Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Farries, Michael A. and David J. Perkel. Electrophysiological Properties of Avian Basal Ganglia Neurons Recorded In Vitro. J. Neurophysiol. 84: 2502-2513, 2000. The forebrains of mammals and birds appear quite different in their gross morphology, making it difficult to identify homologies between them and to assess how far they have diverged in organization. Nevertheless one set of forebrain structures, the basal ganglia, has been successfully compared in mammals and birds. Anatomical, histochemical, and molecular data have identified the avian homologues of the mammalian basal ganglia and indicate that they are very similar in organization, suggesting that they perform similar functions in the two classes. However, the physiological properties of the avian basal ganglia have not been studied, and these properties are critical for inferring functional similarity. We have used a zebra finch brain slice preparation to characterize the intrinsic physiological properties of neurons in the avian basal ganglia, particularly in the input structure of the basal ganglia, the striatum. We found that avian striatum contains a cell type that closely resembles the medium spiny neuron, the principal cell type of mammalian striatum. Avian striatum also contains a rare cell type that is very similar to an interneuron class found in mammalian striatum, the low-threshold spike cell. On the other hand, we found an aspiny, fast-firing cell type in avian striatum that is distinct from all known classes of mammalian striatal neuron. These neurons usually fired spontaneously at 10 Hz or more and were capable of sustained firing at very high rates when injected with depolarizing current. The existence of this cell type represents an important difference between avian striatum and mammalian dorsal striatum. Our data support the general idea that the organization and functional properties of the basal ganglia have been largely conserved in mammals and birds, but they imply that avian striatum is not identical to mammalian dorsal striatum.
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INTRODUCTION |
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At first glance, the mammalian
telencephalon bears little resemblance to the telencephalon of birds
and other tetrapods. The mammalian telencephalon consists of a laminar
cerebral cortex overlying masses of gray matter collectively known as
the basal ganglia, while the forebrains of other tetrapods show few
signs of this laminar organization. Indeed, the prevalent view before the 1960s was that little or nothing in the avian brain is homologous to the mammalian isocortex and held that most of the avian
telencephalon consists of striatum, the largest component of the basal
ganglia (reviewed in Striedter 1997). Thus, the
organization of the mammalian telencephalon seemed to differ radically
from that of the avian telencephalon.
We now know that the brains of birds and mammals are much more alike
than was originally believed. Only a small part of the avian brain,
known as the "paleostriatal complex," is actually a homologue of
the mammalian basal ganglia (Karten and Dubbeldam 1973;
Medina and Reiner 1995
). The other parts of the avian
telencephalon (called neostriatum, archistriatum, and hyperstriatum)
are homologues of mammalian pallium, i.e., the cerebral cortex,
claustrum, and pallial amygdala (Karten 1991
;
Puelles et al. 1999
; Striedter 1997
). The
exact homologies between the various parts of the mammalian pallium and
structures of the avian brain are still unclear (Striedter 1997
), but comparison of the basal ganglia of the two classes seems comparatively straightforward. The main components of the basal
ganglia in mammals are the striatum, which receives input from the
cerebral cortex, and the globus pallidus, which receives GABAergic
input from the striatum and sends GABAergic projections to the midbrain
and thalamus. From anatomical (Karten and Dubbeldam 1973
; Kitt and Brauth 1982
; Medina and
Reiner 1997
; Veenman et al. 1995
), histochemical
(Medina and Reiner 1995
), and molecular (Puelles
et al. 1999
; Smith-Fernandez et al. 1998
)
evidence we know that the avian homologues of the mammalian striatum
are the paleostriatum augmentatum (PA) and the lobus parolfactorius
(LPO), and the avian homologue of the globus pallidus is the
paleostriatum primitivum (PP; see Fig.
1). The organization, projections, and neurochemical properties of the basal ganglia seem to be conserved between mammals and birds to a remarkable degree. This suggests that
the basal ganglia of birds perform functions similar to those of the
mammalian basal ganglia, using similar mechanisms.
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However, anatomical and neurochemical organization do not fully
determine how a neural structure works. Equally important are the
intrinsic physiological properties of its neurons. Neurons in the basal
ganglia of mammals have a characteristic set of physiological properties that have been well studied, particularly in the striatum (Jiang and North 1991; Kawaguchi 1993
;
Kita et al. 1984
; Nisenbaum and Wilson
1995
). Although the anatomical organization of the basal
ganglia of birds and mammals has been largely conserved, their
functions may still have diverged substantially through divergence in
the physiological properties of their neurons. The goal of this study
is to examine the intrinsic physiological properties of neurons in the
avian basal ganglia and assess their similarity to neurons of the
mammalian basal ganglia. To address this issue, we have recorded from
neurons in the zebra finch basal ganglia in a brain slice preparation,
using the whole cell technique in current-clamp mode.
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METHODS |
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Preparation of brain slices
Adult zebra finches were obtained from a local supplier; this
study also employed juvenile zebra finches (31-71 days old) that were
bred in our colony. Adult zebra finches were housed three to five per
cage, while juvenile zebra finches were housed in cages containing only
their parents and siblings. Birds were kept on a 13:11 h light:dark
cycle. Slices were prepared as described by Stark and Perkel
(1999), except for the composition of the artificial
cerebrospinal fluid (ACSF) used during slicing (see following text).
The procedures were approved by the Institutional Animal Care and Use
Committee at the University of Pennsylvania. Briefly, birds were
anesthetized with isoflurane and killed by decapitation. The brain was
rapidly removed and placed in ice-cold ACSF containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgSO4, 2.5 CaCl2, 1 NaH2PO4, 16.2 NaHCO3, 11 D-glucose, and 10 HEPES.
Parasagittal or coronal brain slices, 300 µm thick, were cut with a
vibrating microtome and collected in ACSF heated to 30°C and
subsequently allowed to cool to room temperature. The ACSF used for
collecting slices and recording differed slightly from the ACSF used
during slicing
the ACSF used for collecting and recording had 26.2 mM NaHCO3 and no HEPES, but was otherwise identical
to the ACSF used for slicing. All solutions were bubbled with a 95%
O2-5% CO2 mixture.
Identification of PA, PP, and LPO in living brain slices
A thin layer of fibers, the lamina medullaris dorsalis (LMD; see
Fig. 1), marks the boundary between the paleostriatal complex and the
overlying neostriatum. PA and LPO lie immediately ventral to the LMD,
which is clearly visible in living, unstained brain slices. The
approximate boundary between PA and LPO in coronal slices lies at a
"kink" in the LMD, where it turns more ventrally as one moves from
lateral to medial portions of the slice. Recordings targeting PA were
made substantially lateral to this boundary; LPO recordings were made
substantially medial to it. In parasagittal slices, no border could be
seen between PA and LPO, and thus some cells recorded in these slices
could not be definitively localized to PA or LPO. Accordingly, these
cells are excluded from analyses comparing PA and LPO. The LPO of zebra
finches contains a specialized region known as "area X," found only
in songbirds; cells in this area are not included in this study and
will be described elsewhere. PP was identified as a densely fibrous
region ventromedial to PA. Kuenzel and Masson (1988, pp.
10, 59, and 62) illustrate how PP can be identified in unstained
coronal slices. In practice, however, we found that this fibrous region
marks only the approximate location of PP in living slices; when the
location of recorded neurons was confirmed with Nissl staining, many
cells recorded while targeting PP proved to be in PA. Nevertheless
cells recorded while targeting PA or LPO were always found in the
targeted structure.
Electrophysiological recording
During experiments, slices were placed in a recording chamber
and superfused with ACSF heated to 25-27°C. We recorded from neurons
using the "blind" whole cell method (Blanton et al.
1989). Pipettes had a resistance of 5-9 M
and were filled
with a solution containing (in mM) 120 K methylsulfate, 10 HEPES, 2 EGTA, 8 NaCl, and 2 Mg ATP, pH 7.2-7.4, osmolarity 275-285 mOsm. In
most cases, 0.5% neurobiotin (Vector Laboratories, Burlingame, CA) was
included in the pipette solution to permit visualization of the
recorded neuron. Signals were amplified using an Axoclamp 2B (Axon
Instruments, Foster City, CA) followed by a Brownlee Model 410 amplifier (Brownlee Precision, Santa Clara, CA). Signals were low-pass
filtered at 1-3 kHz, digitized at twice (or more) the filter cutoff
frequency with a National Instruments (Austin, TX) digitizing board,
and acquired using a custom data-acquisition program written in LabVIEW (National Instruments). Membrane potentials were corrected for a liquid
junction potential of +5 mV. The only drug used in this study,
4-aminopyridine (4-AP), was obtained from Research Biochemicals (Natick, MA) and was bath-applied.
Histological procedures and morphological measurements
After recordings using neurobiotin in the pipette solution, slices were immersion-fixed in paraformaldehyde (4% in 0.1 M phosphate buffer) and kept at 4°C for at least 4 h. Slices were then transferred to a cold sucrose solution (30% in 0.1 M phosphate buffer) and stored at 4°C for several hours to several days. Slices were resectioned to 60 µm thickness with a freezing microtome and processed for visualization with an avidin/biotin/horseradish peroxidase complex (ABC Elite Kit, Vector Laboratories) followed by a reaction using the Vector VIP peroxidase substrate kit (Vector Laboratories). In most cases in which the slices were counterstained with cresyl violet (Nissl stain), diaminobenzidine was used as the peroxidase substrate instead of the VIP kit since the VIP kit produces a purple precipitate that may be obscured in a Nissl-stained background. Labeled neurons were examined with a ×40 objective (×100 in some cases), soma diameters along major and minor axes were measured, and the number of primary dendrites was counted. Cell diameters given in the text are averages of the major and minor axis diameters.
Analysis and measurement of electrophysiological parameters
We analyzed our recordings to measure basic electrophysiological
parameters such as input resistance, action potential threshold, etc.
We encountered difficulty in defining the input resistance for many
neurons because the membrane resistance depended strongly on their
membrane potential (the same problem occurs in mammalian striatal
neurons, see Nisenbaum and Wilson 1995). Since different neurons of the same class had different resting membrane potentials, they could exhibit dramatically different input resistances as calculated from their response to a small current pulse from rest. Such
neurons might have exactly the same current-voltage relationship when
brought to the same baseline membrane potential yet display very
different input resistances simply because they happened to be resting
at different potentials. To circumvent this problem, we define a
cell's input resistance as the maximum slope of its current-voltage
(I-V) curve at membrane potentials below
50 mV. We exclude
the portion of the I-V curve more depolarized than
50 mV
to minimize artifacts produced by depolarization-activated depolarizing
currents such as those carried by Na+ and
Ca2+. The I-V curve is generated from
the steady-state voltage deflection produced by current
pulses of 500- to 1,000-ms duration. Note that this excludes from the
I-V curve the "ramping responses" many of our neurons
produced when injected with depolarizing current pulses of sufficient
magnitude (see Fig. 2A for an example). Voltage traces shown
in figures are typically averages of two to six traces evoked by the
same current pulse, except for traces containing action potentials.
We measured various aspects of action potentials using custom software written in IGOR (WaveMetrics, Lake Oswego, OR). The beginning of an action potential (AP) is defined as the averaged times of the maxima of the waveform's second and third derivatives. This definition was chosen because it provided the best match with our visual judgement of where the AP begins. The AP threshold is defined as the membrane voltage at this initiation point; the AP amplitude is the voltage at the peak minus the threshold voltage; the AP duration is its width at half height, the afterhyperpolarization (AHP) peak is the voltage minimum attained after the AP peak but before a point set by the user; the AHP time to peak is the time of this minimum minus the time when the membrane potential crosses the AP threshold on descent from the AP peak. For each cell, the measurements of five APs (where possible) were averaged to produce the final AP measurements for that cell. Only the first AP fired during a current pulse was used in these averages unless more APs were needed to achieve the standard five APs per cell. We also measured the "delay to first spike," defined as the time from the onset of a 500-ms depolarizing current pulse to the occurrence of the first AP in traces where only one AP was fired (if no such traces were available in a recording, we made this measurement on traces with the fewest APs).
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RESULTS |
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Intrinsic electrophysiological properties of the principal cell type of PA and LPO
The most common neuronal type we recorded in PA and LPO
(n = 31 cells, recorded from 14 birds) had a resting
potential of 74 ± 11 (SD) mV and an input resistance of 383 ± 203 M. We recovered 20 neurons of this type filled with
neurobiotin and found that they have irregularly shaped somata
(9.3 ± 1.8 µm diam) with four to seven primary dendrites. The
dendrites were covered with spines (Fig.
2C), and so we designate these
cells as "spiny neurons" (SNs). Spontaneous postsynaptic potentials
were sometimes observed, but these cells never fired spontaneous action
potentials when healthy. When these cells were injected with pulses of
hyperpolarizing current, all exhibited time-independent inward
rectification, i.e., a rapid decrease in membrane resistance (Fig. 2,
A and B). A few SNs also displayed a small
time-dependent component of this hyperpolarization-activated inward
rectification (n = 3, data not shown). We quantified
this inward rectification as the ratio of the minimum membrane
resistance occurring on hyperpolarization to the input resistance as
defined in METHODS. Among the SNs for which this
measurement could be made (n = 26), the ratio was
0.35 ± 0.23.
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When injected with depolarizing current pulses, all SNs responded with a gradual, ramp-like increase in membrane potential (Fig. 2A). With a sufficiently strong or long-duration depolarizing current pulse, the ramping response ended with action potentials that were substantially delayed relative to the onset of the pulse (Figs. 2A and 3). We quantified this delayed spiking by measuring the time from the onset of a 500-ms depolarizing current pulse to the occurrence of the first spike, in traces where only one spike was fired (or, if no such traces were available, in traces where the smallest number of spikes were fired in that recording). The delay to first spike in SNs was 378 ± 70 ms; this measurement for other cell types is given in Table 1. After firing the first spike, these cells usually fire repetitively, occasionally with some accommodation in firing rate (Fig. 3A). As stronger depolarizing pulses were injected, these cells fired action potentials at higher rates (Fig. 3B) with a shorter delay from onset of the pulse to the first spike (Fig. 3C). Additional electrophysiological parameters of SNs and other avian cell types are summarized in Table 1.
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In the properties described above, these cells show no qualitative
differences from the principal cell type of mammalian striatum, the
medium spiny neuron (MSN). In particular, they share two defining properties: fast inward rectification in response to hyperpolarizing current and a ramping response to depolarizing current
(Nisenbaum and Wilson 1995). In mammalian MSNs, the
ramping response is mediated by an A-type K+
current that rapidly activates on depolarization and then gradually inactivates, allowing a slow membrane depolarization. This current is
blocked by 4-AP, and in mammalian MSNs 4-AP eliminates both the ramping
response and the delay in spiking (Nisenbaum and Wilson 1995
; Nisenbaum et al. 1994
). To further explore
the mechanistic similarities between mammalian and avian spiny neurons,
we examined the effect of 4-AP on avian striatal SNs. Bath application
of 100 µM 4-AP blocked the ramping response and the delayed spiking in these cells (Fig. 4, n = 6/6 cells from 4 birds; delay to first spike went from 449 ± 47 to 221 ± 76 ms on application of 4-AP, a statistically
significant difference, paired Wilcoxon test, P = 0.03). This suggests that an A-type current is responsible for the
ramping response and delayed spiking seen in avian striatal SNs, as it
is in mammals.
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Lack of age-related changes in the properties of spiny neurons in PA and LPO
Neurons in some regions of the zebra finch brain undergo changes
in their physiological properties as the birds mature (Boettiger and Doupe 1998; Bottjer et al. 1998
;
Livingston and Mooney 1997
; Stark and Perkel
1999
; White et al. 1999
). To examine the
possibility of age-related changes in PA and LPO, we divided the cells
into two major age groups
a juvenile group recorded from birds aged 31-46 days old (5 birds) and an adult group from birds at least 80 days old (8 birds). We compared SNs recorded in the two age groups, the
only cell type common enough to permit comparison (n = 10 juvenile SNs, n = 16 adult SNs; 5 SNs recorded from
a 72-day-old bird were excluded from this comparison). There were no
qualitative differences between the two groups (e.g., both exhibited
fast inward rectification, ramping depolarization, and delayed spiking)
nor were there any significant differences in major
electrophysiological properties (comparisons summarized in Table
2). For this reason, the data from these
two age groups are combined in all other analyses.
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Properties of a striatal interneuron cell type recorded in PA and LPO
Some of the neurons we recorded were not of the spiny neuron type,
differing in physiological properties or morphology. Four of these
neurons exhibited unusual firing properties resembling a mammalian
striatal interneuron type, the "low-threshold spike" cell [LTS,
also known as "PLTS" (Kawaguchi 1993), recorded from 4 birds]. Three of these neurons rested at relatively depolarized potentials (
50 ± 7 mV); the fourth fired spontaneously roughly once every 5 s. We examined the current-voltage relationship of these neurons by hyperpolarizing them to approximately
80 mV and
delivering current pulses from that potential. These cells had an input
resistance of 388 ± 243 M
. When injected with hyperpolarizing current pulses, these neurons exhibited time-dependent inward rectification to varying degrees, exemplified by a "sag" in the membrane potential near the onset of the current pulse (Fig.
5A). Depolarizing current
pulses elicited a plateau-like spike lasting hundreds of milliseconds,
typically crowned by one to three fast action potentials appearing at
the beginning of the plateau-like potential (Fig. 5, A and
B). This persistent spike was the defining characteristic of
these neurons and was triggered at a membrane potential of
58 ± 2 mV. This spike (and the accompanying fast action potentials) could
also be triggered on rebound following a hyperpolarizing current pulse
(Fig. 5A). We recovered only one of these neurons filled
with neurobiotin; this cell had aspiny dendrites, unlike the SN cell
type (Fig. 5C).
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Intrinsic electrophysiological properties of "anomalous" spiny neurons in PA and LPO
Another group of cells that differed from the SN type were morphologically indistinguishable from SNs (Fig. 6D shows an example) but possessed somewhat different physiological properties. These neurons, like SNs, displayed fast inward rectification in response to hyperpolarizing current pulses (minimum resistance:input resistance ratio of 0.18 ± 0.07, n = 15) and produced a ramping response to depolarizing current pulses but were distinguished from ordinary SNs by their tendency to fire an action potential at the onset of suprathreshold current pulses (Fig. 6A). When injected with larger depolarizing current pulses, such cells either failed to fire more action potentials or fired additional action potentials that were unusually broad and of smaller amplitude. We recorded a total of 15 of these cells (from 11 birds); of these, 7 exhibited the firing properties of "normal" SNs during some part of the recording (i.e., at some point in the recording, they switched from the "normal" state to the "anomalous" state, or vice versa). Such behavior has been recorded in mammalian medium spiny neurons after intracellular dialysis (C. Wilson, personal communication).
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A second population of "anomalous" spiny neurons (n = 9 cells from 8 birds) possessed fast inward rectification (minimum resistance:input resistance ratio of 0.41 ± 0.13) but showed no signs of a ramping response to depolarizing current pulses (Fig. 6B). These cells were generally capable of firing multiple action potentials during strong depolarizing current pulses that were not markedly different from healthy Na+ action potentials (6 of 8, Fig. 6C). In addition, these cells produced a small "bump" in membrane potential near the beginning of sufficiently strong depolarizing current pulses (Fig. 6B). In one case, a neuron with these properties switched to a "normal" SN state, i.e., began showing a ramping response to depolarizing current pulses and fired action potentials delayed relative to the onset of the current pulse (Fig. 6E).
Properties of an aspiny, fast-firing cell type in PA and LPO
We identified one other cell type in avian striatum that was
distinct from the SN class and yet did not closely resemble any of the
striatal interneuron types found in mammals (Fig.
7). These cells had an input resistance
of 414 ± 205 M. Almost all of these cells (7 of 8, recorded
from 7 birds) were spontaneously active, firing action potentials at
regular intervals in the absence of any current injected through the
recording electrode (Fig. 7C, average rate: 14 ± 5 Hz). This spontaneous activity sometimes ceased during the recording
but was always visible prior to break-in, i.e., could be observed
extracellularly while the recording pipette was forming a seal with the
cell's membrane. We therefore hypothesize that the spontaneous
activity does not result from changes brought about by the whole cell
recording but that the cessation of this activity occurring a few
minutes after break-in does result from such changes. To
study the current-voltage relation of these neurons, we silenced their
spontaneous activity by continuously injecting hyperpolarizing current,
bringing the cells to a baseline potential near
75 mV. From this
baseline potential, hyperpolarizing current pulses evoked a sagging
response in membrane voltage, indicating the presence of time-dependent
inward rectification (Fig. 7A, n = 7 of 8).
Subthreshold depolarizing current pulses often evoked a depolarizing
bump (Fig. 7A), generally of longer duration than those seen
in anomalous SNs. Suprathreshold current pulses evoked regular spiking,
with a higher spike rate at the onset of the pulse that declined to a
constant rate for the remainder of the pulse (Fig. 7B).
Firing rate increased roughly linearly with current amplitude, and most
of these cells were capable of sustained firing at high rates (>60 Hz
for at least 500 ms, 4 of 6 tested; Fig. 7D). We recovered
three of these neurons filled with neurobiotin and found that two
possessed thin, beaded, aspiny dendrites (Fig. 7E, both in
LPO). One filled cell of this type, recorded in PA, possessed thicker
dendrites (0.5-2 µm diam), but little else can be said about the
morphology of this cell because of the poor quality of the fill. We
refer to these neurons as the aspiny, fast-firing (AF) cell type.
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Although the AF neuron does not closely resemble any cell type
identified in mammalian dorsal striatum, it might be a substantially modified version of one of the striatal interneuron types. One such
interneuron type is the cholinergic "long-lasting
afterhyperpolarization" (LA) cell (Kawaguchi 1993). To
help evaluate the possibility that the AF cells are the avian
counterpart of striatal cholinergic interneurons, we compared the
morphology of our neurobiotin-labeled AF neurons with zebra finch
striatal cells immunostained for choline acetyltransferase (ChAT), the
enzyme that synthesizes acetylcholine. ChAT immunostaining labels the
somata and proximal dendrites of these presumptively cholinergic
neurons, so we compared the thickness of their proximal dendrites (~2
µm from the soma). Our two "thin dendrite" AF cells have
dendritic diameters of 0.2-0.6 µm. The dendrites of ChAT-positive
cells are clearly thicker, averaging 1.8 µm diam (range: 1-3 µm;
SD: 0.4 µm; n = 20 cells from 2 birds). Our "thick
dendrite" AF cell recorded in caudal PA was morphologically more
similar to ChAT-positive cells, but because there are apparently no
ChAT-positive cells in caudal PA (Li and Sakaguchi 1997
;
Medina and Reiner 1994
; Zuschratter and Scheich
1990
; personal observations), it is unlikely that this cell was cholinergic.
Comparison of PA and LPO
Karten and Dubbeldam (1973) have stated that PA and
LPO are "strikingly different" in cytology and hodological
relationships. We wondered whether these anatomical differences were
accompanied by physiological differences in their neurons. All avian
striatal cell types we identified (SN, LTS, AF) were found in both
structures. We compared the physiological properties of their SNs (the
only cell type common enough to permit comparison) and found a
statistically significant difference in their input resistance: LPO SNs
have higher input resistances (614 ± 212 M
, n = 5 vs. 300 ± 129 M
, n = 16; Mann-Whitney
test, P = 0.009), consistent with Karten and
Dubbeldam's observation that LPO contains smaller cells on average. The only other statistically significant difference was a
longer time to peak for AHPs of LPO SNs (3.46 ± 0.50 vs.
2.82 ± 2.30 ms, P = 0.04), which could be due to
a longer membrane time constant resulting from the higher input
resistance of LPO SNs. A full list of the comparisons made between PA
and LPO SNs is shown in Table 2. It is perfectly possible that there
exist physiological differences between the two structures in
properties we did not examine, but there were no qualitative
physiological differences uncovered by this study.
Intrinsic electrophysiological properties of putative PP neurons
We attempted to record neurons in PP, the avian homologue of the globus pallidus. Obtaining recordings in this region is difficult because the density of cells in PP is low and the density of fibers is high. Furthermore, PP is a relatively small and narrow region whose borders are not clearly visible in unstained slices (see METHODS). Many of the cells recorded while targeting PP were of the striatal SN type. In all cases where we could confirm the location of such cells using the Nissl stain (5 of 9 SN neurons recorded while targeting PP), they proved to be in PA or on the PA-PP border. Because the Nissl stain degraded the quality of our neurobiotin fills, we could not confirm the locations of all recorded cells in this way. We recorded other cell types while targeting PP that may be representatives of bona-fide PP neurons, but we obtained only a handful of such recordings, and in no case could we be absolutely certain that these cells were in PP. Thus, we offer these "PP recordings" with reservations; some of these neurons may have been in PA.
Two putative PP neurons (recorded from different birds) spontaneously
fired bursts of 12-18 action potentials every 5-10 s (Fig.
8A). The membrane potential of
these neurons was virtually impossible to controlmembrane potential
oscillations persisted even when these cells were hyperpolarized by
continuous current injection. These cells show some signs of
time-dependent inward rectification and tend to fire a burst on rebound
following the end of hyperpolarizing current pulses (Fig.
8B). The action potentials of these cells were distinctive
in that each spike was usually followed by an afterdepolarization (Fig.
8C); they were the only cells we recorded in the avian basal
ganglia that displayed this property. Neither of these bursting cells
was recovered after filling with neurobiotin, so their morphology is
unknown.
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Another group of putative PP cells (n = 3 cells
from 3 birds) spontaneously fired action potentials at regular
intervals (Fig. 9C) and
generally resembled the AF cell type of PA and LPO. Like AF neurons,
these cells displayed time-dependent inward rectification when
hyperpolarized (Fig. 9, A and D). One of these
cells displayed a prominent depolarizing bump in membrane potential
when injected with a depolarizing current pulse from a hyperpolarized
baseline potential (Fig. 9E). Another cell (1 of 2 tested)
could sustain firing rates exceeding 60 Hz for at least 500 ms when
injected with a strongly depolarizing current pulse (Fig.
9B). One cell was filled with neurobiotin well enough to
show the morphology of the dendrites; this cell is shown in Fig.
9F. These three "PP" cells resemble a cell type
identified in the rat entopeduncular nucleus ("type I" cells of
Nakanishi et al. 1990), the rat homologue of the
internal segment of the globus pallidus. This is consistent with the
notion that these neurons are avian pallidal cells, but their
physiological similarity to the AF cell type of PA and LPO raises
doubts concerning their true location.
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Unclassified neurons recorded in the paleostriatal complex
We recorded 11 neurons (5 in PA, 3 in LPO, 3 when targeting PP) that we could not classify as a member of any of the cell types described above. Five of these cells bore some resemblance to cholinergic LA or GABAergic "fast-spiking" (FS) interneurons identified in mammalian striatum (3 LA, 2 FS). However, they did not exhibit physiological properties consistent enough to warrant classification into distinct types. The unclassified cells could be singular examples of rare cell types (possibly including types not identified in mammals), damaged examples of the types described above (although some of these neurons lacked any overt physiological signs of damage), or cells drawn from a highly variable population of neurons that cannot be divided into distinct types. Further recordings might clarify the nature and significance of these cells.
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DISCUSSION |
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Our main results are that avian striatum contains two cell types that are virtually identical to cell types found in mammalian striatum (SNs and LTS cells). However, avian striatum also contains one cell type (the AF cell) that has not been identified in mammalian striatum. Thus the intrinsic physiological properties of avian striatal neurons are very similar, but not identical, to those in mammals.
Similarities between avian and mammalian striatum
The most common cell type we recorded in avian striatum, the spiny
neuron, displayed two characteristic physiological properties that it
shared with the principal cell type of mammalian striatum, the medium
spiny neuron. First, these cells have fast inward rectification so that
their membrane resistance rapidly decreases as they are hyperpolarized.
Second, these cells exhibit a ramping response and delayed spiking when
depolarized, known to result primarily from the gradual inactivation of
an A-type K+ current in mammals (Nisenbaum
and Wilson 1995). Since the ramping response of our SNs shows
similar temporal characteristics and is also blocked by 4-AP, an A-type
K+ current probably contributes to the ramp in
birds as well. Both the fast inward rectification and the A-type
K+ current can be expected to profoundly
influence the response of these neurons to synaptic input
(Wilson 1995
; Wilson and Kawaguchi 1996
).
We also identified one rare class of neuron, the LTS cell, that closely
resembles an interneuron type identified in mammalian striatum
(Kawaguchi 1993). The defining feature these cells share with their mammalian counterparts is the ability to generate a plateau-like spike that can last for more than 100 ms, generally accompanied by a few conventional fast action potentials near the onset
of this persistent spike. The mammalian and avian LTS cells also share
a varying degree of time-dependent inward rectification and the
tendency to fire on rebound following the end of hyperpolarizing current pulses. We did not observe any qualitative differences between
avian and mammalian LTS cells. The LTS cells of mammals contain NADPH
diaphorase (Kawaguchi 1993
), an enzyme used in the synthesis of nitric oxide. This may also be true of avian LTS cells
because avian PA and LPO contain NADPH diaphorase-positive neurons
(von Bartheld and Schober 1997
;
Wallhausser-Franke et al. 1995
).
Although we did identify one mammalian striatal interneuron type in
avian striatum, the mammalian striatum has two additional physiological
classes of interneuron that we did not unambiguously observe in birds:
the cholinergic LA neuron and the GABAergic FS neuron (Kawaguchi
1993). However, one cannot conclude that these types are absent
in birds; these cells comprise only a tiny fraction of the neurons
found in the striatum and thus should be rarely encountered with the
blind recording technique we used. Indeed, much of avian striatum (all
of LPO, perhaps some of PA) does have a population of presumptively
cholinergic neurons containing ChAT (Medina and Reiner
1994
; Zuschratter and Scheich 1990
). In summary,
the data currently available provide no reason to believe that the LA
and FS cell types are absent in birds.
Differences between avian and mammalian striatum
We found two prominent differences between avian and mammalian striatum. First, we recorded cells that were morphologically indistinguishable from SNs and yet possessed some physiological properties that have not been recognized in mammalian striatal spiny neurons. We favor the hypothesis that these ASNs are actually SNs that have been damaged or are in an otherwise altered state. In support of this idea, we note that these cells did share some physiological properties with SNs: almost all ASNs displayed fast inward rectification when hyperpolarized and many displayed a ramping response to depolarization. Furthermore, some ASNs exhibited the physiological properties of normal SNs for some time, switching to or from an anomalous state during the recording. ASNs sometimes exhibited other properties associated with unhealthy neurons, such as an inability to fire repetitively or a propensity to fire abnormally small and broad spikes. Finally, there are unpublished observations of ASN-like behavior in mammalian striatum; such behavior is sometimes seen in medium spiny neurons after intracellular dialysis (C. Wilson, personal communication). Nevertheless, it is possible that ASNs represent a population of cells not present in mammalian striatum.
A second difference we discovered between mammalian and avian striatum
is the presence of an AF cell type in birds. These cells have no
obvious counterpart in mammalian striatum, but we considered the
possibility that they are a modified version of the cholinergic LA
interneurons or GABAergic FS interneurons of mammals. Mammalian LA
neurons are spontaneously active in vivo (Wilson et al.
1990) and can be spontaneously active in vitro (Bennett
and Wilson 1999
; but see Kawaguchi 1992
and 1993
). In addition, both avian AF cells and
mammalian LA cells display time-dependent inward rectification when
hyperpolarized (Kawaguchi 1992
, 1993
). However, avian AF
cells fire spontaneously at higher rates than do mammalian LA cells in
vitro and in vivo, and AF cells lack the distinctive long-lasting AHP
of LA cells. Most importantly, AF cells appear morphologically distinct
from mammalian LA cells and ChAT-immunoreactive cells in
avian striatum. Thus AF cells are unlikely to be the avian counterpart
of cholinergic LA interneurons. AF cells could also be a modified
version of the mammalian FS interneuron, but the differences between
the two types are substantial: FS cells do not fire spontaneously in
slices, do not display time-dependent inward rectification, and when
injected with depolarizing current produce pauses in firing not seen in
AF cells (Kawaguchi 1993
).
An alternative explanation for the presence of AF cells in avian
striatum is that they are functionally globus pallidus (GP) neurons. AF
cells closely resemble a cell type recorded in the rat entopeduncular
nucleus, the rat homologue of the internal segment of the GP.
Specifically, both types are spontaneously active in brain slices,
exhibit time-dependent inward rectification, are capable of sustained
firing at high rates, and display rebound firing following
hyperpolarizing current pulses (Nakanishi et al. 1990).
AF cells also resemble a cell type we have tentatively identified in
PP, the avian homologue of the GP. Further support for this idea comes
from the projections of a specialized region of the LPO found only in
songbirds, known as area X. Area X contains the same morphological and
physiological cell types we have reported here in PA and LPO, including
the AF cell type (Farries and Perkel 1998
). Unlike
mammalian striatum, but like the mammalian and avian GP, area X
projects directly to the thalamus (Nottebohm et al. 1976
), and those projection neurons are not the
spiny neuron type that comprises the projection neurons of mammalian
striatum. Instead, the projection neurons of area X are a sparsely
distributed population of GABAergic neurons that morphologically
resemble the AF cell type (Luo and Perkel 1999
). This
thalamic projection could be a specialization limited to the song
system, but there is evidence that the LPO of nonsinging birds projects
to thalamus as well (Székely et al. 1994
). The
notion that the striatum can harbor "pallidal" cells may seem
strange, but it is not unprecedented. The cholinergic interneurons of
mammalian striatum are actually born in the anlage of the GP (the
medial ganglionic eminence) and later migrate into the striatum
(Olsson et al. 1998
). In addition, there are reports
that a small subset of the nigral-projecting neurons of mammalian
striatum are not MSNs, and morphologically resemble neurons
of the GP (Bolam et al. 1981
; Fisher et al.
1986
).
Conclusions
Previous studies have revealed that the mammalian and avian basal
ganglia are remarkably similar in their anatomical and neurochemical organization (Medina and Reiner 1995). The present study
builds on this work by demonstrating a strong resemblance in the
physiological properties of their neurons. Together these results
suggest that the basal ganglia of these two vertebrate classes process
their inputs in a similar manner, using similar mechanisms. However, we
also identified an apparent difference between the basal ganglia of
mammals and birds: the presence of the AF cell type in avian striatum.
From the data currently available, the functional significance of this
difference is unclear; there are several possibilities. First, the AF
cell could represent a new avian striatal cell type or a highly
modified version of a cell type found in mammals; this raises the
possibility of at least some divergence in basal ganglia function
between birds and mammals. Second, the AF cell could be a pallidal cell
type that has migrated into avian striatum. This scenario suggests that
avian and mammalian basal ganglia might have diverged somewhat in
anatomical organization but remain functionally equivalentthe
pallidal AF cells might receive input from striatal SNs and project to
the standard GP targets (e.g., thalamus), as the example of area X
suggests. Third, it is conceivable that the zebra finch striatum is not
representative of the striatum of all birds; perhaps only a subset of
avian taxa have diverged from an ancestral condition that lacks the AF
cell type. Finally, we may be comparing avian PA and LPO to the wrong
mammalian structures. Different regions of striatum tend to receive
inputs from different regions of pallium e.g., isocortex projects to
dorsal striatum and the pallial amygdala projects to ventral striatum
(de Olmos and Heimer 1999
). There is little doubt that
PA and LPO are striatal, but they may not be homologous to mammalian
dorsal striatum.
This last possibility is supported by comparisons of mammalian and
avian pallium; most of the avian pallium appears to be derived from
ventral and lateral pallium (Puelles et al. 1999; Smith-Fernandez et al. 1998
; Striedter et al.
1998
) and thus would be homologous to the pallial amygdala,
piriform cortex, and endopiriform claustrum of mammals (not
isocortex). If this is true, birds and mammals have undergone expansion
of different pallial regions, which may have been accompanied by
differential expansion of their associated striatal regions. Thus, the
avian striatum may be dominated by homologues of ventral
striatal regions such as the nucleus accumbens and striatal amygdala,
structures that receive input from lateral and ventral pallium. The
physiology of these structures has not been as thoroughly studied as
that of dorsal striatum; it is conceivable that a mammalian homologue
of the AF cell type exists in these regions.
In summary, the results reported here are consistent with the
hypothesis that PA and LPO are homologous to mammalian striatum. However, our results also suggest that it may be premature to conclude
that PA and LPO are homologous to dorsal striatum; PA and
LPO may be more akin to ventral striatum. Parts of the ventral striatum
(particularly the extended amygdala) deviate from the canonical pattern
of striatal connections and organization (de Olmos and Heimer
1999). It remains to be seen if dorsal and ventral striatum are
functionally equivalent, i.e., perform the same kind of processing on
different kinds of information. Similarly, we cannot yet say to what
degree the avian and mammalian basal ganglia are functionally
equivalent, although our data suggest a substantial resemblance.
Additional comparative studies measuring cellular physiological
properties will be necessary to obtain a full understanding of how
basal ganglia function has changed during vertebrate evolution.
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ACKNOWLEDGMENTS |
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We thank L. Stark for the use of ChAT immunostained material from zebra finches. We also thank C. Wilson, D. Jaeger, M. Schmidt, D. Contreras, and J. Cardin for helpful comments during preparation of the manuscript.
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FOOTNOTES |
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Present address and address for reprint requests: D. J. Perkel, Depts. of Zoology and Otolaryngology, University of Washington, 1959 NE Pacific St. HSB BB1165, Box 356515, Seattle, WA 98195-6515 (E-mail: perkel{at}u.washington.edu).
Received 28 April 2000; accepted in final form 28 July 2000.
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REFERENCES |
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