Department of Physiology and Pharmacology, The University of Queensland, Brisbane Qld 4072, Australia
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ABSTRACT |
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Bengtson, C. Peter and
Peregrine B. Osborne.
Electrophysiological Properties of Cholinergic and Noncholinergic
Neurons in the Ventral Pallidal Region of the Nucleus Basalis in Rat
Brain Slices.
J. Neurophysiol. 83: 2649-2660, 2000.
The ventral pallidum is a major source of output
for ventral corticobasal ganglia circuits that function in translating
motivationally relevant stimuli into adaptive behavioral responses. In
this study, whole cell patch-clamp recordings were made from ventral
pallidal neurons in brain slices from 6- to 18-day-old rats.
Intracellular filling with biocytin was used to correlate the
electrophysiological and morphological properties of cholinergic and
noncholinergic neurons identified by choline acetyltransferase
immunohistochemistry. Most cholinergic neurons had a large whole cell
conductance and exhibited marked fast (i.e., anomalous) inward
rectification. These cells typically did not fire spontaneously, had a
hyperpolarized resting membrane potential, and also exhibited a
prominent spike afterhyperpolarization (AHP) and strong spike
accommodation. Noncholinergic neurons had a smaller whole cell
conductance, and the majority of these cells exhibited marked
time-dependent inward rectification that was due to an h-current. This
current activated slowly over several hundred milliseconds at
potentials more negative than 80 mV. Noncholinergic neurons fired
tonically in regular or intermittent patterns, and two-thirds of the
cells fired spontaneously. Depolarizing current injection in current
clamp did not cause spike accommodation but markedly increased the
firing frequency and in some cells also altered the pattern of firing.
Spontaneous tetrodotoxin-sensitive GABAA-mediated
inhibitory postsynaptic currents (IPSCs) were frequently recorded in
noncholinergic neurons. These results show that cholinergic pallidal
neurons have similar properties to magnocellular cholinergic neurons in
other parts of the forebrain, except that they exhibit strong spike
accommodation. Noncholinergic ventral pallidal neurons have large
h-currents that could have a physiological role in determining the rate
or pattern of firing of these cells.
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INTRODUCTION |
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The ventral pallidum was first identified in the
rat as a subcommissural extension of the globus pallidus (Heimer
and Wilson 1975). By virtue of the dense projection it receives
from the nucleus accumbens (Heimer et al. 1991
;
Nauta et al. 1978
; Zahm and Heimer 1990
),
the ventral pallidum is a major source of output for ventral
corticobasal ganglia circuits that are organized in parallel to dorsal
basal ganglia-thalamocortical pathways involved in sensorimotor and
associative functions (Alexander and Crutcher 1990
;
Alexander et al. 1986
; Groenewegen et al.
1990
; Parent and Hazrati 1995
; Smith et
al. 1998
). In contrast to their dorsal counterparts, ventral
striatopallidal pathways receive "limbic" afferent input from the
cortex, hippocampus, and amygdala and are considered to play a role in
translating motivationally relevant stimuli into adaptive behavioral
responses (Kalivas 1993
; Kalivas et al.
1999
; LeMoal and Simon 1991
;
Mogenson et al. 1990
).
The ventral pallidum in the rat contains two main populations of
neurons. The majority are GABA neurons (i.e., immunoreactive for
glutamic acid decarboxylase) (Gritti et al. 1993), which
have a somatodendritic morphology that is shared with the main cell group found in the dorsal pallidum (Iwahori and Mizuno
1981
; Millhouse 1986
; Nambu and
Llinás 1997
; Pang et al. 1998
; Park
et al. 1982
). Intracellular filling has separated these neurons
into those with simple axons and few intranuclear branches, or those
with complex axons and extensive intranuclear arborizations, which may
correspond to projection and interneurons, respectively (Kita
and Kitai 1994
; Pang et al. 1998
). Targets
supplied by ventral pallidal projection neurons include both structures
that are traditionally included in the "extrapyramidal motor
system" (entopeduncular nucleus, substantia nigra, and mesencephalic
locomotor area) and "limbic" circuits (ventral tegmental area,
mediodorsal nucleus of the thalamus, medial prefrontal cortex,
basolateral amygdala, and entorhinal cortex) (Bevan et al.
1996
; Groenewegen et al. 1993
; Haber et al. 1985
, 1993
; Maurice et al.
1997
; O'Donnell et al. 1997
; Zahm and
Heimer 1990
; Zahm et al. 1996
). About 23% of
the cell population in the rat ventral pallidum are neurons that belong
to the magnocellular forebrain cholinergic system (Gritti et al.
1993
), which has been studied extensively in relation to its
function in cognitive processing and putative role in disease processes
such as schizophrenia and Alzheimer's disease (e.g.,
Détári et al. 1999
; Page and
Sofroniew 1996
). In the rat these choline acetyltransferase
(ChAT) immunoreactive neurons intersperse with noncholinergic neurons
through a contiguous volume of basal forebrain that extends through the
medial septum, nucleus of the diagonal band of Broca, substantia
innominata, and the pallidal complex (Gritti et al.
1993
; Grove 1988
; Mesulam et al.
1983
; Rye et al. 1984
; Schwaber
et al. 1987
). The cholinergic neurons in the ventral pallidum
also receive synaptic input from the amygdala (Zaborszky et al.
1984
) and nucleus accumbens (Bolam et al. 1986
;
Zaborszky and Cullman 1992
) and project to the cerebral cortex, amygdala, and lateral habenula (Carlsen et al.
1985
; Ingham et al. 1985
; McKinney et al.
1983
; Mesulam et al. 1983
; Parent 1979
).
Three types of neuron have been identified in the ventral pallidum by
intracellular microelectrode recording in vivo (Lavin and Grace
1996). These were distinguished by electrophysiological criteria relating to action potential discharge patterns and responses to afferent fiber stimulation. Although no anatomic characterization was done in this study, two of these types (A and B) were tentatively identified as noncholinergic neurons based on electrophysiological similarities to neurons that have been morphologically characterized in
the globus pallidus (Kita and Kitai 1994
; Nambu
and Llinás 1997
) and entopeduncular nucleus
(Nakanishi et al. 1991
). More recently, ventral pallidal
neurons with similar patterns of firing identified by extracellular
recording in vivo, have been shown to be noncholinergic by using
juxtacellular staining in combination with immunohistochemistry for
ChAT (Pang et al. 1998
). The third type of neuron (type
C) identified in ventral pallidum may be cholinergic. The strongest
line of evidence supporting this conclusion is that these neurons fire
in bursts that are associated with a low-threshold calcium spike. This
pattern of firing is characteristic of magnocellular cholinergic
neurons as shown by many studies that have recorded from
immunohistochemically identified cells in other forebrain areas (rat:
Gorelova and Reiner 1996
; Markram and Segal
1990
; Sim and Allen 1998
; guinea-pig:
Alonso et al. 1996
; Griffith and Matthews
1986
; Khateb et al. 1992
, 1993
,
1995
).
Our aim in this study was to integrate the findings of previous studies
of pallidal neurons by correlating the electrophysiological and
morphological properties of identified noncholinergic and cholinergic
neurons. To do this, patch-clamp techniques were used to record from
ventral pallidal neurons in brain slices. The cells were
intracellularly filled and later immunolabeled for ChAT. Voltage-clamp
recording was used to characterize the expression of inward
rectification, which has been found to be diagnostic for cholinergic
neurons situated elsewhere in the rat magnocellular forebrain complex
(Gorelova and Reiner 1996; Markram and Segal 1989
; Sim and Allen 1998
). Current-clamp
recording was used for comparison with previous electrophysiological
studies in the pallidum and associated areas.
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METHODS |
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Brain slice preparation and recording
Transverse brain slices (200-250 µm thick) containing the ventral pallidum were prepared from 6- to 18-day-old Wistar rats that had been anesthetized by halothane inhalation and decapitated. The slices were submerged in ice-cold artificial cerebrospinal fluid (ACSF, containing in mM: 125 NaCl, 2.5 KCl, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, 25 glucose, and 25 NaHCO3) equilibrated with 95% O2-5% CO2, and cut with a tissue slicer (Campden Instruments). The slices were kept for recording, in a holding chamber, submerged in ACSF at RT (25°C).
Electrophysiological recordings were made from slices continuously
superfused with ACSF (32°C) that were placed in a chamber (0.75 ml
volume) mounted on a fixed-stage upright microscope (Zeiss Axioskop).
Differential interference contrast/infrared (DIC/IR) optics and
a charge-coupled device camera (Javelin) enabled neurons to be seen on
a video monitor. Patch-clamp recordings were made in the whole cell
configuration using an Axopatch 1D amplifier (Axon Instruments). The
electrodes (4-8 M) were filled with a potassium methylsulphate
based solution (containing, in mM, 135 KCH3SO4, 8 NaCl, 10 HEPES,
2 MgATP, and 0.25 NaGTP) that was used to minimize changes in the
action potential waveform with current-clamp recording (Sah and
Isaacson 1995
; Velumian et al. 1997
). The
solution also included 0.3% biocytin (Neurobiotin, Vector
Laboratories) and was adjusted to a pH of 7.3 with KOH, and an
osmolarity of 270-290 mosmol/l. Corrections were made for a calculated
liquid junction potential of the solution of
10 mV (Barry
1994
). All data were obtained from recordings where the series
resistance was maintained below 20 M
, which was monitored at regular
intervals throughout each experiment.
Intracellular filling and immunohistochemistry
Brain slices containing biocytin-filled neurons were fixed overnight (at 4°C) in 4% paraformaldehyde in phosphate buffer (PB: 0.1 M, pH 7.4), rinsed in PB, and placed for 4 days in 0.3% triton X-100 and 0.01% sodium azide in PB. After being rinsed in PB and placed for 60 min in 10% normal horse serum and 0.1% triton X-100 in PB, the slices were then incubated for 24 h at RT in a primary antiserum raised in goat against choline acetyltransferase (ChAT, 1:500, Chemicon). The slices were rinsed in PB and incubated for 1 h at RT in either 1) Cy3-conjugated donkey anti-goat IgG (1:1,000, Jackson Immunoresearch Laboratories) and FITC-conjugated streptavidin (1:1,000, Sigma Aldrich) or 2) FITC-conjugated rabbit anti-goat IgG (1:400, Sigma Aldrich) and Cy3-conjugated streptavidin (1:2,000, Sigma Aldrich). All antisera and streptavidin-conjugates were diluted in 1% normal horse serum and 0.3% triton X-100 in PB. An additional group of biocytin-filled neurons in 10 brain slices were single-stained using a Vectastain Elite ABC kit (Vector Laboratories) using 3',3'diaminobenzidene as the chromagen.
Drugs used
Barium chloride, bicuculline, cesium chloride (Sigma);
tetrodotoxin citrate (Tocris); and ZD7288 (Research Biochemicals) were made up as stock solutions in deionized water. Picrotoxin (Research Biochemicals) was made up as a stock solution in DMSO. All drugs were
diluted in artificial cerebrospinal fluid (ACSF) and applied by
superfusion. The final concentration of DMSO in the superfusate was
0.1%, which had no direct effects.
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RESULTS |
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Identification and morphological characteristics of cholinergic and noncholinergic ventral pallidal neurons
In this study we successfully recovered 49 biocytin-filled cells that had previously been electrophysiologically characterized in brain slices. Thirty-one of these were also subjected to choline acetyltransferase (ChAT) immunohistochemistry, which identified 13 ChAT-immunopositive and 18 ChAT-immunonegative neurons. Another eight small cells were filled that did not have neuronal morphologies (see final paragraph of this section); six of these were immunostained and shown to be ChAT-negative.
Three filled ChAT-positive neurons in fixed brain slices are shown in the top row of paired micrographs in Fig. 1. Recordings from this type of neuron were most commonly made in caudal areas of the ventral pallidum (Fig. 2). The filled neurons were typically found in close proximity to other ChAT-positive neurons, forming small clusters of two to five cells (e.g., Fig. 1A, left and right panels) or occasionally larger groups of 10 or more cells (e.g., Fig. 1A, middle panel). However, all filled cholinergic neurons were located some distance away from a large body of many hundred ChAT-immunopositive neurons that occupied an area of basal forebrain ventral to the pallidum. Measurements of soma size (made from the video monitor at the time of recording) were used to construct the frequency histogram in Fig. 3, which shows that cholinergic neurons had the largest somata (29 ± 1.5 µm, mean ± SE, n = 12) of the neurons we recorded from. The morphology of a representative ChAT-positive neuron is shown by the camera lucida drawing on the left of Fig. 4. These neurons typically had multipolar somata with four to seven thick, tapering primary dendrites that extended <100 µm before branching. In six fills, spines were visible on the soma, as well as forming a moderate covering on primary and higher order dendrites. In five cases, filled axons projected 200-1,000 µm from the soma in a ventral (n = 3) or medial (n = 2) direction, one of which had a short axon branch.
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Three examples of filled ChAT-immunonegative neurons are shown by the pairs of photomicrographs in Fig. 1B. Recordings were made from these noncholinergic neurons at all rostrocaudal levels of the ventral pallidum (Fig. 2). These cells had smaller somata than cholinergic neurons (Fig. 3; 14 ± 1.4 µm; range: 5-24 µm, n = 18; P < 0.001, Mann-Whitney U, n = 18, 12). A camera lucida drawing of a representative noncholinergic neuron is shown on the right of Fig. 4. These neurons typically had triangular somata with two to four thick tapering primary dendrites that extended less than 100 µm before branching. Spines were visible on both primary, and higher order dendrites in 13 of the fills. In eight cases, axons originating from primary dendrites that extended 200-1,200 µm within the 200-µm-thick brain slices. In three cases, axon branches were observed. The orientation of the projecting axons was ventral (n = 2), dorsomedial (n = 3), or both dorsal and ventral after bifurcating close to the soma (n = 2).
Eight cells were filled that had characteristics more typical of glial
cells (Fig. 4), particularly oligodendrocytes (e.g., Vernadakis
and Roots 1995). They had small somata (5.9 ± 0.5 µm; range: 4-8 µm) and multiple stubby primary processes, which branched to form an extensive tertiary arborization that failed to extend beyond
100 µm of the soma. Consistent with this identification, we were
unable to evoke action potentials in these cells in current clamp.
Cholinergic and noncholinergic ventral pallidal neurons express different inwardly rectifying currents
Only two of the ventral pallidal neurons we filled did not exhibit
inward rectification in voltage clamp in response to hyperpolarizing voltage steps from a holding potential of 60 mV. Representative recordings from a cholinergic and a noncholinergic neuron are shown in
Fig. 5, along with the corresponding
current-voltage (I-V) relationships for the
quasi-instantaneous and steady-state currents measured respectively at
the time points shown by the open and closed circles. In cholinergic
neurons the major inwardly rectifying current had fast activation
kinetics (Fig. 5A), whereas in noncholinergic neurons the
major current activated slowly over several hundred milliseconds
(
= 600 ± 230 ms at
100 mV, and 290 ± 80 ms at
120 mV; Fig. 5B).
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The values for GHOLD (slope
conductance at 60 to
80 mV), GINST
("instantaneous" conductance at
100 to
120 mV), and
GSS ("steady-state" conductance at
100 to
120 mV) were calculated from I-V relationships
and are summarized in Table 1. In
cholinergic neurons (n = 13) the conductance of the
fast inward rectifier current (equal to
GINST
GHOLD), was 4.5 ± 0.8 nS, and
the conductance of the time-dependent inward rectifier current (equal
to GSS
GINST) was 0.6 ± 0.2 nS. In
noncholinergic neurons (n = 18), the instantaneous
inward rectifier conductance was smaller (0.7 ± 0.2 nS;
Mann-Whitney U, P < 0.001, n = 13, 18), but the time-dependent current was larger
(2.1 ± 0.4 nS, P < 0.001, n = 13, 18) and contributed to a greater proportion of the total
conductance (41% cf. 5% of GSS).
These data are summarized in Fig. 6 in
which the instantaneous and time-dependent inward rectifier
conductances are plotted against the resting conductance,
GHOLD. Figure 6A shows that
in 10 of the 13 cholinergic neurons the large conductances of the fast inward rectifier and holding currents separate these neurons from the
remainder of the population. One noncholinergic neuron also fell in
this cluster, which based on its size (20 µm) and pattern of firing
in current clamp (see next section) could represent an
immunohistochemical false-negative. Figure 6B shows that in 14 of 18 noncholinergic neurons the conductance of the time-dependent inward rectifier was equal to or greater than the conductance of the
holding current. Only one cholinergic neuron fell into this group. Two
noncholinergic neurons (
, Fig. 6) did not exhibit rectification.
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In separate experiments, we tested the effects of channel blocking
drugs applied in the extracellular solution. Fast inward rectifier
currents were completely blocked by 200 µM barium (n = 4; Fig. 7A). Time-dependent
inward rectifier currents were not affected by barium
(n = 6) but were completely blocked by cesium ions
(n = 7; Fig. 7B) or by 30 µM ZD 7288 (n = 2), which has been reported to be a selective
blocker of h-current channels (BoSmith et al. 1993).
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Large, frequent spontaneous postsynaptic currents (PSCs) were
frequently observed in neurons that had strong time-dependent rectification (Fig. 8). These appeared to
be caused by the activity of other neurons in the slices as they were
abolished by 1 µM tetrodotoxin (n = 7). In most cases
the PSCs were blocked by 60 µM bicuculline (n = 11)
or 100 µM picrotoxin (n = 8) and reversed polarity
between 50 and
60 mV. However, in three neurons, spontaneous PSCs
were recorded that were not blocked by picrotoxin and reversed polarity
at potentials negative to
80 mV. In a further three neurons, apparent
excitatory postsynaptic currents (EPSCs) were recorded that reversed
polarity at potentials positive to
30 mV. Spontaneous PSCs were also
observed in cholinergic neurons but were less frequent and were not
analyzed in this study.
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Cholinergic and noncholinergic neurons show different patterns of action potential firing
Previous in vivo and in vitro studies have used intracellular
microelectrodes to make record membrane potential recordings in dorsal
and ventral pallidal neurons (Kita and Kitai 1994;
Lavin and Grace 1996
; Nambu and Llinás
1994
, 1997
) To further characterize the
properties of ventral pallidal neurons and facilitate comparison with
these earlier studies, current-clamp recordings were made from eight
cholinergic neurons and nine noncholinergic neurons.
Cell-attached recording identified spontaneous firing in only 2 of 13 cholinergic neurons. Immediately after establishing whole cell
recording, these cells had a mean resting membrane potential of
65 ± 2.7 mV (n = 13). In current-clamp,
hyperpolarizing current injection produced inward rectification, as
shown in Fig. 9A. Depolarizing
current injection evoked one to two action potentials near to threshold
of
41 ± 1 mV (n = 8), which accommodated during 1- to 1.4-s pulses (Fig. 9, A and C). The spikes
typically arose off a depolarizing plateau potential and had a mean
amplitude of 60 ± 9 mV and a half-width of 1.3 ± 0.1 ms.
They were followed by a large afterhyperpolarization (AHP) that was
23 ± 1.7 mV in amplitude and 288 ± 24 ms in duration.
Further depolarizing current injection increased the number of action
potentials from 1-2 spikes/s to 2-7 spikes/s, and the instantaneous
frequency (between the 1st spike pair) to maxima of 6-13 Hz. Spike
frequency accommodation was maintained during a 1-s pulse in seven of
eight neurons. One ChAT-immunonegative neuron exhibited a pattern of
action potential firing that was indistinguishable from the ChAT
immunopositive neurons. This cell had a large h-current (5.2 nS) but
had resting and inward rectifier conductances (8.7 and 3.5 nS,
respectively) that were similar to cholinergic neurons.
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Spontaneous firing was seen in 10 of 18 noncholinergic neurons with
cell-attached recording. Immediately after establishing whole cell
recording, the resting membrane potential of the cells was 53 ± 2.8 mV and less polarized than in cholinergic neurons (Mann-Whitney
U, P = 0.006, n = 13, 18).
Current-clamp recordings were made from eight neurons, four of which
fired spontaneous action potentials in continuous or irregular
patterns. Hyperpolarizing current injection produced a depolarizing sag
that developed slowly during a 1-s pulse at potentials negative to
80
mV, as shown in Fig. 9B. Depolarizing current injection
evoked action potentials that showed little or no spike frequency
adaptation. Action potentials measured near threshold (
45 ± 5 mV, n = 9) had a mean amplitude of 70 ± 3.5 mV
and half-width of 1.2 ± 0.2 ms and were followed by a prominent
AHP that was
18 ± 2.6 mV in amplitude (n = 8) and 133 ± 11 ms (n = 8) in duration. Graded
increases in the depolarizing currents increased the spike number from
around 1-6 spikes/s to 7-29 spikes/s and the instantaneous frequency
of the first spike pair from 1-2 Hz to rates over 15 Hz (Fig.
9D).
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DISCUSSION |
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To our knowledge this is the first electrophysiological study of the ventral pallidum to be performed in vitro. Patch-clamp recordings were made from noncholinergic and cholinergic neurons, which were identified by intracellular filling and immunolabeling for ChAT. This allowed us to use both voltage- and current-clamp recording to define the electrophysiological properties of these two neuron types.
Inward rectification in pallidal neurons
Prior to this study, voltage-clamp recording had not been used to
characterize pallidal neurons. We found that cholinergic and
noncholinergic neurons exhibited different types of inward rectification. In cholinergic neurons, there was a large inward rectifier current that had fast activation kinetics and was blocked by
barium, which suggests that it was carried by inward rectifier potassium channels (Gay and Stanfield 1977;
Standen and Stanfield 1978
; Uchimura et al.
1989
). In most noncholinergic neurons (13/18) there was a large
h-current that caused time-dependent rectification and was blocked by
cesium ions and the selective antagonist ZD7288 (BoSmith et al.
1993
; Mayer and Westbrook 1983
; Pape
1996
). Although h-currents were present in some cholinergic
neurons, they contributed to no more than 20% of the whole cell
conductance, whereas in the main group of noncholinergic neurons, the
h-current comprised 40-60% of the whole cell conductance. A group of
six neurons (2 cholinergic and 4 noncholinergic) either did not
rectify, or had inward rectifier currents with small conductances. Some
of these could represent additional electrophysiological subtypes of
pallidal neuron, or alternatively they could be cells damaged by brain slicing.
Previous studies, using intracellular microelectrodes and current-clamp
recording, provide conflicting reports on the expression of inward
rectification by pallidal neurons. Lavin and Grace
(1996) did not identify inward rectification in the rat ventral
pallidum using in vivo recording, whereas in guinea pig brain slices
Nambu and Llinás (1994)
identified fast inward
rectification but no time-dependent inward rectification in globus
pallidus neurons. However, in rat brain slices, time-dependent
rectification is exhibited by neurons in the entopeduncular nucleus
(homologous to the external globus pallidus in primates) and substantia
nigra pars reticularis (Nakanishi et al. 1987
,
1990
; Stanford and Lacey 1996
), which
have been reported to be similar to globus pallidus neurons
(DeLong and Georgopoulos 1981
; Iwahori and Mizuno
1981
; Nambu and Llinás 1994
). Although
time-dependent inward rectification may disappear in adult ventral
pallidal neurons (we used immature rats), we believe that the kinetics
and voltage dependence of the h-current in these cells could make it
difficult to identify using in vivo recording. In our recording
conditions the h-current activated at potentials negative to
80 with
an activation time constant of 600 ms at
100 mV, and 290 ms at
120
mV. Lavin and Grace (1996)
used current pulses around
200 ms in duration that hyperpolarized the neurons to around
90 mV as
it is difficult to deliver longer, larger pulses in vivo (A. Grace,
personal communication). The depolarizing sag that is characteristic of
the h-current would be difficult to resolve under these conditions (cf.
Fig. 9B). It is interesting to note that one of the two
mouse h-current channel proteins that have been recently cloned
(Ludwig et al. 1998
; Santoro et al. 1998
)
has slow activation kinetics (
activation: 241 ms, cf. 98 ms),
similar to the h-current in pallidal noncholinergic neurons.
Anomalous or fast inward rectification is an identifying characteristic
of magnocellular cholinergic neurons in other areas of the forebrain
cholinergic complex. This has been shown for histochemically identified
neurons in the medial septum and nucleus diagonal band of Broca complex
in rat (MS/DBB) (Gorelova and Reiner 1996;
Markram and Segal 1990
) and in more caudally located
nucleus basalis neurons in guinea pig (Alonso et al.
1996
; Griffith and Matthews 1986
; Khateb
et al. 1992
). It is not clear why anomalous inward
rectification was not exhibited by ventral pallidal neurons in vivo
(Lavin and Grace 1996
), although it is possible that
recordings were not made from cholinergic neurons in this study. The
difference in age could account for this result, although inward
rectification is maintained in other magnocellular cholinergic neurons
in both adult rat and guinea pig (Griffith 1988
;
Griffith and Matthews 1986
; Khateb et al.
1992
; Markram and Segal 1990
). Inward
rectification is also exhibited by all three types of globus pallidus
neurons identified in adult guinea pig (Nambu and Llinás
1994
).
Electrophysiological properties of pallidal neurons identified by current-clamp recording
Noncholinergic pallidal neurons identified by
ChAT-immunohistochemistry have been characterized by extracellular
recording in vivo combined with juxtacellular labeling (Pang et
al. 1998). This study identified two neuron types: cells with
axons that had few or no axon branches and fired in random or regular
patterns at around 13 Hz (type I), and cells with extensive axonal
arborisations that fired at a faster rate of 36 Hz (type II). Ventral
pallidal neurons have also been classified according to
electrophysiological criteria by intracellular recording in vivo
without reference to morphological or histochemical characteristics
(Lavin and Grace 1996
). Three types of neuron were
distinguished according to action potential discharge patterns and
responses to afferent fiber stimulation. Type A fired a tonic discharge
at 8.7 spikes/s and had long duration action potentials with no AHPs.
Type B fired at a rate of 14.5 spikes/s, and exhibited a slow ramplike
depolarization that preceded a short-duration spike and a prominent
afterpolarization. Type C fired in couplets or bursts and showed
accommodation. A similar scheme had previously been used to classify
globus pallidus neurons in guinea pig brain slices (Nambu and
Llinás 1994
, 1997
). Although not
completely identical, there is a general correspondence between the
type A, B, and C classes in rat, and respectively, type III, II, and I
classes in guinea pig (see Table 3 in Pang et al. 1998
).
We found in rat brain slices that the main characteristics
distinguishing cholinergic ventral pallidal neurons in current-clamp recordings were a more hyperpolarized resting membrane potential, the
longer duration of the spike AHP, and strong spike accommodation in
response to depolarizing current injection. Previous studies of
cholinergic neurons in other parts of the magnocellular complex have
shown that a slow AHP is a characteristic of these cells. However,
strong spike accommodation is not normally seen, and instead
depolarization from the resting membrane potential elicits continuous
single spikes in a slow rhythmic pattern (Alonso et al.
1996; Gorelova and Reiner 1996
; Griffith
1988
; Griffith and Matthews 1986
; Khateb
et al. 1992
, 1993
, 1995
;
Markram and Segal 1990
; Sim and Allen
1998
). The accommodation exhibited in our recordings could
result from the use of patch-clamp recording. In rat cholinergic
neurons in the medial septum and diagonal band of Broca (MS/DBB), a
slow AHP opposes spike frequency adaptation, which is rarely observed
in these cells (Gorelova and Reiner 1996
). However,
spike frequency adaptation is revealed if the AHP is shortened by using
channel blockers (e.g., cadmium or apamin), applying serotonin (5-HT),
or increasing the internal calcium concentration by altering the
buffering capacity of the electrode solution. Under these conditions
the normal slow (2-4 Hz) regular pattern of firing is lost, resulting
in high-frequency discharge in more complex patterns. In contrast,
although we saw accommodation in cholinergic pallidal neurons, they
maintained a slow regular rate of firing when mutiple spikes were
elicited by depolarizing current injection. Furthermore, although the
AHP was shorter in the ventral pallidum than in the MS/DBB (mean: 228 ms, cf. 375 ms) it was larger in amplitude (23 mV, cf. 10 mV). The
recording solution we used had no added EGTA or BAPTA and has been
shown previously to minimally affect the measurement of AHPs in other cells (Sah and Isaacson 1995
; Velumian et al.
1997
).
The type C/type I pallidal neurons respectively identified in rat and
guinea pig have been put forward as candidate cholinergic neurons. This
is based in part on the morphology of the guinea pig neurons
(Nambu and Llinás 1997) but more particularly on identification of high-frequency burst firing (Lavin and Grace 1996
; Nambu and Llinás 1994
,
1997
). This pattern of firing is produced in some
cholinergic neurons when depolarized by current injection from a
hyperpolarized resting membrane potential and is related to the
activity of a low-threshold calcium current (Alonso et al.
1996
; Khateb et al. 1992
). Although we did not see burst firing in pallidal cholinergic neurons, the effects of
depolarization from hyperpolarized potentials was not rigorously tested. However, it also should be noted that burst firing is not
necessarily a diagnostic characteristic for all cholinergic magnocellular neurons in the rat, because it is rarely observed in
MS/DBB cholinergic neurons (Gorelova and Reiner 1996
).
In general the type C neurons identified in rat ventral pallidum in
vivo correspond poorly with the cholinergic neurons we identified in vitro. They lack a prominent AHP, do not have a different input resistance to type A or B cells, and only two of four neurons showed
spike accommodation (Lavin and Grace 1996
). In contrast, type I neurons in the guinea pig globus pallidus do have an AHP (although it is relatively short in duration; mean: 93 ± 89 ms), show strong spike accommodation, and maintain a low frequency and
number of spikes during tonic firing in response to increased current
injection (Nambu and Llinás 1994
). This class also
has a low input resistance and has been morphologically identified by
intracellular filling as large neurons that are similar in appearance
to cholinergic neurons (Nambu and Llinás 1997
).
We found that noncholinergic neurons had action potential waveforms in
vitro that are most similar to the type B neurons of Lavin and
Grace (1996), which have a short-duration spike AHP and exhibit
a depolarizing ramp preceding the spike. We did not observe neurons
without an AHP, which is the identifying characteristic of type A
neurons. This may be a feature only seen in vivo, because the type III
globus pallidus neurons identified by in vitro recording in guinea pig
have an AHP but are otherwise very similar to the type A cells
(Nambu and Llinás 1994
,
1997
). In the rat, both type A and type B
neurons can be antidromically activated by stimulation from the
mediodorsal nucleus of the thalamus, which is strong evidence that both
types include GABA projection neurons. This is further supported by the
morphological identification of the counterparts of these cell types in
guinea pig (i.e., types II and III) (Nambu and Llinás
1997
).
We recorded spontaneous inhibitory postsynaptic currents (IPSCs) in
many noncholinergic neurons that were blocked by tetrodotoxin and
GABAA antagonists. The transverse (coronal)
orientation of the slices used for this study would disconnect most
extranuclear cell bodies from their pallidal terminals, especially
those in the nucleus accumbens, which is the major source of inhibitory input to the ventral pallidum. Therefore it is highly likely that these
tetrodotoxin-sensitive IPSCs involve synaptic transmission from the
terminals of spontaneously firing GABA neurons within the pallidum.
Golgi and intracellular staining studies in the pallidum have
identified intranuclear axons that form en passant and terminal boutons
(Iwahori and Mizuno 1981; Millhouse 1986
; Park et al. 1982
). Two basic patterns of axon branching
are seen, which may identify projection and local circuit neurons,
respectively: axons with infrequent or no intranuclear branches and
axons that form a more complex arborization that often does not extend
outside the dendritic field (Kita and Kitai 1994
;
Pang et al. 1998
). Stimulation of the
mediodorsal nucleus of the thalamus in vivo produces short-latency monosynaptic inhibition in type A and B pallidal neurons, which has
been attributed to release from axon collaterals in response to
antidromic activation of the soma (Lavin and Grace
1996
).
Concluding remarks
We show in this study that cholinergic ventral pallidal neurons
can be identified in brain slices by their large size in combination with strong inward rectification seen in voltage-clamp recording. Similarly, the major population of noncholinergic neurons can be
distinguished by a large h-current, which contributes to a major
proportion of the whole cell conductance. The ability to discriminate
these two neuron types using electrophysiological criteria will
simplify future studies of the synaptic physiology and pharmacological
properties of these neurons. The electrophysiological properties of the
magnocellular cholinergic neurons in the ventral pallidum largely
conform to the patterns seen in cholinergic neurons located in other
parts of the forebrain complex. The main outstanding feature shown by
the pallidal neurons is strong spike accommodation, but it remains to
be established if this is a feature that is maintained into adulthood.
The presence of a large h-current in pallidal noncholinergic neurons is
likely to be of major physiological significance. In addition to having
a well-characterized role in pacemaking, h-currents can also
participate in shaping more complex patterns of cell firing such as
rhythmic bursting (see review by Luthi and McCormick
1998). Furthermore, these currents are modulated by
intracellular cAMP and are a potential target for endogenous
transmitters such as dopamine and opioids that couple to adenyl cyclase
signaling, have been localized to the pallidum, and have been shown to
affect the firing of ventral pallidal neurons in vivo (e.g.,
Napier et al. 1991
; Napier and Maslowski-Cobuzzi
1994
; Mitrovic and Napier 1995
,
1996
).
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ACKNOWLEDGMENTS |
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The authors thank Dr. Janet Keast for assistance and facilities provided by her laboratory; Dr. Steven Robinson for making available Perl's stained rat brain sections; and Prof. David Adams and Drs. Trevor Day and David Vaney for equipment used in this study. We also thank Drs. John Williams and Janet Keast for reading and commenting on the manuscript.
This work was supported by project grant 971126 from the National Health and Medical Research Council of Australia (P. B. Osborne) and an Australian Postgraduate Award (C. P. Bengtson).
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FOOTNOTES |
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Present address and address for reprint requests: P. Osborne, Prince of Wales Medical Research Institute, High St. Randwick NSW 2031, Australia.
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 13 July 1999; accepted in final form 24 January 2000.
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REFERENCES |
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