Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Spiro, John E., Matthew B. Dalva, and Richard Mooney. Long-range inhibition within the zebra finch song nucleus RA can coordinate the firing of multiple projection neurons. The zebra finch forebrain song control nucleus RA (robust nucleus of the archistriatum) generates a phasic and temporally precise neural signal that drives vocal and respiratory motoneurons during singing. RA's output during singing predicts individual notes, even though afferent drive to RA from the song nucleus HVc is more tonic, and predicts song syllables, independent of the particular notes that comprise the syllable. Therefore RA's intrinsic circuitry transforms neural activity from HVc into a highly precise premotor output. To understand how RA's intrinsic circuitry effects this transformation, we characterized RA interneurons and projection neurons using intracellular recordings in brain slices. RA interneurons fired fast action potentials with steep current-frequency relationships and had small somata with thin aspinous processes that extended throughout large portions of the nucleus; the similarity of their fine processes to those labeled with a glutamic acid decarboxylase (GAD) antibody strongly suggests that these interneurons are GABAergic. Electrical stimulation revealed that RA interneurons receive excitatory inputs from RA's afferents, the lateral magnocellular nucleus of the anterior neostriatum (LMAN) and HVc, and from local axon collaterals of RA projection neurons. To map the functional connections that RA interneurons make onto RA projection neurons, we focally uncaged glutamate, revealing long-range inhibitory connections in RA. Thus these interneurons provide fast feed-forward and feedback inhibition to RA projection neurons and could help create the phasic pattern of bursts and pauses that characterizes RA output during singing. Furthermore, selectively activating the inhibitory network phase locks the firing of otherwise unconnected pairs of projection neurons, suggesting that local inhibition could coordinate RA output during singing.
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INTRODUCTION |
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Birdsong, like human speech, requires precise
control of vocal and respiratory muscles. The neural circuits for
birdsong are localized to a well-defined set of interconnected brain
nuclei, affording an opportunity to discover the neural mechanisms
enabling this precise vocal control. Here we investigate the intrinsic circuitry of the forebrain nucleus RA (robust nucleus of the
archistriatum), the sole output of the telencephalic song circuitry. RA
intrinsic circuitry is of general interest because it transforms higher level neural activity from HVc [used here as the full name of the
nucleus, following Fortune and Margoliash (1995)] into a highly precise premotor output (Yu and Margoliash 1996
).
RA has an obligatory role in the central control of learned song, as
has been shown with lesion studies, chronic recordings, and
microstimulation experiments (Nottebohm et al. 1976;
McCasland 1987
; Vu et al. 1994
; Yu
and Margoliash 1996
; see Margoliash 1997
for a
review). RA receives synaptic input from the song nucleus HVc and from
the lateral magnocellular nucleus of the anterior neostriatum (LMAN),
an area essential to normal song development but not to adult song
production (Fig. 1A)
(Bottjer et al. 1984
; Scharff and Nottebohm
1991
). The axons of RA projection neurons in ventral RA project
topographically to the hypoglossal motoneurons that innervate muscles
of the syrinx, the avian song organ, and those in the dorsal RA project
to areas in the lateral medulla that control respiration (Fig.
1B) (Vicario 1993
, 1994
;
Wild 1993a
,b
, 1994
).
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At rest, RA projection neurons display a tonic, pacemaker-like
activity. During singing, this tonic pattern changes to a highly phasic
pattern so precise that it predicts note identity (Yu and Margoliash
1996). The input to RA from HVc lacks these qualities; it is instead
more tonic and is predictive of syllable identity, independent of the
particular notes that make up the syllable (Yu and Margoliash
1996
). Because the extrinsic afferents to RA are purely
excitatory (Canady et al. 1988
; Kubota and Saito
1991
; Mooney 1992
; Mooney and Konishi
1991
), RA inhibitory circuits are likely to transform incoming
neural activity to an appropriate output for the brain stem motor
circuitry. Indeed, cells staining positive with an anti-GABA antibody
have been detected in RA (Grisham and Arnold 1994
;
Sakaguchi 1996
), and electrical stimulation of HVc and
LMAN axons produces monosynaptic excitatory postsynaptic potentials
(EPSPs) and polysynaptic inhibitory postsynaptic potentials (IPSPs) in
RA projection neurons; these IPSPs are blocked by the GABAA
receptor antagonist bicuculline (Mooney 1992
). The
intrinsic physiology, connectivity, and morphology of RA interneurons,
as well as their role in the transformation of afferent activity, however, have remained unknown.
Using high-resistance, dye-filled electrodes, we were able to record
directly from interneurons to examine their intrinsic properties and
synaptic inputs and to study their morphology. To map the spatial
distribution and functional properties of interneuronal input onto RA
projection neurons, we used scanning laser photostimulation (Dalva and Katz 1994; Katz and Dalva
1994
; Sawatari and Callaway 1996
). We show that
RA interneurons can provide fast feed-forward and feedback inhibition
to RA projection neurons and therefore could help create the pattern of
bursts and pauses that characterizes RA output during singing. As
activation of the inhibitory network can coordinate the firing of
multiple projection neurons, and the interneurons extend functional
processes across areas of RA that control either breathing or syringeal
muscles, inhibition could also serve to coordinate breathing with
syringeal control during singing.
Preliminary data have been presented in abstract form (Spiro et
al. 1996).
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METHODS |
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Tissue preparation and solutions
Photostimulation and intracellular recording and labeling
experiments were performed with in vitro brain slices made from male
zebra finches (Estrildidae: Taeniopygia guttata) ranging in
age from 26 to 372 posthatch days in accordance with a protocol approved by the Duke University Institutional Animal Care and Use
Committee. The details of the slice preparation procedure have been
described previously (Mooney 1992; Mooney and
Konishi 1991
). Briefly, the bird was decapitated following
ketamine injection (0.05 ml im) followed by methoxyflurane (Metofane;
Mallinckrodt Veterinary, Mundelein, IL) inhalation anesthesia. The
brain was removed and chilled in artificial cerebrospinal fluid (ACSF)
(4°C, equilibrated with 95% O2-5% CO2),
then cut in half along the midsagittal sinus, or blocked transversely
for coronal sectioning. A vibratome was then used to cut 300- to
400-µm-thick either sagittal or coronal slices through RA (slice
orientation depended on the fiber stimulation protocol used, see
Electrophysiological recordings; sharp electrodes), which
were immediately transferred to an interface-type holding chamber,
where they were maintained at room temperature for 60-90 min.
ACSF consisted of (in mM) 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose. Equiosmolar sucrose was substituted
for NaCl during the tissue preparation stage. All reagents for ACSF
were obtained from Mallinckrodt Chemical; D-gluconic acid
and picrotoxin were from Sigma.
1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulfonamide (NBQX), R()-2-amino-5-phosphonovaleric acid
(D(
)-APV), and lidocaine N-ethyl bromide
quaternary salt (QX-314), were from RBI (Natick, MA).
Electrophysiological recordings; sharp electrodes
Sharp intracellular recordings of RA interneurons and projection
neurons were made in an interface-type chamber (30°C; Medical Systems, Greenvale, NY) using borosilicate glass pipettes
(BF-100-50-10, Sutter Instruments, Fountain Valley, CA) pulled to a
resistance of 150-200 M and filled with 2 M K-acetate and 5-10%
neurobiotin (Vector Laboratories, Burlingame, CA). RA is clearly
visible under transillumination, facilitating electrode placement.
Interneuron recordings were made in slices from male birds ranging in
age from 49 to 168 days post hatch (113 ± 22 days, mean ± SE). Seven interneurons in seven slices from six birds were used for
electrophysiological analysis; seven additional interneurons were held
too briefly for electrophysiological analysis but were filled
adequately with neurobiotin for morphological analysis. Projection
neuron recordings were made in slices from birds ranging from 26 to 372 days posthatch age (mean, 158 ± 38 days; not significantly
different from the interneuron donor pool, P = 0.4, 2-tailed t-test). Ten cells in nine slices from seven birds
were used for electrophysiological analysis. A total of >400 RA
neurons were recorded from in the course of this study; projection
neurons were encountered at a much higher frequency compared with
interneurons (~30:1). Intracellular potentials were amplified with an
Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) in bridge
mode, digitized at 10 kHz (low-pass filtered at 3 kHz) using a National
Instruments data acquisition board (AT-MI0-16E2; Austin, TX) controlled
by custom LabView software (National Instruments) written by F. Livingston and R. Neumann. In some experiments, concentric bipolar
stimulating electrodes [200-µm outer pole (33-gauge stainless
steel), 25-µm inner pole (platinum); FHC, Bowdoinham, ME], were
placed just dorsal to RA to stimulate the RA-projecting HVc axons
orthodromically (100 µs, ~100 µA, Axon Instruments isolator-10
stimulus isolation unit). Similar bipolar electrodes were also placed
in the RA outflow tract to antidromically stimulate the local axon
collaterals of RA projection neurons, or within RA to stimulate
interneurons directly. In coronal slices, stimulating electrodes were
also used to activate RA-projecting LMAN axons orthodromically
(Mooney 1992
). Data analyses were performed off-line
using LabView and Origin software (Microcal, Northampton, MA).
Statistical analyses were performed using JMP IN software (SAS
Institute). All values reported are means ± SE.
Input resistance was calculated from the steady-state voltage
deflections following the injection of 200-pA current pulses throughout the recording. Because interneurons sometimes did not fire
action potentials spontaneously, measurements of spike characteristics for both cell classes were made during +200-pA current pulses; for each
neuron, measurements from at least two action potentials were averaged.
Comparisons of spontaneously spiking interneurons (n = 4) and projection neurons (n = 4) revealed similar
relationships (data not shown). Action potential width was measured at
half maximal amplitude, afterhyperpolarization (AHP) amplitude is
relative to action potential threshold voltage, and delay to the peak
of AHP is relative to the time of action potential threshold. The AHP
recovery reflects the slope of the initial (2 ms) depolarization following the peak of the AHP. The firing frequency versus current relationship was calculated as the slope of a linear fit to a plot of
intracellular current injection amplitude versus average instantaneous
firing frequency of the first two action potentials.
Cross-correlation analysis
Simultaneous intracellular current-clamp recordings were made from RA projection neurons using the two channels of an Axoclamp 2-B, while a separate site in RA was periodically stimulated with a concentric bipolar stimulating electrode. To isolate monosynaptic IPSPs, excitatory transmission was blocked by adding D-APV (50 µM) and NBQX (5 µM) to the bath. Membrane potential waveforms were passed through a second-order digital Bessel filter, the DC component was subtracted, and then the cross-correlation function was calculated with the use of a 300-ms sliding window with a 240-ms overlap between successive windows, using customized software written in Labview by J. Spiro and F. Livingston. The successive correlograms were then used to generate two-dimensional correlograms using Scion Image (Scion Corporation, Frederick, MD) with colors coding for the amplitude of the cross-correlation function.
Scanning laser photostimulation
The techniques used here have been described previously
(Katz and Dalva 1994). Briefly, slices from young male
zebra finches (30-47 days posthatch; mean, 40 days; 14 cells in 14 slices from 8 birds) were submerged in a recording chamber perfused
with ACSF (room temperature) supplemented with 100 µM CNB-caged
glutamate [L-glutamic
-(
-carboxy-2-nitrobenzyl)
ester (Molecular Probes)]. Young tissue was used because of its
superior viability in the submersion recording chamber, the increased
ease in obtaining whole cell recordings compared with older tissue, and
also to avoid light scattering caused by the heavy myelination of adult tissue.
Whole cell recordings from RA projection neurons were made with patch
pipettes (4-8 M) filled with an internal solution consisting of (in
mM) 100 cesium gluconate, 10 EGTA, 5 MgCl, 40 HEPES, 2 sodium-ATP, 0.3 sodium-GTP, and 1 QX-314, with 0.5% neurobiotin, pH 7.25. Whole cell
currents were measured with an Axopatch 1D intracellular amplifier
(Axon Instruments), and current traces were digitized at 2-10 kHz
after low-pass filtering at half the sampling rate. Data acquisition
was performed with a TL-1 interface (Axon Instruments). Custom
acquisition and analysis software (M. Dalva) permitted linking of
specific electrophysiological responses to specific sites of photostimulation.
The recording chamber was mounted in a fixed position over a Zeiss microscope nose piece resting on a motorized x-y-z translation stage, the position of which was computer controlled. A dissecting microscope was used to guide the placement of patch electrodes within RA. After obtaining a whole cell recording, the laser light was scanned across the tissue systematically (50-µm increments, 2-s delay between flashes), usually over an area that included all of RA, and at least some of the archistriatal tissue immediately surrounding it. Photostimulation (i.e., the release of glutamate from the caged compound) was achieved by illuminating the tissue with a Coherent Enterprises Argon ion laser (50 mW, continuous wave, model 622) transmitted through a ×40 Nikon fluor objective (1.3 NA) to the specimen. The duration of the illumination was controlled by opening briefly (5-20 ms) a mechanical shutter (Uniblitz) placed in the light path. In most cases, the same region was scanned at least twice, to determine both the reproducibility of the result and also to measure the reversal potential of the evoked postsynaptic currents (PSCs). The numbers of PSCs from repeated scans were averaged to generate both the histograms and the stimulation maps. Fiducial marks were placed in the tissue by injecting fluorescent latex microspheres into several distinct points around RA at the end of the experiment; these spots were then illuminated with laser light, and their positions were recorded along with the photostimulation map. These marks permitted alignment of photostimulation maps with histological sections produced in the neurobiotin processing.
Synaptic events were discriminated and counted using a previously
described algorithm (Dalva and Katz 1994). Briefly, the instantaneous derivative was calculated for each point in the first 70 ms after the stimulus. PSCs were defined by two zero crossings and a
slope of >2 SD larger than the average derivative during the last 150 ms of the trace. If a synaptic event was detected at short latency (50 ms), then all synaptic events were counted that occurred within a
200-ms postflash window.
In some cases, RA was remapped in the presence of picrotoxin (PTX). Blocking GABAA currents with PTX has no effect on the size of photostimulation-evoked glutamatergic currents (M. B. Dalva and L. Katz, unpublished observations). The recirculating perfusion system used for photostimulation experiments prevented wash out of PTX and recovery of photostimulation-evoked inhibitory postsynaptic currents (IPSCs).
Histology of RA neurons and Sholl analysis
Individual RA neurons were filled with neurobiotin using either
depolarizing current pulses (~0.5 nA, 500 ms, 50% duty cycle), or
simply by diffusion (whole cell recordings). Slices were then fixed in
4% paraformaldehyde in 0.025 M sodium phosphate buffer (PB) for a
minimum of 12 h at 4°C, resectioned on a vibratome at 75 µm,
and the neurobiotin was then visualized by standard techniques
[Vectastain ABC, dilution of 1:1,000 followed by application of 0.05%
diaminobenzidine (DAB), 1.5 × 105
H2O2 in PB enhanced with 1% cobalt chloride
and 1% nickel ammonium sulfate added to the DAB solution]. A stained
cell was categorized as a projection neuron if it had either an axon
that left the nucleus or thick, radially symmetrical spinous dendrites,
or as an interneuron if it had thin, aspinous neurites and lacked a projection axon. Camera lucida drawings and photomicrographs were made
with a Zeiss Axioskop using a ×63 oil-immersion objective. Areal
measurements for either individual RA cell bodies or for the entire RA
were made by tracing the borders of either the largest cross section of
the soma or the nucleus with a camera lucida, then scanning the drawing
into a computer and using Scion Image software (Scion Corporation) to
convert pixels into square micrometers. No corrections were made for
tissue shrinkage.
To measure the extent of an individual cell's processes within RA, a
Sholl-like analysis (Sholl 1956) was performed from
camera lucida drawings of filled cells. We counted the number of
intersections between processes and each of a series of evenly spaced
concentric circles (50-µm increments beginning at 100-µm radius)
surrounding the cell body. No distinction was made between dendrites
and axon collaterals for either cell type because it was difficult to
distinguish between these two types of processes for interneurons.
GAD immunohistochemistry
Adult zebra finches were anesthetized with a lethal dose (60-80 µl) of Equithesin (0.85 gm of chloral hydrate, 0.21 gm of pentobarbital sodium, 0.42 gm of MgSO4, 1.8 ml of 100% ethanol, and 8.6 ml of propylene glycol to a total volume of 20 ml with H2O) and then perfused transcardially with 0.9% saline for 5 min followed by 4% paraformaldehyde in 0.025 M PB for 30 min. The brain was removed from the skull and postfixed in 4% paraformaldehyde and 20% sucrose in 0.025 M PB overnight at 4°C, then blocked sagittally and resectioned at 30 µm on a freezing microtome. Sections were collected into Tris-buffered saline (TBS), blocked in 4% normal rabbit serum (NS) for 30 min at room temperature (RT), rinsed in TBS (3 × 25 min), then incubated in antiserum to GAD (1:2,000 in TBS, 48 h at 4°C) made in sheep against partially purified rat brain GAD. The antiserum was provided from a source (1440-4) developed at the National Institutes of Health by Drs. Irwin J. Kopin, Wolfgand Oertel, Donald E. Schmechel, and Marcel Tappaz. Effective use in immunocytochemistry was greatly aided through the laboratory of E. Mugnaini (University of Connecticut, Storrs). The sections were rinsed in TBS (3 × 15 min), then incubated with biotinylated rabbit anti-sheep IgG (Vector) at 1:1,500 in TBS at RT for 60 min, rinsed in TBS (3 × 15 min), and transferred to Vector Elite ABC reagent for 1 h at RT. After a standard DAB reaction (see Histology of RA neurons and Sholl analysis), the sections were mounted on subbed slides, dehydrated through alcohols, cleared in xylene, and coverslipped.
Western blot
Tissues for Western blot analysis (i.e., cerebellum, forebrain,
liver) were removed from an adult male zebra finch previously anesthetized with Metofane and killed by decapitation. The tissues were
then homogenized immediately in a 10-fold volume of Ca2+-
and Mg2+-free PBS (pH 7.4) with 5 mM EGTA, 0.5% sodium
dodecyl sulfate (SDS) and DNase (2,000 units). Western blots were
performed as detailed in Gutman et al. (1997). Briefly,
protein concentration of the homogenates was determined using the DC
Protein Assay Kit (Bio-Rad). Samples were incubated in 2 times
SDS-Laemmli buffer, and proteins were separated with
SDS-polyacrylamide gel electrophoresis (7.5% gel) with 20 µg of
protein loaded per lane. They were then transferred to a polyvinylidene
fluoride membrane, blocked in Blotto [5% dried milk in TBS, pH 7.6, with 0.05% Tween (Surfact Amps-20, Pierce, Rockford, IL)], and
incubated in primary antibody (NIH 1440 anti-GAD, 1:500) for 1 h
(dilutions up to 1:7,000 yielded similar results). Membranes were then
washed and incubated with secondary antibody for 1 h
(HRP-conjugated anti-sheep IgG diluted 1:2,500, Boehringer Mannheim).
The secondary antibody was then visualized using a chemiluminescent
substrate exposed to Hyperfilm. Similar blots were also made using a
commercial anti-GAD antibody (Chemicon AB108, 1:2,000) using an
HRP-conjugated anti-rabbit IgG as the secondary (1:5,000).
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RESULTS |
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Interneuron physiology: intrinsic
We recorded intracellularly from RA interneurons and made direct comparisons of their electrophysiological and morphological properties to those of projection neurons (see Table 1 for all statistical comparisons between interneurons and projection neurons). This new cell type was encountered at very low frequency (~1 of every 30 stable recordings), had intrinsic electrophysiological properties that differed from projection neurons, and was subsequently confirmed morphologically to be a RA interneuron (see Interneuron morphology). We recorded from seven of these cells long enough to collect electrophysiological and morphological data; seven other cells were held too briefly to analyze electrophysiologically, but were filled adequately with neurobiotin for morphological analysis.
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RA interneurons were characterized by fast action potentials
(half-height width 0.40 ± 0.02 ms; Fig.
2, A and B) and by
very high-frequency trains of action potentials with varying interspike intervals in response to depolarizing currents (254 Hz/nA; Fig. 2,
A and C). RA interneurons fired few or no action
potentials at their resting potential (66 ± 3 mV). In contrast,
RA projection neurons fired broader action potentials (0.83 ± 0.11 ms; Fig. 2, A and B), lower frequency (90 Hz/nA), and more regular trains of action potentials in response to
depolarizing currents, and displayed spontaneous pacemaker-like action
potentials (~15 Hz, Fig. 2, A and C)
(Mooney 1992
). Although neither interneuron nor projection neuron action potential trains accommodated significantly with low-amplitude currents, some accommodation occurred with larger
currents in both cell types but was not a reliable measure for
distinguishing cell type. Although interneurons and projection neurons
differed markedly in their responses to similar depolarizing currents,
they did not differ significantly in their input impedances (Table 1).
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During spike trains, a sigmoidal depolarizing phase preceded each interneuron action potential, whereas a smooth monotonic trajectory preceded each projection neuron action potential (Fig. 2B). Differences were also evident in the slope of the recovery from the peak of the afterhyperpolarization: interneurons recovered with a much steeper slope than did projection neurons (2.4 ± 0.4 vs. 0.4 ± 0.1 mV/ms). These differences in spike shape and the marked difference in firing rates in response to similar depolarizing currents (Fig. 2C) permitted us to readily distinguish between these cell types during recordings.
Interneuron morphology
Intracellular staining revealed that these two electrophysiologically distinct cell types also had marked morphological differences. Interneurons had small cell bodies (136 ± 12 µm2) with extensive processes that were thin and aspinous and often appeared beaded (Figs. 3, A and B, and 5A). Despite the great extent of these processes, they did not exit RA. Swellings could be detected in certain processes that resembled presynaptic specializations. In contrast to the interneurons, the projection neurons had a larger average soma size (204 ± 12 µm2), thick spiny dendrites, and an axon that clearly exited along the rostral-ventral border of RA (Fig. 3A, arrow). Projection neuron axons also elaborated a thin axon collateral that branched extensively in a region similar to the parent cell's dendritic field (Fig. 3A). Sholl analysis confirmed that the extent of an average interneuron's processes was more than twice that of a typical projection neuron: although in all cases the dendrites and axon collaterals of projection neurons were restricted to within a 200-µm radius of the cell body, interneuronal processes extended in both dorsal-ventral and rostrocaudal directions up to 500 µm away from the cell body (Fig. 4). Thus, in contrast to a projection neuron, a single interneuron extends across large portions of RA. Although subregions of RA project preferentially to syringeal or respiratory motoneurons (Fig. 1), we did not detect any measurable systematic difference in morphology with a neuron's position in the nucleus.
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GAD immunohistochemistry
We suspected that the interneurons studied here were GABAergic
neurons based on their comparatively smaller soma size relative to
projection neurons, consistent with previous studies of
GABA-immunoreactive RA neurons (Grisham and Arnold 1994;
Sakaguchi 1996
). To test this idea, we stained zebra
finch tissue for GAD, the synthetic enzyme for GABA, and compared the
morphology of immunopositive cells with those interneurons identified
physiologically and filled intracellularly with neurobiotin (attempts
to label neurobiotin-filled interneurons directly with GAD were
unsuccessful). This antibody labeled cells and processes diffusely
throughout the zebra finch brain, including dense labeling of
cerebellar Purkinje cells, a well-documented population of GABAergic
neurons. This staining produced especially dense labeling of fine
processes in RA. These GAD-positive processes, often beaded, strongly
resembled the processes that were labeled by neurobiotin during
interneuron recordings (Fig. 5),
suggesting that the fast-spiking aspinous interneurons that we recorded
from here are GABAergic.
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Western blots of both homogenized cerebellum and forebrain of an adult
male zebra finch were used to provide additional confirmation of the
specificity of the antibody in avian tissue (Fig. 5C). As a
negative control, liver was subjected to identical analysis. The
antibody clearly labeled a protein doublet at 61 and 59 kD in the
cerebellum and forebrain, without evidence of labeling in the liver, in
agreement with published studies using the same antibody in rat (the
species to which it was generated). A doublet of the same molecular
weight was also observed using another polyclonal anti-GAD antibody in
zebra finch [this study, data not shown, and in a recent report (Luo
and Pertel 1999)], and with other anti-GAD antibodies in a variety of
other vertebrate species (e.g., Gottlieb et al. 1986
).
Interneuron physiology: synaptic
To determine the sources of synaptic input onto RA interneurons
directly, we made intracellular recordings from them and stimulated fibers originating from HVc and LMAN, as well as antidromically activating RA projection neuron axon collaterals. Previous studies have
shown that electrical stimulation of HVc and LMAN axons can elicit
disynaptic IPSPs from RA projection neurons and can trigger the release
of 3H-GABA from RA (Mooney 1992;
Sakaguchi et al. 1987
), suggesting that these extrinsic
inputs directly excite GABAergic interneurons in RA. Here, consistent
with earlier studies, stimulation of the HVc axons elicited compound
PSPs in interneurons (Fig.
6A). Such interneurons could
fire an action potential on the shortest latency PSP (mean latency from
the stimulus artifact = 2.8 ms, rise time = 7.5 mV/ms;
n = 3 cells), which is consistent with the existence of
a direct excitatory projection from HVc onto RA interneurons. Similar
results were obtained by stimulating LMAN fibers, as well as RA
projection neuron collaterals (Fig. 6A; mean latencies = 2.1, 1.2 ms; rise times = 7.4, 14.0 mV/ms, respectively). In
fact, a single interneuron could receive input from both LMAN and HVc (Fig. 6A).
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In addition to excitatory inputs, RA interneurons receive inhibitory
inputs, because spontaneous hyperpolarizing synaptic events were
commonly observed while the cell was at its resting potential (Fig.
6B). Given the excitatory nature of RA's afferents (Kubota and Saito 1991; Mooney and Konishi
1991
), these inhibitory inputs are likely to arise from other
inhibitory neurons within RA. Due to the difficulty in obtaining long,
stable recordings from interneurons, and the use of an interface
chamber for maximum brain slice viability, however, we were unable to
describe the neurotransmitter receptors involved in synaptic
transmission onto interneurons in more detail.
In summary, similar to RA projection neurons, RA interneurons receive synaptic inputs from the two sources of afferent drive to RA, HVc, and LMAN, as well as from intrinsic sources, including axon collaterals of projection neurons and other inhibitory interneurons. Thus RA interneurons receive synaptic inputs that could enable them to participate in both feed-forward and feedback processes in RA.
Photostimulation
The extensive processes of RA interneurons suggested to us that
they could participate in long-range inhibitory processes within RA. To
test this idea, photostimulation coupled with whole cell recordings
from RA projection neurons was used to assess the spatial extent of RA
inhibitory networks. To discriminate evoked inhibitory postsynaptic
currents (IPSCs) from excitatory postsynaptic currents (EPSCs), we set
the holding potential between 40 and
30 mV, where GABAA
receptor-mediated IPSCs are outward and EPSCs are inward-going.
Photostimulation within RA elicited robust IPSCs from all RA projection
neurons tested (14 cells in 14 slices from 8 birds; Fig.
7, A and B). For
each cell, we stimulated 585 ± 56 points centered roughly on the
recording site and covering an area of 0.54 ± 0.06 mm2; this stimulated area was ~50% greater than the area
occupied by RA in these slices (0.35 ± 0.03 mm2). The
areas providing synaptic input onto a single projection neuron extended
dorsoventrally and rostrocaudally through a major portion of the
nucleus. The spatial distribution of locations that evoked IPSCs from
all recorded cells is shown in Fig. 8, where the percentages of stimulated sites from which IPSCs could be
evoked are plotted as a function of distance from the recording site.
Although the density of sites providing input drops off with distance,
a given RA neuron can receive synaptic input from a large portion of
the nucleus, because >10% of the sites stimulated at 400-500 µm
from the recording site still evoked IPSCs. We suspect that spontaneous
IPSCs generated false positives at a certain low frequency, as for some
sites clearly outside of the borders of RA (Fig. 7). That these are
indeed false positives and not indicative of actual connections onto RA
neurons comes from other lines of evidence (see Fig.
9) (and see Mello et al.
1998), which suggest that sites in the surrounding
archistriatum do not make synaptic contacts onto RA projection neurons.
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To determine whether the IPSCs elicited by photostimulation were GABA mediated, we applied the noncompetitive GABAA receptor antagonist PTX (50 µM) to the slice after first mapping responses in control conditions. Subsequent mapping in the presence of PTX revealed that the evoked IPSCs were blocked completely, confirming that they were mediated by GABAA receptors (n = 3; Fig. 7C). To measure the effect of 50 µM picrotoxin on the size of evoked events more quantitatively, we made whole cell recordings of identically prepared slices with the same concentration of picrotoxin, but used electrical stimulation of an RA afferent (LMAN) instead of photostimulation to evoke polysynaptic IPSCs. In this configuration (unlike photostimulation), we could measure the amplitude of an evoked IPSC over many trials and compare it with the amplitude evoked after the application of picrotoxin. As with the responses evoked by photostimulation, electrically evoked IPSCs were essentially completely eliminated 5 min after the introduction of picrotoxin (they were reduced in amplitude by 95 ± 2%; the remaining current was within the noise level of the recordings; n = 3 cells; data not shown). These results indicate that neurons local to RA provide GABAergic inhibitory input onto both near and distant RA projection neurons.
We did not evoke large excitatory responses in the projection neurons
with photostimulation, which was somewhat surprising given that local
excitatory connections have been demonstrated in RA. For example,
putative excitatory synaptic profiles of local origin are seen at the
electron microscope (EM) level on RA projection neuron dendrites in
canaries (Canady et al. 1988), and antidromic activation
of zebra finch RA projection neurons elicits excitatory synaptic
currents in other RA projection neurons, presumably through local axon
collaterals (Perkel 1995
). The lack of evoked excitation can be partially explained by our choice of holding potentials, which
increased the driving force for chloride-mediated responses and
decreased that for excitatory responses. Furthermore, we were able to
exclude the possibility that excitatory responses were simply masked by
otherwise robust inhibitory inputs, because photostimulation in the
presence of GABAA blockers did not reveal any large
excitatory responses.
Electrical stimulation in RA
Because the variable latency from the laser flash to the evoked response (see DISCUSSION) prevented an unequivocal determination that the long-range responses evoked here by photostimulation were monosynaptic, we also used focal electrical stimulation to map the RA inhibitory circuit. To directly activate monosynaptic IPSPs, and prevent indirect activation of IPSPs by intervening excitatory pathways, slices were bathed in the glutamate receptor antagonists D-APV (50 µM) and NBQX (5 µM). An intracellular recording from a projection neuron was then maintained while a number of sites within RA and in the surrounding archistriatum were stimulated sequentially using a small concentric bipolar electrode (Fig. 9A). Recordings were made from projection neurons near the borders of RA to maximize the potential distance between the stimulating and recording electrodes (n = 3 recordings from 3 slices). IPSPs could be elicited at all sites tested in RA, even those where recording and stimulating electrodes were maximally separated (Fig. 9A, e.g., position f). Consistent with a monosynaptic input, these IPSPs were elicited at short latencies and with low stimulus currents. As a further test of their monosynaptic nature, individual sites also were stimulated at a high frequency (50-100 Hz), which would be expected to cause failure in polysynaptic pathways. However, these evoked IPSPs followed the stimulus 1:1, even at high frequency of stimulation (~65 Hz; Fig. 9B; n = 3 cells). At even higher stimulus frequencies the PSPs did not fail, but rather the deflection of the voltage trace was maintained, and it became difficult to resolve individual PSPs due to capacitive artifacts. To ensure that such electrical stimulation at more distant sites within RA was indeed focal, and not merely activating inhibitory cells proximal to the recorded cell via current spread, the stimulating electrode was moved to positions closer to the recording electrode, but just outside the borders of RA (i.e., Fig. 9A, positions a and b). At these sites, no PSPs were evoked even with extremely high currents (500-1,000 µA). Taken together with the photostimulation data and the cellular anatomy of the interneurons, these results are consistent with long-range inhibitory circuits in RA.
Local GABAergic inhibition phase locks RA projection neuron firing
The anatomic structure of RA's inhibitory circuitry could be
especially well-suited to coordinate the firing of spatially and
functionally distinct sets of RA projection neurons. In other systems,
GABAergic inhibitory circuits resembling those described here can
function to phase lock the firing of otherwise unconnected neurons
(Cobb et al. 1995; Stopfer et al. 1997
).
To test whether the inhibitory network that we have described here
could serve a similar function, we made intracellular recordings from
pairs of projection neurons and selectively activated the local
inhibitory network with electrical stimulation by including glutamate
receptor antagonists in the bath (NBQX and D-APV; see
METHODS). No evidence of synaptic or electrical coupling
was observed between these cell pairs (n = 4; for the 3 cell pairs later recovered histologically, the somata were separated by
200-400 µm in the sagittal plane, mean = 283 µm), or between
other pairs (n = 20) examined in the absence of the
glutamate receptor antagonists. RA projection neurons displayed a
spontaneous, pacemaker-like action potential discharge at rest, as
previously described in vitro (Mooney 1992
; present study) and in vivo (Yu and Margoliash 1996
). Here,
pairwise recordings revealed that these action potential trains were
uncorrelated (n = 4 cell pairs; Fig.
10, 2 bottom traces). In
contrast, stimulus trains (~10-20 Hz) generated IPSPs that were
strongly phase locked to each stimulus; the effect of the IPSPs was to
modulate each projection neuron's pacemaker-like activity and strongly
entrain the subsequent action potential. Thus the action potential
firing of the two cells became strongly phase locked to each other,
resulting in high degrees of cross-correlation (n = 4 cell pairs; Fig. 10, 2 top traces). We computed the mean
area underneath the rectified cross-correlation function for each dual
projection neuron recording for three time points before the stimulus
was turned on, and for three time points while the stimulus was on, and
compared those numbers as a relative measure of the change in the
degree of correlation. For the four cell pairs, the mean increase in
area was 63% (range: 5-115%).
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DISCUSSION |
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General conclusions
Understanding the neural mechanisms for birdsong requires
elucidating the cell types and connectivity of vocal premotor areas. The intrinsic circuitry of the song nucleus RA is especially important in this regard because of its role in generating highly precise outputs
for the coordinated control of breathing and syringeal muscles during
singing (Suthers 1997; Yu and Margoliash
1996
). Toward this goal, we have explored RA's intrinsic
circuitry and detected a class of RA interneuron that provides
GABAergic inhibition to RA projection neurons. RA interneurons are well
suited to transform afferent activity from HVc into an appropriate
premotor output because they 1) can provide inhibition that
might enable the transition in RA projection neurons from tonic to
phasic activity during singing, 2) form long-range
connections and thus link disparate parts of RA, and 3) can
coordinate the firing of multiple RA projection neurons.
Evidence for two distinct classes of neurons in RA
Using sharp intracellular recording in RA, we detected a class of
interneuron distinguished electrophysiologically and morphologically from RA projection neurons. Interneurons fired brief action potentials with a steep current-frequency relationship, and fired only
sporadically at rest. In contrast, projection neurons fired broader
action potentials, had shallower current frequency relationships, and were spontaneously and rhythmically active at rest (see also
Mooney 1992). Interneurons had small somata and long
aspinous thin processes that projected widely throughout RA without
exiting the nucleus. Projection neurons had larger somata, thick spiny
dendrites, and an axon that exited the nucleus.
For several reasons we believe that the interneurons we recorded from
are GABAergic: consistent with GABA-positive cells in previous studies
(Sakaguchi 1996), the average soma area of interneurons recorded here was on average 33% smaller than that of projection neurons. In addition, the interneurons we encountered had thin and
beaded processes closely resembling those processes that stain positive
for GAD. Finally, the interneurons that we recorded are also remarkably
similar in morphological and physiological properties to putative
interneurons of another song control nucleus, HVc (Dutar et al.
1998
; Kubota and Taniguchi 1998
), and to
well-described GABAergic interneurons of the mammalian neocortex. Like
RA interneurons, neocortical basket cells have dendrites that are
aspinous and beaded, fire very short-duration action potentials, and
are capable of firing at high frequencies (Azouz et al.
1997
; Thomson and Deuchars 1997
).
Although the intrinsic properties and morphology of RA neurons supports
the classification into at least two cell types, interneuron and
projection neuron, this classification is not exhaustive. Based on
Golgi material, Gurney (1981) also detected two classes of neuron in the zebra finch RA, spiny and aspinous. In contrast to our
observations that the aspinous neurons did not project an axon that
exited the nucleus, however, Gurney reported that in several examples,
both types of neurons sent an axon outside of the nucleus. Also, in
another songbird, the canary, DeVoogd and Nottebohm
(1981)
distinguished four classes of neurons based on Golgi
staining; these included small nonspiny neurons and neurons with thick
spiny dendrites, resembling the interneurons and projection neurons
described in this report. In addition, however, they reported two other
classes of spiny neuron, one like projection neurons that we have
filled near the borders of RA, whose dendrites were asymmetric and
directed inward toward the center of RA, and another cell class that
was moderately spiny with finer dendrites. In short, our classification
of RA neurons into projection and interneurons may need to be further
expanded. Recent evidence in the mammalian hippocampus suggests that
subsets of interneurons defined by morphological, physiological, or
pharmacological properties often do not coincide, suggesting that
interneurons may not be easily categorized into distinct groups, or the
number of groups may be very large (Parra et al. 1998
).
Evidence from photostimulation and electrical stimulation for functional long-range GABAergic inhibition in RA
The photostimulation data presented here provide direct functional
evidence that long-range inhibition acts in RA via GABAA receptors on projection neurons. Focally uncaging glutamate evoked IPSCs in a projection neuron even when stimulating 400-600 µm from
the projection neuron's cell body. Because inhibitory currents were
usually the first evoked responses that we observed, the long-range
inhibitory responses elicited here by photostimulation were likely to
be monosynaptic. However, as was the case in previous studies in cortex
(Dalva and Katz 1994; Sawatari and Callaway 1996
), we observed a variable latency from the laser flash to the evoked response, probably due to variability in the time required for uncaged glutamate to diffuse to the presynaptic cell and reach a
high enough concentration to cause spiking. This feature, along with
low maximal rates of stimulation, makes an unequivocal distinction between mono- versus polysynaptic pathways problematic with this technique. We were also restricted to using juvenile birds for this
analysis for technical reasons outlined in METHODS. More direct evidence that the long-range inhibition studied here is monosynaptic, and also is present in adult birds, comes from electrical stimulation experiments conducted in the presence of glutamate receptor
antagonists, which confirmed that short-latency IPSPs could be evoked
from projection neurons even while stimulating at distant sites within
RA. These IPSPs could follow at high frequencies of stimulation,
characteristic of a monosynaptic connection. When the results of
photostimulation and electrical stimulation experiments are taken
together with the anatomic structure of RA interneurons, they provide
strong evidence for long-range monosynaptic inhibition within RA.
Further studies combining cell fills and EM level analysis will be needed to develop a more quantitative description of the connections in the nucleus, such as how many projection neurons are contacted by a single interneuron, and the number of interneurons that are contacted by a given projection neuron's collaterals.
Roles of inhibitory circuits in RA
TRANSITION FROM TONIC TO PHASIC FIRING AND INCREASE IN PRECISION.
Here we show that axons from HVc and LMAN provide direct excitatory
input onto RA interneurons, confirming and extending earlier electrophysiological (Mooney 1992) and biochemical
(Sakaguchi et al. 1987
) evidence for feed-forward
GABAergic pathways within RA. These same interneurons also are likely
to participate in feedback inhibition, because antidromic activation of
RA projection neuron axon collaterals can also elicit short-latency
EPSPs. Based on their intrinsic firing properties and synaptic
connections, we speculate that RA interneurons transform afferent HVc
activity into a more temporally precise pattern of RA projection neuron firing that is then relayed to vocal and respiratory motoneurons. Although RA projection neurons are tonically active when the bird is
silent, they display highly phasic bursts of action potentials during
singing that alternate with sharp transitions to periods of total
silence (Yu and Margoliash 1996
). As the pattern of HVc activity during singing lacks this phasic quality (Yu and
Margoliash 1996
), and HVc terminals in RA are excitatory (this
study; Kubota and Saito 1991
; Mooney
1992
; Mooney and Konishi 1991
), local inhibitory interneurons could mediate the tonic to phasic transition in RA. Some
longer time scale features of this inhibition could also be achieved by
other mechanisms, including slow afterhyperpolarizations of RA
projection neurons (Spiro, unpublished observations), and GABAB receptor-mediated IPSPs, as detected in HVc
(Schmidt and Perkel 1998
). Nonetheless, the intrinsic
properties of the RA interneurons described here are especially
well-suited for the rapid initiation of inhibition. The fast action
potentials and steep current-frequency relationships of RA interneurons
would allow fast feed-forward inhibition that could sharpen the slower monosynaptic EPSPs evoked by HVc and LMAN axon terminals. This mechanism could reduce jitter by narrowing the time window during which
correlated afferent activity drives RA neurons to fire.
LINKING FUNCTIONALLY SPECIALIZED SUBDOMAINS.
RA interneurons have neurites up to 400 µm long, and projection
neurons have dendritic processes that extend 150 µm from their somata
(Fig. 4), suggesting that activity in an interneuron could influence a
projection neuron up to ~550 µm distant, which is in close
agreement with the results achieved with photostimulation and
electrical stimulation. These long-range synaptic connections span
spatially disparate regions of RA (i.e., dorsal vs. equatorial and
ventral RA) that ultimately project to respiratory or syringeal motoneurons. Therefore the interneurons could serve as one neural substrate for coordinating breathing and syringeal muscles observed during singing (see Suthers 1997 for a review). These
interneurons might also play a similar role in coordinating different
syringeal muscles, because interneurons also link equatorial and
ventral regions of RA, which innervate distinct pools of hypoglossal
motoneurons that control either ventral or dorsal syringeal muscles
(Fig. 1B) (Vicario 1991
; Vicario and
Nottebohm 1988
).
COORDINATION OF ACTIVITY IN RA.
Beyond a transient suppression of RA projection neuron firing,
the present study reveals that RA interneurons can also coordinate the
firing of otherwise unconnected and previously unsynchronized output
neurons. This role of intrinsic circuitry is directly analogous to that
of GABAergic interneurons in other systems (Cobb et al. 1995; Stopfer et al. 1997
). For example, using
dual recordings of hippocampal pyramidal cells and minimal stimulation
in the presence of glutamate receptor blockers to selectively activate inhibitory interneurons, Cobb et al. (1995)
demonstrated
that interneurons can transiently phase lock previously unsynchronized pyramidal cell firing. In sensory systems, such synchronization has
been shown to be important for encoding and discriminating different
stimuli (Stopfer et al. 1997
). In the vocal premotor system, such synchronized firing could be used to encode and coordinate the integrated movements of the vocal and respiratory systems necessary
to birdsong. For example, interneurons receiving excitatory input from
HVc during singing could transiently create discrete functional domains
of RA projection neurons by phase locking their activity; such an
activity pattern could be the neural code for a particular note. A
different activity pattern from HVc onto another interneuron might
sculpt a new domain of activity for the subsequent note. Such a
transient reconfiguration of circuitry is analogous to that which
occurs in the stomatogastric ganglion under the influence of various
neuromodulators (for review, Harris-Warrick and Marder
1991
), albeit at a much slower time scale.
ROLE IN LEARNING.
Both note duration and structure are learned features of birdsong.
Because RA is the first site within the descending vocal motor pathway
where temporally precise neural coding for these features emerges, it
is strongly implicated as a locus for synaptic modification underlying
song learning. Excitatory synapses on the interneurons that we describe
here are a potentially important cellular site within RA for these
changes, especially if they display use-dependent synaptic plasticity,
as described for excitatory inputs onto inhibitory interneurons in
other systems, for example in the hippocampus (Maccaferri et al.
1998; McMahon and Kauer 1997
). Because single
inhibitory interneurons in RA can exert a widespread influence on
projection neurons, changes in excitatory drive onto a single neuron
could significantly alter the firing pattern of many RA projection
neurons, thus strongly influencing song quality. The interaction of
LMAN and HVc inputs on RA interneurons during song learning may be an
important aspect of such a process.
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ACKNOWLEDGMENTS |
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We thank L. Katz for providing the scanning laser photostimulation equipment used in these experiments, D. Fitzpatrick, L. Katz, J. Kauer, F. Livingston, and S. White for providing thoughtful discussion and reading early drafts of the manuscript, and M. Booze and R. Stacy for expert assistance with the histology. We also thank C. R. Gutman and B. Matthew for expert assistance in performing the Western blots.
This work was supported by National Institutes of Health Grants T32 NS-07370 and F32 DC-00333 to J. E. Spiro, and by NIH Grant R01 DC-02524 and McKnight, Klingenstein, and Sloan Foundation awards to R. Mooney.
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FOOTNOTES |
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Address for reprint requests: R. Mooney, Dept. of Neurobiology, Box 3209, Duke University Medical Center, Durham, NC 27710.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 November 1998; accepted in final form 4 February 1999.
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REFERENCES |
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