Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee Memphis, Memphis, Tennessee 38163
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ABSTRACT |
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Ogura, Mitsuhiro and
Hitoshi Kita.
Dynorphin Exerts Both Postsynaptic and Presynaptic Effects in the
Globus Pallidus of the Rat.
J. Neurophysiol. 83: 3366-3376, 2000.
The opioids contained in
striato-pallidal axons are thought to play a significant role in motor
control. We examined post- and presynaptic effects of the kappa
()-receptor agonist dynorphin A (1-13) (DYN13) on the globus
pallidus (GP) neurons in rat brain slice preparations using the whole
cell recording method. DYN13 hyperpolarized and decreased the input
resistance of approximately one-quarter of neurons examined. All of
these DYN13-sensitive neurons had medium-sized somata, large aspiny
dendrites and generated repetitive firing without strong accommodation.
The hyperpolarization was blocked by barium and was independent of TTX
and intracellular chloride levels. The hyperpolarization was also
selectively blocked by the
-antagonist nor-binaltorphimine
dihydrochloride but not by the mu- or delta-antagonists. These data
suggested that DYN13 activates barium-sensitive potassium currents in
some GP neurons. Low- and high-intensity stimulation of the neostriatum
(Str) evoked long- and short-latency GABAergic responses, respectively.
Previous data suggested that the long- and the short-latency responses were due to activation of the striato-pallidal axons and the local collaterals of pallido-striatal axons, respectively. DYN13 diminished the amplitude of both the short- and long-latency GABAergic responses in all the neurons tested. The effects of DYN13 on GABAergic
postsynaptic responses were also selectively blocked by a
-antagonist. To investigate whether the effects were pre- or
postsynaptic, the effects of DYN13 on spontaneous inhibitory
postsynaptic potentials (IPSPs) and TTX-independent
miniature-inhibitory postsynaptic currents (IPSCs) were examined.
DYN13 decreased the frequency, but not the amplitude, of spontaneous
IPSCs and calcium-dependent miniature-IPSCs. However, DYN13 did not
alter the cadmium-insensitive miniature-IPSCs. These results suggested
that DYN13 suppressed GABA release from presynaptic terminals. This
possibility was tested using a paired-stimulation test. DYN13 reduced
the probability of evoking IPSCs to the first stimulation and greatly
increased the success probability to the second stimulus. The amplitude of successfully evoked IPSCs was not changed with DYN13. DYN13 did not
affect the excitatory postsynaptic potentials (EPSPs) or the response
to iontophoretically applied GABA and glutamate. Together, these
results suggest that DYN released from striato-pallidal axons controls
the activity of GP neurons 1) by directly hyperpolarizing a
population of neurons and 2) by presynaptically inhibiting
GABA release from striato-pallidal and intrapallidal terminals.
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INTRODUCTION |
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The globus pallidus (GP) of the rodent is
homologous to the external segment of the pallidum of the primate. The
GP is a large nucleus located caudomedially to the neostriatum (Str).
The existence of rich reciprocal connections between the GP and other
basal ganglia nuclei implies that the GP plays a significant role in controlling the neuronal activity of the entire basal ganglia (reviews
by Chesselet and Delfs 1996; Kita 1994a
;
Kita et al. 1999
; Mink 1996
; Smith
et al. 1998
). Anatomical and morphological studies suggest that
the activity of GP neurons is controlled by three major inputs: Str
inhibitory inputs, subthalamic excitatory inputs, and intrinsic
collateral inhibitory inputs (Chesselet and Delfs 1996
;
Kita 1994a
; Mink 1996
). The
striato-pallidal fibers form the largest number of terminals in the GP
and terminate mainly on the dendritic shafts of GP neurons
(Falls et al. 1983
; Okoyama et al. 1987
).
Intracellular staining and immunohistochemical studies indicated that
the striato-pallidal terminals could be classified into two major
groups: approximately two-thirds of the terminals contain GABA and
enkephalin and belong to the Str neurons projecting only to the GP; the
other one-third contain GABA, dynorphin (DYN) and substance P and
belong to the collateral axons of Str neurons projecting to the
entopeduncular nucleus and the substantia nigra (Gerfen and
Young 1988
; Kawaguchi et al. 1990
; Lee et
al. 1997
; Penny et al. 1986
). The subthalamic
axons form the second greatest number of terminals in the GP. These
axons terminate on the somata and dendrites of GP neurons. Stimulation
of the subthalamo-pallidal pathway evokes powerful excitatory responses
in GP neurons (Kita 1994a
; Kita and Kitai
1991
). The third largest number of terminals in the GP are
formed by the local-collateral axons of GABAergic GP projection neurons
(Kita 1994b
). Intracellular staining studies have shown
that all GP projection neurons have local-collateral axons
(Bevan et al. 1998
; Kita and Kitai 1994
;
Nambu and Llinas 1997
). These collateral axons may
strongly inhibit GP neurons because they terminate on the somata and
proximal dendrites of GP neurons (Kita 1994b
).
Several possible roles of opioids contained in the Str efferent fibers
have been described recently. DYN and enkephalin are endogenous ligands
of kappa ()- and mu (µ)-opioid receptors, respectively
(Brookes and Bradley 1984
; Chavkin et al.
1982
; Raynor et al. 1994
). Both types of
receptors are found in the GP (Mansour et al. 1988
;
Morris and Herz 1986
; Sharif and Hughes
1989
). A recent study indicated that µ-opioid receptor
agonists presynaptically inhibit GABAergic transmission in the GP of
rat brain slice preparations (Stanford and Cooper 1999
).
Several studies suggested that DYN may also play a prominent role in
controlling the neuronal activity of the GP. Intrapallidal injection of
-selective agonists caused a slowing of the contralateral head turn
movement evoked by Str electrical stimulation (Slater and
Longman 1980
). Intrapallidal injection of
-agonists
decreased apomorphine-induced circling behavior of the rats that had
received unilateral deafferentation of nigro-striatal dopaminergic
fibers (Slater 1982
). In rats with unilateral dopamine
lesions, administration of apomorphine resulted in a marked increase in
the expression of mRNA for DYN in the lesioned site of the Str
(Gerfen et al. 1991
). Systemic or iontophoretically applied morphine or DYN decreased unit activity of the GP in
anesthetized rats (Huffman and Felpel 1981
;
Huffman and Frey 1989
; Napier et al.
1983a
,b
). These behavioral and unit recording studies all suggest that DYN may play a significant role in controlling the activity of GP neurons.
The aim of this study was to explore the underlying mechanism of these
-agonist actions cited above using whole cell recordings in brain
slice preparations. Specifically, we examined the effects of the
-agonist dynorphin A (1-13) (DYN13) on the neuronal membrane and
GABAergic and glutamatergic synaptic transmissions in the GP. The
results indicate that DYN13 exerts both post- and presynaptic effects
in the GP. Preliminary accounts of these results have appeared
previously (Ogura and Kita 1998
).
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METHODS |
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Slice preparations
Sprague-Dawley juvenile rats (15-21 days old, 28-45 g) of both
sexes were used. The physiological responses to current injection and
synaptic stimulation and the morphology of the juvenile GP neurons
reported herein were very similar to those reported for adults GP
(Bevan et al. 1998; Kita and Kitai 1991
,
1994
; Nambu and Llinas 1997
). Furthermore, the
properties of immunohistochemical staining for enkephalin, glutamate
decarboxylase, and parvalbumin in the GP of juvenile rats (H. Kita,
unpublished observation) were similar to those of adult rat GP
(Kita 1994b
; Kita and Kitai 1994
).
Animals were anesthetized (ip) with a mixture of Ketamine (85 mg/kg)
and Xylazine (15 mg/kg) and were perfused through the heart with cold
oxygenated artificial cerebrospinal fluid (ACSF). After decapitation,
the brains were rapidly removed and blocks containing the GP were
obtained. Parasagittal slices (300 µm thick) were cut from the blocks
on a Vibroslice (Campden, UK) in ice-cold ACSF. The slices were
incubated in ACSF at 37°C for 1 h before recording. The
composition of ACSF (in mM) was 124 NaCl, 5.0 KCl, 1.24 KH2PO4, 26 NaHCO3, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose.
Recording and electrical stimulation
The slices were transferred to a recording chamber with
oxygenated ACSF continuously perfused at a flow rate of 2 ml/min. The
temperature of the recording chamber was kept at 34°C. Whole cell
patch recording pipettes with a tip diameter of about 1.5 µm were
pulled from 1.5 mm, thin wall, borosilicate glass capillaries on a
horizontal electrode puller (P-87, Sutter Instruments, Navato, CA). Two
kinds of electrolytes were used to fill the pipettes. For inhibitory
postsynaptic potential (IPSP) and inhibitory postsynaptic current
(IPSC) recordings, the pipettes were filled with high-Cl electrolyte
containing (in mM) 90 K-gluconate, 50 KCl, 10 HEPES, 2 Mg-ATP, and 0.2 Na-GTP, 0.2% Neurobiotin, with pH adjusted to 7.2 with KOH. The
chloride equilibrium potential of the cells was calculated to be 25
mV by the Nernst equation when the cytoplasm of the recorded cells was
fully equilibrated with 50 mM chloride. For EPSP recordings, the
pipettes were filled with low-Cl electrolyte containing (in mM) 135 K-gluconate, 5 KCl, 10 HEPES, 2 Mg-ATP, and 0.2 Na-GTP, 0.2%
Neurobiotin, with pH adjusted to 7.2. The resistance of these recording
pipettes ranged from 4.0 to 8.0 M
. Neurons and recording pipettes
were visualized using an infrared-differential interference contrast
microscope Reichert Diastar (Leica, Deerfield, IL) with a ×40 water
immersion objective (Carl Zeiss, Thornwood, NY) and a CCD camera
model-6412 (COHU).
Current- and voltage-clamp recordings were obtained using an amplifier
IR183 (Neurodata Instruments, New York, NY) and an electrometer
AXOPATCH 200B (Axon Instruments, Foster City, CA), respectively. The
output of the amplifiers was monitored with an oscilloscope D51
(Tektronix, Beaverton, OR) and a chart recorder WindoGraf (GOULD
Instrument Systems, Valley View, OH). Neurons with the resting membrane
potential more negative than 60 mV were deemed acceptable for
recording. All data were digitized with an NEURO-CORDER model DR-484
(Neurodata Instruments) and stored on videotape. A Macintosh computer
with the data analysis program Oscilloscope, written and generously
provided by Dr. C. J. Wilson, was used for data analysis.
To evoke postsynaptic responses, electrical stimulation (0.2 ms in duration) was applied through a bipolar electrode. The electrode was placed in the Str or the internal capsule to evoke IPSPs/IPSCs or excitatory postsynaptic potentials (EPSPs), respectively. To isolate GABAergic responses from glutamatergic ones, the N-methyl-D-aspartate (NMDA) receptor antagonist, 3-(2-carboxypiperzin-4-yl)-propyl-1-phosponic acid (CPP, 10 µM), and the non-NMDA receptor antagonist, 6,7-dinitroquinozaline-2,3-dione (DNQX, 25-50 µM), were applied to the bath. To isolate glutamatergic responses, the GABAA receptor blocker bicuculline methiodide (50 µM) was applied to the bath. The efficacy of DNQX to block the glutamatergic response was tested on the response to iontophoretically applied glutamate. A concentration of 25-50 µM DNQX was required to block the response to <10% of the control amplitude. Based on this data, the routine concentration of DNQX used in this study was 50 µM. A similar method was used to determine the concentrations of CPP and bicuculline methiodide. DNQX (50 µM), CPP (10 µM), and bicuculline methiodide (50 µM) did not alter the membrane potential, the input resistance, or the shape of the action potentials of GP neurons. To record action potential-independent miniature-IPSCs (mIPSCs), tetrodotoxin (TTX, 1 µM) was applied in addition to the glutamate blockers. The glutamate and GABAA antagonists and TTX were obtained from Research Biochemicals International (RBI, Natick, MA).
Iontophoretic application of GABA and glutamate
Two-barreled pipettes, one barrel containing GABA (0.1 M, pH 4.8) or monosodium L-glutamate (0.1 M, pH 7.0) and the other saline, were placed approximately 30 µm from the cells. An ejection current pulse with an intensity of 5-30 nA and duration of 30-100 ms was applied between the drug and the saline containing barrels using a constant current pump NEURO PHORE BH-2 (Medical System Crop, Greenvale, NY).
Opioid receptor ligand
The following opioid receptor ligands were used: the -agonist
DYN13 (0.1-20 µM); nonselective antagonist naloxone hydrochloride (5-10 µM);
-receptor selective antagonist nor-binaltorphimine dihydrochloride (nor-BNI, 1-5 µM);
-receptor selective antagonist naltrindole hydrochloride (1-2 µM); and the µ-receptor selective antagonist
D-Phe-Cys-Tyr-D-trp-Orn-Thr-Pen-Thr-NH2
(CTOP, 1-4 µM). These drugs, obtained from RBI, were dissolved in
deoxygenated water as concentrated stocks. The stocks were aliquoted
and stored at
20°C. They were thawed and diluted immediately before use.
Histology
After recording, the slices were fixed overnight with a mixture
of 4% paraformaldehyde and 0.2% picric acid. The fixed slices were
rinsed several times with buffered saline, incubated overnight with
avidin-biotinhorseradish peroxidase (HRP) complex (1% in buffered
saline with 0.4% Triton-X 100), rinsed, and then reacted with
diaminobenzidine. The slices were postfixed with 0.5% osmium tetroxide, infiltrated with a plastic resin, and mounted onto glass
slides. The stained neurons were drawn under the microscope BH2
(Olympus, Tokyo, Japan) equipped with a drawing tube and a ×60 dry objective.
Statistics
All group data were expressed as a means ± SE, and
analyzed statistically using a Student's t-test or an
ANOVA. The Kolmogorov-Smirnov test (Press et al. 1986)
was used to determine the statistical significance of the amplitude
distributions of spontaneous IPSCs.
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RESULTS |
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DYN13 hyperpolarizes some GP neurons
Bath application of DYN13 (1 µM) hyperpolarized 15 of 61 GP
neurons that were recorded using the current-clamp method under three
different recording conditions (Table 1).
Fourteen and 38 neurons were recorded with the low- and high-Cl
electrolyte containing pipettes, respectively. Nine others were
recorded with the high-Cl electrolyte containing pipettes in the
presence of TTX (1 µM). DYN13 (1 µM) hyperpolarized approximately
one-quarter of neurons in every condition (Table 1). The DYN13-induced
hyperpolarization was accompanied by a decrease (16.2 ± 3.6%,
n = 15) in the input resistance at the resting membrane
potential (Fig. 1). The hyperpolarization was reversed by washing (n = 7), by the nonselective
opioid antagonist naloxone (5 µM, n = 4), or by the
-opioid receptor selective antagonist nor-BNI (0.2 µM,
n = 4; Fig. 1A), but not by the
-antagonist naltrindole (1 µM, n = 3) or the
µ-antagonist CTOP (1 µM, n = 3). DYN13 depolarized
one of the neurons recorded with a high-Cl electrolyte containing
pipette (Table 1). Washing reversed the depolarization, but no further
tests were possible because of a clogged recording pipette.
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All of the DYN13-sensitive neurons generated sustained firing without
strong accommodation on injection of supra-threshold current pulses
(Fig. 1B). Stimulation of DYN13-insensitive neurons generated either sustained firing or a short train of firing with strong accommodation. Intracellular staining with Neurobiotin revealed
that the neurons with sustained firing were of medium size (12.3 ± 0.2 × 27.8 ± 0.5 µm, mean ± SE,
n = 15) and had two to four thick, slowly tapering,
smooth dendrites (an example shown in Fig.
2). Distal dendrites had occasional small
spines. The neurons with strong accommodation had smaller (9.8 ± 0.2 × 24.2 ± 1.0 µm, n = 9) somata and
had two to five thin dendrites with spines (data not shown) (see
Kita and Kitai 1994; Nambu and Llinas 1997
).
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The current-voltage relationship curves obtained from the
DYN13-sensitive neurons before and after DYN13 application crossed at
85.2 ± 1.7 mV (n = 15), which is near the
potassium-equilibrium potential of 83 mV estimated by using the
Nernst equation. This suggested the possibility that DYN13 activated
inwardly rectifying potassium channels in these neurons. To test this
possibility, barium chloride (100 µM) was applied to the slice before
DYN13. Barium caused a depolarization (5.7 ± 0.6 mV,
n = 22) and an increase (147-208%) in the input
resistance of all 22 GP neurons tested. None of these barium
chloride-treated neurons were hyperpolarized by DYN13.
DYN13 diminishes inhibitory postsynaptic response
Stimulation of the Str evoked GABAergic responses in GP neurons of
slices perfused with ACSF containing DNQX (50 µM) and CPP (10 µM).
Recent anatomical studies have revealed that a large number of GP
neurons project to the Str and that all of these neurons have local
collateral axons (Bevan et al. 1998; Kita and Kitai 1994
; Kita et al. 1999
; Nambu and
Llinas 1997
). Thus it can be expected that Str stimulation
activates both striato-pallidal fibers and local collaterals of
pallido-striatal fibers. Str stimulation induced responses were
recorded in the initial part of the study with a current-clamp
amplifier (n = 24) and later with a voltage-clamp amplifier (n = 73). Str stimulation at threshold
intensity evoked very small (<3 mV or 10 pA) IPSPs (n = 22) and IPSCs (n = 64) with a latency of 7-10 ms in
86 of 97 GP neurons tested. When the stimulus intensity was gradually
increased, the response amplitude gradually increased, and the latency
gradually shortened to about 6 ms (example in Fig. 4A). When
the stimulus intensity was increased to some value, the response
amplitude steeply increased, and the latency steeply decreased to about
4 ms. In the majority (81 of 86) of the neurons, the long-latency
component blended into the preceding short-latency component and could
not be isolated. In five neurons, however, the late component was large
enough so that the amplitude could be measured from the crest of the
short-latency response. Data obtained from these five neurons are
presented below. In 11 of 97 neurons, Str stimulation at threshold
intensity evoked short-latency (approximately 4 ms) large IPSPs
(n = 2) and IPSCs (n = 9) without the
long-latency IPSCs seen with other neurons. The short-latency response
was very stable with time and could be tested with DYN13. The
long-latency response to low-intensity stimulation could not be tested
reliably with DYN13 because of large fluctuations in the amplitude and
a tendency to lose the amplitude with time.
Bath application of DYN13 (0.1-20 µM) reduced the amplitude of short-latency IPSPs and IPSCs of all GP neurons (n = 62) tested (Fig. 3, A and B). Figure 3C shows the time course of the IPSP amplitude change. The maximal effect was obtained within 10 min after initiation of DYN13 (1 µM) application and was reversed gradually by washing the tissue. More than 1 h of washing was often required to reverse the response to the control level. The DNY13 effect was dose dependent and reached near maximum at 5 µM (Fig. 3D). The dose-response curve for DYN13 was fitted with a Langmuir isotherm of the form: IPSC = IPSCsensitive (1 + [A]/EC50)-n + IPSCresistant, where IPSCsensitive was the blockable postsynaptic responses, [A] was the agonist concentration, and IPSCresistant was the portion of the responses resistant to modulation. The curve best fit using a least square method with EC50 = 0.68, n = 1.4, and IPSCresistant = 15%.
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In five neurons, Str stimulation evoked complex IPSCs with two distinguishable short- and long-latency components (Fig. 4A). The long-latency IPSCs induced by low stimulus currents had small amplitudes. With an increase in the stimulus intensity, stable complex IPSCs with two latency components, approximately 4 and 6 ms, were evoked (an example shown in Fig. 4A). DYN13 (1 µM) decreased the amplitude of the short-latency IPSCs by 57.6% and the long-latency IPSCs by 35.6% (Fig. 4C). The reduction in amplitude was greater for the short-latency IPSPs, and the difference in the reduction between the short- and long-latency IPSPs was statistically significant (P < 0.0002, paired t-test).
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To verify the receptors involved in the DYN13 effect on the GABAergic
response, various opioid antagonists were tested. A nonselective opioid
receptor antagonist naloxone (5 µM) and the -selective antagonist
nor-BNI (0.2 µM) completely antagonized the DYN13 effect. However,
the
-antagonist naltrindole (1 µM) and the µ-antagonist CTOP (1 µM) had no effect (Fig. 5). Without DYN13, naloxone, Nor-BIN, naltrindole, and CTOP had no effect on the
evoked IPSCs.
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DYN13 failed to diminish the response to iontophoretically applied GABA
The effect of DYN13 on GABA receptors at the postsynaptic membrane was studied using the iontophoretic GABA application method. Two-barreled pipettes, one barrel containing 0.1 M GABA and the other containing saline, were placed approximately 30 µm from the somata of the recording neurons. Iontophoresis of GABA by constant current pulses (5-30 nA in amplitude and 30-100 ms in duration) between the GABA and the saline containing pipette induced 13- to 25-mV depolarizations in the neurons when recordings were made with high-Cl electrolyte containing pipettes (n = 10). The response was sensitive to bicuculline methiodide (50 µM) and thus was considered GABAA receptor mediated (Fig. 6). Of 10 neurons tested, 8 were DYN insensitive and two DYN sensitive. In the eight DYN-insensitive neurons, bath application of DYN13 (2 µM) did not change the response to exogenous GABA (Fig. 6). In the two DYN-sensitive neurons, DYN13 (2 µM) decreased the response to GABA by approximately 10% (data not shown). The reduction was much smaller than that observed in the synaptically induced GABAergic responses. This small reduction might be due to an increase of the membrane conductance at distal dendrites. These results indicate that DYN13 does not change the GABA sensitivity of the postsynaptic membrane.
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DYN13 decreases the frequency of spontaneous IPSCs
All the GP neurons examined exhibited spontaneous
GABAergic IPSCs. The amplitudes of spontaneous IPSCs were as high as
220 pA when the membrane potential was clamped at the resting membrane potential of 65 to
75 mV. We analyzed spontaneous IPSCs that were
obtained from DYN-insensitive neurons and that had the amplitudes exceeding two times the noise level (i.e., typically 1-3 pA). The rise
time of these spontaneous IPSCs was 1.7 ± 0.05 ms
(n = 20). DYN13 (1 µM) diminished the frequency of
the spontaneous IPSCs (Fig.
7C) without changing their
mean amplitude (Fig. 7D) or the amplitude distribution (Fig.
7E). The decrease in frequency was fully reversible with
washing (Fig. 7C) and was also completely blocked by nor-BNI
(0.2 µM; n = 3, data not shown). These results and
the iontophoretic GABA application experiment described above suggest
that DYN13 suppresses presynaptic GABA release but not postsynaptic
GABA sensitivity. To investigate this further, the following
experiments were performed.
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DYN13 decreases the frequency of calcium-dependent mIPSCs
Action potential-independent mIPSCs were recorded from GP neurons
using the high-Cl electrolyte containing pipettes in the presence of
TTX (1 µM), CPP (10 µM), and DNQX (50 µM). Under these conditions, the amplitudes of mIPSCs were as high as 150 pA, and the
frequency was <1 Hz (Fig. 8,
A and D). In five neurons, application of DYN13
(1 µM) resulted in an insignificant decrease (9%, P = 0.07, paired t-test) in the frequency with no change in
the amplitude. Application of cadmium (200 µM) reduced the frequency
of mIPSCs by approximately 60% without altering the amplitude. Thus
the insignificant reduction of mIPSCs frequency by DYN13 might be explained if only the Ca-dependent portion of mIPSCs was modulated by
DYN13. To depolarize synaptic terminals and increase the occurrence of
Ca-dependent mIPSCs, the extracellular potassium concentration was
raised to 20 mM by substituting a portion of NaCl with KCl in ACSF
(Doze et al. 1995). The somatic membrane of the
recording neurons was clamped at its resting membrane potential in
control ACSF. Switching to the high-potassium ACSF did not change the amplitude but did increase the frequency of mIPSCs in all seven neurons
tested (Fig. 8, A, B, and E). DYN13 (1 µM) decreased the frequency of mIPSCs in all these neurons (Fig. 8,
A and F), without changing their mean amplitude
(Fig. 8B) or amplitude distributions (Fig. 8C).
Washing the slices with the high-potassium ACSF reversed the DYN13
effect (Fig. 8, A and B). The application of
cadmium (200 µM) to the high-potassium ACSF diminished the frequency, but not the amplitude, of mIPSCs by approximately two-thirds below that
measured in control ASCF (Fig. 8, A and B). DYN13
had no effect on the cadmium-insensitive mIPSPs (Fig. 8, A
and B). These results suggest that DYN13 inhibits
Ca-dependent, but not quantal, GABA release from terminals.
|
DYN13 decreases the success probability of IPSCs evoked by Str stimulation
To gain additional information about the presynaptic action of DYN13, the effect of DYN13 on the IPSCs with paired Str stimulation was examined. As mentioned above, Str stimulation with threshold intensity induced large IPSCs with a latency of approximately 4 ms in eight neurons. In five of the eight neurons, IPSCs with a constant latency and fairly constant amplitude could be evoked (Fig. 9A). The amplitude and the latency were unchanged over a wide range of stimulus intensities (e.g., 35-90 µA for the neuron shown in Fig. 9). In control ACSF, the success probability and the amplitude of the successfully evoked IPSCs to the first and second stimulus with an interstimulus interval more than 10 ms were very similar (Fig. 9, A, B, and D). Thus the probability ratio (i.e., success probability to the 2nd/1st stimulus) was approximately one (Fig. 9C). In the presence of DYN13 (1 µM), the success probability of evoking IPSCs to the first stimulus decreased significantly (Fig. 9B). The success probability to the second stimulus was higher than that to the first stimulus with an interstimulus interval of 10-60 ms. We chose the inter-stimulus interval of 25 ms in this study because it yielded the highest success probability to the second stimulus in three neurons tested with various inter-stimulus intervals. Thus the probability ratio was significantly higher in the presence of DYN13 than in control ACSF (Fig. 9B). DYN13 did not reduce the amplitude of successfully evoked IPSCs (Fig. 9D).
|
DYN13 does not modulate presynaptic glutamatergic release or postsynaptic glutamate sensitivity
Stimulation of the internal capsule evoked glutamatergic responses in GP neurons. DYN13 (1-2 µM) did not affect the EPSPs of 11 DYN-insensitive GP neurons. Three others that were DYN sensitive were hyperpolarized (5.3 ± 2.3 mV), and their input resistance was decreased (16.4 ± 6.4%). In these DYN-sensitive neurons, DYN13 decreased the amplitude of EPSPs by 39.9 ± 12.3%.
The effect of DYN13 on the postsynaptic glutamate receptors was studied
using iontophoretic glutamate application with appropriate receptor
blockers. The method of iontophoretic application of glutamate was
similar to that used for GABA application. Both CPP-sensitive NMDA
responses and DNQX-sensitive
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate
responses evoked in DYN-insensitive neurons (n = 8)
were unaffected by DYN13 (1 µM). These results suggest that DYN13
does not change the glutamate sensitivity of the postsynaptic membrane.
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DISCUSSION |
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This is the first report of DYN13 effects on single GP neurons in slice preparations using the whole cell recording technique. The results indicate that DYN13 exerts both post- and presynaptic effects on GP neurons.
Postsynaptic effects of DNY13
DYN13 caused a hyperpolarization and a decrease in the input
resistance in approximately one-quarter of the GP neurons. The hyperpolarization was thought to be due to an activation of
-receptors because only the
-receptor-selective antagonist, not
- or µ-antagonists, blocked the response. The hyperpolarization
was insensitive to TTX and was independent of the intracellular
chloride concentration. The current-voltage relationship curves before
and after DYN13 application crossed at approximately
85 mV, very
close to the predicted potassium equilibrium potential of these
neurons. These results suggested that the postsynaptic effect of DYN13
involved a potassium conductance. Direct pharmacological examination of the response was difficult because only a quarter of GP neurons were
DYN sensitive and because it was difficult to maintain long recordings
for repeated application of DYN13 to single neurons. However, we tested
the effect of barium on neurons that were not tested for DYN
sensitivity. The result that DYN13 hyperpolarized none of 22 barium-treated neurons suggested that the hyperpolarization was due to
the activation of a barium-sensitive inwardly rectifying potassium
conductance. Activation of the potassium conductance by
-agonists
has been reported in rat bulbospinal neurons (Hayar and Guyenet
1998
) and in guinea pig substantia gelatinosa neurons (Grudt and Williams 1993
). It has been also shown that
-receptors and inwardly rectifying potassium channels coexpressed by
Xenopus oocytes formed functional couplings
(Henry et al. 1995
; Ikeda at al.
1995
). The hyperpolarization of GP neurons by DYN13 was consistent with the results of a unit recording study in anesthetized rats showing that iontophoretically applied
-agonists depressed spontaneous firing and discharges to iontophoretically applied glutamate in some GP neurons (Huffman and Frey 1989
).
All DYN13-sensitive GP neurons were medium-sized aspiny neurons,
and all were capable of firing continuously without strong spike
accommodation. These neurons are known to project to the subthalamic
nucleus and the substantia nigra. Some of these neurons project to the
Str as well (Bevan et al. 1998; Kita and Kitai 1994
; Nambu and Llinas 1997
). It should be noted
that DYN13 failed to hyperpolarize some aspiny neurons that shared very
similar morphological and physiological properties with the
DYN13-sensitive neurons.
DYN13 effects on GABAergic synaptic transmission
DNY13 greatly reduced the amplitude of postsynaptic GABAergic
responses in all of the GP neurons tested. This effect was most likely
mediated by -receptors because the
-receptor antagonist blocked
the effect, but
- or µ-antagonists did not. A recent report
indicated that µ-agonists had a similar effect in the GP (Stanford and Cooper 1999
). We have confirmed Stanford
and Cooper's results that the µ-agonist
Tyr-D-Ala-Gly-(NMe) Phe-Gly-ol (DAMGO) reduced the
amplitude of IPSCs and that the effect was antagonized by the
µ-agonist CTOP (1 µM) but not by the
-antagonist nor-BNI (Ogura and Kita 1999
). Thus it was unlikely in the
present study that DYN13 and nor-BNI acted as a partial µ-agonist and
antagonist, respectively.
DYN13 failed to decrease the response to iontophoretically
applied GABA. DYN13 decreased the frequency of both spontaneous IPSCs
and high-potassium-induced mIPSCs without changing the amplitude of either. In the paired-stimulation test, DYN13 decreased the success
probability of evoking IPSCs but increased the probability ratio (i.e.,
probability of the 2nd/the 1st stimulation). These results suggest that
DNY13 presynaptically modulates GABAergic transmission. The reduction
of neurotransmitter release by -agonists has been reported in
several brain areas including the glutamatergic mossy fibers in the
hippocampus (Gannon and Terrian 1991
; Wagner et
al. 1993
; Weisskopf et al. 1993
), glutamatergic
inputs to bulbospinal neurons (Hayar and Guyenet 1998
),
dopaminergic fibers in the Str (Mulder et al. 1984
),
glutamate release in Str synaptosomes (Hill and Brotchie
1995
), and the dorsal root ganglion in culture
(Macdonald and Nelson 1978
).
Multiple mechanisms may account for the DNY13 modulation of presynaptic
GABA release in the GP. One mechanism may be to decrease spiking at the
somata of DYN-sensitive GP neurons, hence reducing the number of the
spikes arriving to the local-collateral terminals. Another mechanism
may be due to the direct action of DYN13 on the synaptic terminals.
DYN13 may hyperpolarize the synaptic terminals of DYN-sensitive GP
neurons by activating an inwardly rectifying potassium current as it
did in their somata. The hyperpolarization would indirectly reduce
Ca-inflow to the terminals by enhancing the rate of spike
repolarization, thereby reducing GABA release. Our preliminary results
indicate that barium partially blocked the DYN13 effect on the evoked
IPSCs (M. Ogura and H. Kita, unpublished data). These
preliminary data also suggested that multiple mechanisms may be
involved in the presynaptic action of DYN13. Other possible mechanisms
include an activation of a voltage-gated potassium current
(Muller et al. 1999; Simmons and Chavkin
1996
; Vaughan et al. 1997
) and an inhibition of
the high-threshold voltage-gated calcium channels (Kanemasa et
al. 1995
; Moises et al. 1994
; Rusin et
al. 1997
; Simmons and Chavkin 1996
;
Vaughan et al. 1997
). The result that DYN13 reduced the
frequency but not the amplitude of the high-potassium-induced mIPSCs
is consistent with the possibility that
-agonists might directly
reduce voltage-gated Ca-currents.
Origins of GABAergic responses
It has become increasingly clear that at least one-third of GP
neurons project to the Str (Bevan et al. 1998;
Kita and Kitai 1994
; Kita et al. 1999
).
Thus electrical stimulation of the Str might activate both
striato-pallidal and pallido-striatal GABAergic fibers. The response
observed in most GP neurons to low-intensity Str stimulation was a
long-latency small IPSP or IPSC. A gradual increase in the stimulus
intensity resulted in a large jump in amplitude and a shortening of the
latency to approximately 4 ms. We consider that the long- and the
short-latency responses were mediated by striato-pallidal axons and the
local collateral axons of pallido-striatal neurons, respectively, for
the following reasons: 1) the threshold current needed to
stimulate the somata of Str projection neurons should be lower than
that for the pallido-striatal axons, 2) the striato-pallidal
axons have much slower conduction velocity than the pallido-striatal
axons (Kita 1994a
; Nambu and Llinas
1994
), 3) the striato-pallidal axons form synapses
mainly on the dendrites of GP neurons (Falls et al.
1983
; Okoyama et al. 1987
; Smith et al.
1998
), 4) all the pallido-striatal neurons have
intrinsic collateral axons that form synapses on the somata and
proximal dendrites of GP neurons (Bevan et al. 1998
;
Kita and Kitai 1991
, 1994
; Smith
et al. 1998
), and 5) the latency of the antidromic
spikes recorded in the GP after Str stimulation ranges from 1-3 ms
(Kita and Kitai 1991
; Nambu and Llinas
1994
; Walker et al. 1989
). This latency is
consistent with the short-latency response obtained in this study. It
is also likely that the spontaneous and TTX-independent mIPSCs
originated from the local collateral axons of GP neurons. The
spontaneous IPSCs that had a large amplitude and a short rise time
might have been evoked by the synapses on the somata and proximal
dendrites of the recorded neurons.
We have examined the effects of DYN13 mainly on the short-latency IPSCs because they were large in amplitude and very stable for long periods. The long-latency IPSCs could be examined with DYN13 in five neurons. The results of the present study suggest that DYN13 reduced GABA release from the terminals of both the striato-pallidal and local-collateral axons of GP projection neurons.
Modulation of glutamatergic input by -agonist
The GP receives the major glutamatergic input from the
subthalamic nucleus (Kita 1994a; Kita and Kitai
1991
; Smith et al. 1998
). The subthalamic input
plays a significant role in maintaining the activity and in shaping the
firing patterns of GP neurons. The possibility that the opioids
released from Str terminals hetero-synaptically modulate the
subthalamic input was suggested by an observation of Huffman and
Frey (1989)
. They showed that iontophoretically applied
-agonists, DYN13 and benzomorphan, depressed spontaneous firing and
discharges to iontophoretically applied glutamate in some GP neurons in
anesthetized rats. The present results show that DYN13 does not
modulate EPSPs or the response to the iontophoretically applied
glutamate in DYN-insensitive neurons. DYN13 decreased the amplitude of
EPSPs only in DYN-sensitive neurons. Thus it is likely that DYN13
reduces glutamatergic responses by decreasing the input resistance of
neurons but not by presynaptic modulation.
Functional significance
Possible functional roles of the opioids contained in the Str
spiny projection neurons have begun to be described. Jiang and North (1992) have reported a presynaptic inhibition of
GABAergic transmission by the opioids in rat Str. They observed that
the local stimulation-induced GABAergic response was reduced by
-agonists but not by
- and µ-agonists, and speculated that
-agonists affected local collateral axons of the Str projection
neurons. More recently, Stanford and Cooper (1999)
showed that µ- and
-agonists presynaptically inhibit GABAergic
transmission in the GP. These authors considered 1) that
µ-receptors are located on the terminals of striato-pallidal axons
and local-collateral axons of spontaneously active GP neurons and
2) that
-receptors are located on the local axon
terminals of quiescent GP neurons. We report here that the
-agonist
DYN13 affects the GP in two different manners. One is a direct
inhibitory action on approximately one-quarter of GP neurons by
activating the inwardly rectifying potassium conductance, and the other
is a presynaptic inhibition of the GABAergic terminals of the local collateral axons of GP projection neurons and the striato-pallidal axons.
In the GP, DYN is contained in the collaterals of
striato-entopeduncular and striato-nigral axons. It can be speculated
from the results obtained in the present study that DYN released in the
GP by activation of the Str efferent pathways will have multiple effects on GP neurons. DYN may directly inhibit a small subpopulation of GP projection neurons by activating postsynaptic -receptors. In a
larger subpopulation, DYN and another striatal opioid, enkephalin, may
act to disinhibit GP neurons by presynaptically suppressing GABA
release from both the striato-pallidal and the local-collateral terminals. The results of Stanford and Cooper (1999)
and
the present experiment indicate that the suppression of GABA release by
the striatal opioids is accompanied by a development of the paired pulse facilitation. Thus it can be further suggested that the striatal
opioids might alter response characteristics of GP neurons to
high-frequency burst GABAergic inputs.
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ACKNOWLEDGMENTS |
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We thank Dr. W. E. Armstrong for helpful comments and critical reading during the preparation of the manuscript. We also thank D. Merrick for editing the manuscript.
This study was supported by National Institute of Neurological Disorders and Stroke Grants NS-26473 and NS-36720.
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FOOTNOTES |
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Address for reprint requests: H. Kita, Dept. of Anatomy and Neurobiology, College of Medicine, The University of Tennessee Memphis, 855 Monroe Ave., Memphis, TN 38163.
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 17 August 1999; accepted in final form 15 February 2000.
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REFERENCES |
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