorsoventral axis)
in a 35 d DF bird produced anterograde label over the ventral
two-thirds of RA. Most of the anterograde label was concentrated in the
central part and along the ventral-intermediate border of RA, but
sparser label was also clearly visible along its ventromedial and
ventrolateral borders (Fig. 3B). The dorsal cap, a region
that is occupied by axon arbors from the medial subregion of
lMANcore in normal adult and 35 d birds, was
the only part of RA devoid of label in this bird. In 35 d and
adult control birds, injections into intermediate
lMANcore label axons only within ventrolateral RA
(i.e., as in Fig. 3A). Thus, not only do the terminal
fields of neurons from subregions within lMANcore
occupy a greater proportion of RA in 35 d deaf birds than in
hearing birds, but also the location of anterograde label within RA
indicates that spatial patterns of axonal connectivity within this
circuit do not match those seen in 35 d and adult controls.
The injections shown for adult and 35 d DF birds in Figure 3 are
matched for medial-lateral location within
lMANcore, although the injection in the 35 d
DF bird extended across the dorsoventral axis, whereas the injection in
the adult DF bird occupied the dorsal part of intermediate
lMANcore. However, it is appropriate to compare
the pattern of anterograde label in RA in these birds because the total
volume of these injections was comparable (0.010 mm3 and 0.012 mm3 in the 35 d and adult DF bird,
respectively) and because our previous work demonstrated that the
dorsoventral extent of injections into lMANcore
does not affect the extent or location of anterograde label in RA
(Iyengar et al., 1999). Comparison of these two cases therefore
indicates that comparable injections produce a much larger terminal
field in RA of 35 d DF birds than that of adult DF birds, such
that a much greater proportion of RA is occupied in juvenile deafened birds.
A similar lack of topographic specificity was seen in both DF and WN
birds at 35 d of age. For example, labeled axons arborized throughout ventromedial and ventrolateral subregions of RA in a 35 d WN bird after an injection centered in the intermediate-lateral subregion of dorsal lMANcore (Fig.
3C). The only part of RA that was not occupied by
anterograde label was the dorsal cap, although anterograde label over
RA in this bird was more extensive than in the 35 d DF bird (Fig.
3B). Similar injections into intermediate-lateral lMANcore in normal 35 d and adult birds
label a restricted region within medial and central RA (Fig. 2)
(Iyengar et al., 1999). However, this WN bird did provide an example of
the fact that initial stages of refinement in topographic organization
within the lMANcoreRA circuit were evident in
some 35 d DF and WN birds. Although the ventral two-thirds of RA
was covered by anterograde label in this 35 d WN bird, label was
most intense within medial and central parts of RA.
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Figure 3.
Photomicrographs of anterograde label over coronal
sections of right RA resulting from injections of RDA into right
lMANcore in an adult DF bird, a 35 d DF bird, and a
35 d WN bird. Nissl-defined outlines of RA are depicted by
dashed lines, and injection sites within
lMANcore for each bird are shown in schematics; lateral,
right; dorsal, superior. A, An injection within
dorsal-intermediate lMANcore produced a terminal field
that was restricted to the ventrolateral part of RA in an adult DF
bird. Small amounts of label in medial and dorsal RA represent single
axons that looped through these loci before arborizing within
ventrolateral RA. Similar injections in normal adult controls also
labeled single axons that looped through topographically
"inappropriate" areas before arborizing in the correct location
(cf. Simon and O'Leary, 1992; Iyengar et al., 1999). B,
An injection within the intermediate part of lMANcore that
extended across its dorsoventral axis in a 35 d DF bird produced
anterograde label encompassing the central, ventrolateral, and
ventromedial parts of RA. The dorsal cap region was the only part of RA
that was devoid of label in this bird. C, Anterograde
label extended throughout the ventromedial and ventrolateral subregions
of RA after an injection centered in the lateral-intermediate
subregion of dorsal lMANcore in a 35 d WN bird.
Although the lMANcore terminal field in RA in this bird was
more extensively labeled than in the 35 d DF bird, it did not
extend into the dorsal cap of RA. Scale bar, 200 µm.
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Quantitative analysis
We decided to confirm our qualitative observations by quantifying
the proportion of RA occupied by terminal label in 35 d and adult
hearing-deprived and normal birds. The volume of RA, the volume of
labeled lMANcore axons within RA, and the
proportion of RA occupied by the labeled lMANcore
terminal field were compared across different groups of birds. None of
these measures varied when DF and WN birds were compared with each
other at either 35 d or adulthood (all F values < 1). Therefore, we compared data from DF and WN birds together against
normative age-matched data from Iyengar et al. (1999).
The volume of RA in 35 d DF and WN birds was comparable with that
in normal 35 d birds (F < 1), whereas the volume
of the anterogradely labeled terminal field of
lMANcore neurons within RA was substantially
greater in 35 d DF and WN birds than in control 35 d
birds (Table 3, Fig. 5)
(F(1,20) = 6.44; p = 0.02). Because the overall size of RA was not different in birds with
and without normal hearing, whereas the labeled
lMANcore terminal field was greatly enlarged in
birds deprived of normal auditory experience, there was a dramatic
twofold increase in the proportion of RA occupied by
lMANcore axons in 35 d DF and WN birds
compared with controls (48% vs 22%)
(F(1,20) = 8.47; p = 0.009). The finding that RA volume is unaffected by the lack of normal
patterns of auditory input during the sensitive period for song
learning in zebra finches confirms previous work by Burek et al.
(1991). However, the fact that the projection of
lMANcore neurons to RA in deprived birds is
substantially larger in volume at 35 d compared with age-matched
controls shows that auditory experience regulates the development of
topographic organization in this pathway by influencing the growth of
axon arbors.
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Figure 5.
Comparison between changes in RA volume
(in cubic millimeters), the volume of the lMANcore
terminal field in RA after dye injections in lMANcore (in
cubic millimeters), and the proportion of RA occupied by labeled
lMANcore terminals across normal birds (- -) and
DF and WN birds (- -) during the course of song learning; average
values of means for each group are given in Table 4; error bars
represent SEMs. A, RA volume increases normally between
20 d and adulthood in birds deprived of normal auditory input (cf.
Burek et al., 1991). B, The overall volume of the
labeled lMANcore terminal field increases dramatically
between 20 and 35 d in DF and WN birds, in contrast to the slight
decrease in the volume of the lMANcore terminal field in
normal 35 d birds. By adulthood, the overall volume of the
lMANcore terminal field within RA in DF and WN birds is
comparable with that in normal adults, suggesting that refinement of overall topographic
organization within this circuit is delayed and occurs between 35 d and adulthood. C, Because the volume of the
lMANcore terminal field is much larger in deprived 35 d birds compared with controls, whereas RA volume is comparable between
the two groups, the proportion of RA occupied by anterogradely labeled
lMANcore terminals is significantly larger in 35 d DF
and WN birds compared with normal 35 d birds. By adulthood, the
proportion of RA occupied by the lMANcore terminal field is
comparable in normal or DF and WN birds, because RA volume as well as
the overall volume of the lMANcore terminal field within RA
are comparable in the two groups of birds.
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To study changes occurring within the
lMANcoreRA circuit between the onset of song
learning and 35 d in deprived birds, we compared the pattern of
results in 35 d DF and WN birds with that in normal 20 d
birds. The RA was much larger in volume in 35 d DF and WN birds
than in control 20 d birds
(F(1,28) = 17.4; p < 0.0001), reflecting the pronounced growth of RA that occurs during
early stages of song learning in both normal and deafened birds
(Bottjer et al., 1985; Burek et al., 1991) (Table 3, Fig. 5). The
volume of the terminal field was substantially greater in 35 d DF
and WN birds than in control 20 d birds
(F(1,28) = 4.30; p = 0.04), despite the fact that there is normally a slight decrease in the
volume of the terminal field of lMANcore neurons in RA between 20 d and 35 d in control male zebra finches.
Therefore, depriving juvenile birds of normal auditory input during
vocal learning causes small groups of lMANcore
axon arbors to occupy an expanded amount of postsynaptic territory
within the RA between 20 and 35 d. This increased volume of
anterograde label in 35 d DF and WN birds was somewhat offset by
the increase in overall volume of RA, such that the proportion of RA
occupied by labeled lMANcore axons was
significantly lower in 35 d DF and WN birds compared with
normal 20 d birds (48% vs 65%)
(F(1,28) = 6.67; p = 0.015). These results indicate that the terminal field of lMANcore neurons expands abnormally between 20 and 35 d in hearing-deprived birds, rather than regressing
slightly as in normal birds. Because RA volume increases in both
hearing and deprived birds, the proportion of RA labeled by core axons
does decrease somewhat between 20 and 35 d in deprived birds but
is still much higher in DF and WN birds compared with normal 35 d controls.
In contrast to 35 d DF and WN birds, the proportion of RA occupied
by the anterogradely labeled lMANcore terminal
field in adult DF and WN birds was not significantly different from
that in normal adult birds (35% vs 37%) (Table 3, Fig. 5)
(F < 1). There was no significant difference in the
volume of RA between adult DF and WN birds and normal adults
(F(1,17) = 1.50; p > 0.05) or in the volume of the terminal field of
lMANcore neurons in RA (F < 1).
These results confirm that depriving zebra finches of auditory input
during vocal learning does not prevent the emergence of overall
topographic organization within the lMANcoreRA
circuit by the time birds reach adulthood.
In male zebra finches, the total number of neurons in RA remains
constant throughout song learning. However, RA volume increases during
this period because of a decrease in the density of neurons within this
nucleus (Konishi and Akutagawa, 1985; Bottjer et al., 1986; Herrmann
and Bischof, 1986; E. J. Nordeen and Nordeen, 1988; K. W. Nordeen and Nordeen, 1988). Deafening birds during vocal learning does
not affect the total number of neurons within RA or the increase in
overall volume of this nucleus over the course of vocal learning (Burek
et al., 1991). Our results have confirmed that the increase in RA
volume during song learning in DF and WN birds is comparable with
age-matched controls. However, the lMANcore
terminal field undergoes a significant increase in size between 20 d and 35 d in DF and WN birds, in contrast to the slight decrease
in volume seen in control birds between 20 d and 35 d. Because the total number of projection neurons within
lMANcore remains the same throughout song
learning (Nordeen et al., 1992), any developmental changes in the
volume of the lMANcore terminal field within RA
presumably reflect either exuberant growth of individual axon arbors or
adjacent lMANcore neurons projecting to different
regions rather than highly similar regions within RA. Therefore, our
results suggest that remodeling of individual lMANcore axon arbors within RA between 20 d
and 35 d of development depends on the presence of normal patterns
of auditory input. By the time 35 d DF and WN birds reach
adulthood, the volume of RA increases to match adult controls and the
proportion of RA occupied by the lMANcore
terminal field is not significantly different from control adults (Fig.
5C).
In summary, both quantitative and qualitative analyses of the
lMANcoreRA circuit in DF and WN birds indicate
that preventing normal auditory input during the period of vocal
learning delays but does not prevent the emergence of topographic
organization within the lMANcoreRA circuit.
That is, topographic patterns of connectivity do not emerge in birds
deprived of normal auditory experience between 20 and 35 d, as
they do in normal birds, but do develop some time after 35 d of
age. Interestingly, the emergence of topographic organization was
delayed beyond 35 d within the lMANcoreRA
circuit in both DF and WN birds, although early deafening disrupted the
songs of zebra finches to a greater extent than exposure to chronic
white noise. We do not know whether the greater behavioral disruption
induced by deafening is attributable to lesion-induced changes in the
brain or to a more complete blockade of auditory input than is obtained
via white noise. However, it seems likely that spontaneous patterns of
activity in the lMANcoreRA pathway could be
similar in both DF and WN birds, because lMAN neurons respond best to
song-like sounds and not to pure tones or noise (for review, see Doupe
and Solis, 1997). Thus, patterned activity based on auditory experience
may be replaced by similar patterns of spontaneous activity in both DF
and WN birds.
Topographic organization within the lMANcorearea
X circuit
Mature topographic organization within the
lMANcorearea X circuit is already present in
normal males at the onset of vocal learning (20 d), unlike the
lMANcoreRA circuit, despite the fact that
single lMANcore axons bifurcate and project to
both RA and area X (Nixdorf-Bergweiler et al., 1995; Vates and
Nottebohm, 1995; Iyengar et al., 1999). The
lMANcorearea X circuit displays medial-to-lateral topography, such that neurons in the medial, intermediate, and lateral parts of lMANcore
project to corresponding medial, intermediate, and lateral subregions
of area X. In addition, neurons within the dorsal and ventral parts of
lMANcore project to dorsal and ventral subregions
of area X, respectively, in normal birds.
Qualitative analysis
We found that topographic organization of the
lMANcorearea X circuit in virtually all DF and
WN birds at adulthood (20 of 21 injections; Table 1) was similar to
that seen in age-matched normal birds. For example, an injection
centered in the ventral intermediate-lateral part of
lMANcore in an adult DF bird produced anterograde
label within the ventral intermediate- lateral part of area X (Fig.
4A). Labeled axons from
the injection site in lMANcore (Fig.
4A, arrows) traversed dorsal and
intermediate parts of area X before arborizing in ventral subregions
similar to those labeled in normal adult birds.
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Figure 4.
Injections of RDA into different subregions of
right lMANcore (shown in schematics) produced
anterograde label over specific subregions within coronal sections of
right area X (schematics and photomicrographs) in an adult DF bird and
a 35 d WN bird. Nissl-defined outlines of area X are depicted by
dashed lines; lateral, right; dorsal, superior.
A, An injection centered in ventral-intermediate and
ventrolateral lMANcore in an adult DF bird produced patches
of label within the ventral-intermediate and ventrolateral subregions
of area X. Labeled axons from the injection site in
lMANcore (arrows) traversed the dorsal and
intermediate parts of area X before arborizing exclusively in its
ventral subregion. B, Anterograde label within the
dorsal intermediate subregion of area X (X) was
produced by an injection into the dorsal-intermediate part of
lMANcore of a 35 d WN bird. LPO,
Parolfactory lobe. Scale bar, 200 µm.
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Dye injections into different subregions of
lMANcore in DF and WN birds at 35 d
demonstrated that topographic organization within the
lMANcorearea X circuit was similar to that
seen in normal 35 d and adult birds (18 of 19 injections; Table
1). For example, an injection into the dorsal-intermediate part
of the lMANcore in a 35 d WN bird produced
anterograde label concentrated within the dorsal intermediate subregion
of area X (Fig. 4B).
Quantitative analysis
Our qualitative observations revealed that topographic
organization within the lMANcoreX projection
is unaffected by altering auditory experience, and were also borne out
by quantitative analysis (Table 4). The
proportion of area X occupied by labeled terminals from
lMANcore neurons in adult DF and WN birds (8%)
was not substantially different from that in adult controls (16%)
(F(1,15) = 2.08; p > 0.05). Neither the absolute volume of area X (F < 1)
nor the volume of the lMANcore terminal field in
area X (F(1,15) = 1.45; p > 0.05) was significantly different between the two
groups.
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Table 4.
Summary data for quantitative analysis of label over area X
after injections into lMANcore in control and experimental
birds
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Our previous work showed that spatial patterns of axonal connectivity
within the lMANcoreX circuit were similar in
normal 20 d, 35 d, and adult birds, and quantitative analysis
confirmed that the proportion of area X occupied by the
lMANcore terminal field did not vary in
normal 20 d and adult birds (Table 4) (Iyengar et al., 1999).
Therefore, we did not quantify the proportion of area X occupied by
terminal label in normal 35 d birds (because this value was
unlikely to be significantly different at 35 d, based on the
overall pattern of both qualitative and quantitative results). In the
current study, we therefore compared the proportion of area X covered
by terminal label in 35 d DF and WN birds with that obtained from
normal 20 d and adult birds. Axonal arbors of
lMANcore neurons occupied 20% and 16% of area X
in normal 20 d birds and normal adults, respectively, and 9% in
35 d DF and WN birds (F(2,31) = 5.65; p = 0.05). The absolute volume of the lMANcore terminal field in area X was not
different between normal 20 d birds and 35 d WN and DF birds
(Table 4) (F(1,31) = 1.32; p > 0.05), but as expected, normal adult birds had a
larger volume of label than juvenile DF and WN birds
(F(1,31) = 6.93; p = 0.013). The total volume of area X was larger in 35 d DF and WN
birds compared with 20 d birds
(F(1,24) = 9.35; p = 0.002) and was also larger in normal adults compared with 35 d DF
and WN birds (F(1,22) = 11.80;
p = 0.002).
These results demonstrate that depriving birds of normal auditory input
during song learning does not affect the growth in volume of area X
that occurs during song learning in normal male zebra finches, as has
been demonstrated previously (Bottjer et al., 1985; cf. Burek et al.,
1991). Our results also suggest that auditory experience does not
strongly influence the growth of the lMANcore
terminal field in area X that occurs in normal birds, which acts to
preserve the relatively constant proportion of area X occupied by
lMANcore axon arbors during vocal development
(i.e., 16-20%). In agreement with this idea, the volume of the
lMANcore terminal field in area X was not
significantly different between adult DF and WN birds and normal adult
birds or between 35 d DF and WN birds and 20 d controls.
However, this measure was higher in adult controls than in 35 d DF
and WN birds, raising the possibility that
lMANcore axon arbors may undergo some regression
at 35 d in DF and WN birds. Because we did not quantify the
lMANcore terminal field in area X in control
35 d birds (Iyengar et al., 1999), we do not know whether it
undergoes similar changes in normal birds during vocal development or
whether these changes result from the lack of normal auditory input to
35 d DF and WN birds.
These results indicate that neither the establishment nor the
maintenance of topography within the
lMANcorearea X circuit depends on normal
patterns of auditory input during the sensitive period for song
learning. The increase in the absolute volume of area X that occurs
between 20 d and adulthood in control birds also occurs in both
groups of experimental birds used in this study (cf. Bottjer et al.,
1985; E. J. Nordeen and Nordeen, 1988; K. W. Nordeen and
Nordeen, 1988; Burek et al., 1991). Because the total number of
lMANcore projection neurons remains the same throughout song learning, axon arbors of these neurons within area X
must be remodeled constantly between 20 d and adulthood in normal
birds so that broad patterns of topography are maintained during this
period (Iyengar et al., 1999). Therefore, the results in the present
study suggest that there are no long-term effects of deafening or
chronic exposure to white noise on axon remodeling, which is required
to maintain topographic organization within the
lMANcoreX circuit.
These results also underscore the differences between the
lMANcoreRA and
lMANcorearea X circuits in response to
deafening and white noise exposure in juvenile birds. That is,
emergence of topographic organization within the
lMANcore RA circuit is delayed in 35 d
DF and WN birds. However, patterns of axonal connectivity within the
lMANcorearea X circuit in 35 d deprived
birds are unaffected by altered patterns of auditory input during the
sensitive period for song learning, although individual
lMANcore projection neurons send collaterals to
both RA and area X (Nixdorf-Bergweiler et al., 1995; Vates and
Nottebohm, 1995). This pattern demonstrates the validity of the
labeling method used here: the exact same injections that produced an
expanded pattern of label within RA of 35 d birds did not produce
expanded anterograde label within area X.
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DISCUSSION |
Topographic specificity in the lMANcoreRA
pathway emerges between 20 and 35 d in normal male zebra finches,
during early stages of the sensitive period for vocal learning (Fig. 2)
(Iyengar et al., 1999). We report here that depriving male zebra
finches of normal patterns of auditory input during this period delays the establishment of topographic organization within this circuit. Topographic specificity within this projection emerges some time after
35 d in deprived birds and is comparable with that seen in normal
birds by adulthood, although experimental birds produce highly
disrupted songs (cf. Konishi, 1965; Nottebohm, 1968; Price, 1979). This
finding suggests the possibility that spontaneous patterns of activity
within the lMANcoreRA pathway in DF and WN
birds may be sufficient to produce a fairly refined degree of
topographic order. Of course, it is highly likely that final refinements in the mapping of all axonal projections to and from lMAN
are influenced by sensory experience and contribute to the functional
processes of song learning by influencing the fine precision of
connectivity in these pathways.
Axonal projections between presynaptic neurons and their postsynaptic
targets are organized into topographic maps, which are generally
present at birth and show well-organized, innately determined patterns
of connectivity (O'Leary and Cowan, 1983; Udin, 1985; Young and Rubel,
1986; Catalano et al., 1991; Agmon et al., 1993, 1995; Weliky and Katz,
1997; Crair et al., 1998). The finer details of these maps (at the
level of individual axon arbors and synaptic contacts) are shaped by
experience during a later sensitive period of development and
presumably give rise to the exquisite matching between presynaptic and
postsynaptic neurons seen in adult animals (Wallhaüsser-Franke et
al., 1995; Crair et al., 1998). The results we present here for most
song-control pathways are consistent with other studies showing that
topographic organization is evidenced in nascent axonal projections as
innate patterns of precise axonal connectivity, which are not
influenced by abnormal patterns of sensory experience. However, the
lMANcoreRA circuit represents an interesting
exception, because topographic mapping is not established in the
initially formed projection. The lack of topographic specificity in the
lMANcoreRA projection is similar to the
developing retinocollicular projection in birds and mammals, in which
topographic order is initially lacking and subsequently emerges as
a result of substantial remodeling of axon arbors (Nakamura
and O'Leary, 1989; Simon and O'Leary, 1990, 1992) (for review,
see Roskies et al., 1995).
A striking aspect of the lMANcoreRA projection
is that the emergence of initial topographic organization within this
map is affected by experience (in this case, auditory input). An
influence of experience on patterns of topographic connectivity has
also been detected in barn owls, in which prism rearing during the sensitive period when vision calibrates the auditory map of space causes a remapping of the axonal projection leading to the
space-specific map in the inferior colliculus (Feldman and Knudsen,
1997). In the case of prism-reared barn owls, the remapped axonal
projection persists into adulthood, an effect that is in contrast to
our observation of the emergence of topographic mapping in DF/WN adult birds (cf. Knudsen, 1998). This difference raises an interesting question regarding whether removal of experiential inputs (e.g., deafening) is comparable with directional alterations in experiential inputs (e.g., prism rearing). It seems possible that blocking specific
experiential factors (as we attempted to do in the current study) might
reveal a less profound effect on development of the nervous system than
directional alterations in experience, because in the former case an
instructive influence of experience is absent and innately specified
molecular cues may come to predominate. In contrast, directional
manipulations produce a situation in which an instructive influence of
experience, although altered, is present throughout the sensitive
period (cf. Udin, 1985; Knudsen, 1994). This is an extremely important
point for studies examining the role of experiential factors on broad
patterns of topographic organization, because most such experiments
have blocked experience. If directional manipulations of experience do
in fact provide a more sensitive assay, then the basic tenet that
initial patterns of topographic mapping are not influenced to a
significant extent by experience may be subject to revision.
Effects of altered patterns of auditory input on RA volume and the
lMANcore terminal field within RA during song learning
The results presented herein confirm that depriving juvenile male
zebra finches of normal patterns of auditory input did not affect the
increase in volume of RA that normally occurs between 20 d and
adulthood (Bottjer et al., 1986; Herrmann and Bischof, 1986; Kirn and
DeVoogd, 1989). In contrast, the volume of the terminal field made by
small subgroups of lMANcore neurons within the RA
of deprived birds at 35 d was almost twice as large as that in
normal 35 d birds. Furthermore, comparison of the size of the
lMANcore terminal field within RA in deprived
35 d birds with that in normal 20 d birds revealed a
substantial increase in the volume of lMANcore
axon arbors between 20 d and 35 d in DF/WN birds, which
contrasts markedly with the slight decrease seen between 20 d and
35 d in normal birds (Iyengar et al., 1999). Because the total
number of neurons within lMAN is comparable in juvenile deaf birds and
controls (Burek et al., 1991), any change in the overall volume of the
lMANcore terminal field in RA likely reflects
changes in individual lMANcore axon arbors within
RA. One possibility is that individual axon arbors grow to encompass an
expanded postsynaptic target region within RA. Another possible
mechanism is that axon arbors of adjacent
lMANcore arbors become mistargeted by the lack of
normal auditory input, and therefore project to different subregions
within RA rather than converging on the same subregion. Therefore, our
results suggest that the absence of normal auditory experience during early stages of song learning prevents normal remodeling of
lMANcore axon arbors in RA. By adulthood, the
overall volume of the lMANcore terminal field
within RA as well as the spatial pattern of connectivity seen from
lMANcore to RA are comparable in deprived birds
and in those birds that received normal patterns of auditory input. However, precise patterns of synaptic connectivity formed by individual lMANcore axon arbors in RA may nevertheless be
abnormal in deprived adult birds compared with those in controls, which
could in turn prevent the precise alignment of auditory and motor
information necessary for normal vocal learning (Dave and Margoliash,
2000
TOP
ABSTRACT
INTRODUCTION
