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The Journal of Neuroscience, June 15, 2002, 22(12):4794-4804
Coordinate Release of ATP and GABA at In Vitro
Synapses of Lateral Hypothalamic Neurons
Young-Hwan
Jo and
Lorna W.
Role
Department of Anatomy and Cell Biology, Center for Neurobiology and
Behavior, Columbia University, College of Physicians and
Surgeons, New York, New York 10032
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ABSTRACT |
Autonomic and limbic information is integrated within the lateral
hypothalamus (LH), and excitability of LH neurons is important in the
control of feeding and behavioral arousal. Despite the prominent
expression of P2X-type ATP receptors throughout the hypothalamus, the role of ATP in LH excitability is not known. Perforated-patch-clamp recordings of synaptically coupled neurons from
both embryonic chick and postnatal mouse lateral hypothalamus in
vitro reveal robust stimulus-evoked purinergic synaptic
transmission. Suprathreshold activation elicits reliable and concurrent
release of ATP with GABA. Tetrodotoxin-resistant P2X receptor-mediated events are readily observed at LH synapses from the embryonic chick,
whereas GABA miniature postsynaptic currents (mPSCs) are recorded in innervated LH neurons from either embryonic chicks or
postnatal mice. Two distinct mPSCs are recorded at ATP-GABA cosynapses; one has a monoexponential decay phase and is modulated by
flunitrazepam, and the other has a decay phase that is best fit by a
sum of two exponential functions ( fast and
slow), and only the slow component
is affected by flunitrazepam. Bicuculline does not completely inhibit
all mPSCs. The remaining bicuculline-resistant mPSCs are blocked by
suramin, and their decay phase is briefer than that of GABAergic mPSCs.
Furthermore, at a holding potential intermediate for the reversal
potentials of GABAA and P2X receptors, little or no current
is observed, consistent with concomitant release (and detection) of
GABA and ATP. Together, our data suggest that a subset of spontaneous
and evoked PSCs arise from the concurrent activation of both
GABAA and P2X receptors.
Key words:
cotransmission; feeding; P2X receptor; plasticity; lateral hypothalamus; ATP; GABA; GABAA receptor
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INTRODUCTION |
The lateral hypothalamus (LH) is an
important center for the integration of autonomic and limbic
information. The LH is implicated in the modulation of visceral motor
and sensory pathways, including those underlying feeding (Bernardis and
Bellinger, 1996 ) and behavioral arousal (Chemelli et al., 1999; Hara et
al., 2001; Willie et al., 2001). Animal models with lesions of the
lateral hypothalamic area (LHA) exhibit hypophagia, increased metabolic
rate, and decreased arousal, often resulting in death by starvation
(Willie et al., 2001). The LHA has classically been regarded as an
important component of the autonomic control of feeding behavior, with
extensive projections both within the hypothalamus per se and
throughout the neuroaxis.
The hypothalamus is a prominent region of P2X-type ATP receptor
expression (Xiang et al., 1998; Kanjhan et al., 1999) and, as such,
might constitute a region of robust purinergic synaptic transmission.
Several of the ATP receptor subunits cloned (Brake et al., 1994; Valera
et al., 1994; Chen et al., 1995; Buell et al., 1996; Collo et al.,
1996; Surprenant et al., 1996) are expressed in specific brain regions,
including the LH. Mammalian CNS neurons express
P2X4, P2X6, and to a lesser
degree, P2X2-type purinergic receptors (Buell et
al., 1996; Collo et al., 1996; Kanjhan et al., 1999). A novel ATP P2X
receptor (P2X8) is expressed in embryonic chick
brain (Bo et al., 2000).
Despite the prominent expression of ATP receptor subunits in many
regions of the CNS (Collo et al., 1996; Kanjhan et al., 1999; Norenberg
and Illes, 2000), relatively little is known about purinergic
transmission in the brain. This is primarily because of the
difficulties inherent in the reliable identification of small-amplitude
synaptic currents and the lack of highly selective ATP P2X receptor
antagonists. So far, ATP P2X receptor-mediated transmission has been
detected as a minor component of synaptic activity in the mammalian
habenula (Edwards et al., 1992) and hippocampus (Pankratov et al.,
1998). In contrast, studies of peripheral (autonomic) neurons and of
spinal cord dorsal horn neurons suggest a prominent role of ATP as a
cotransmitter with norepinephrine, acetylcholine, or GABA (Burnstock,
1999; Jo and Schlichter, 1999). The net effect of ATP at such
cotransmitting sites is difficult to evaluate and may also be
complicated in view of recent reports demonstrating cross-inhibition of
ATP P2X receptors with both nicotinic acetylcholine receptors (Khakh et al., 2000) and GABAA receptors (Sokolova et al.,
2001).
Our initial aim was to ascertain whether ATP P2X receptor expression in
the hypothalamus underlies a significant contribution of ATP to
synaptic transmission. To test this idea, we examined 139 purinergic
synapses. We subsequently examined 72 synapses at which we detected
purinergic and GABAergic cotransmission. We found that a subset of
miniature postsynaptic currents (mPSCs) is composed of both
GABAA and P2X receptors and that ATP and GABA may
be costored within the same synaptic vesicle. ATP and GABA cotransmission could play an important role in the control of excitability of LH neurons at early stages of development, when GABA
exerts a depolarizing effect in hypothalamic neurons.
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MATERIALS AND METHODS |
Neuronal cultures. Primary cultures of lateral
hypothalamic neurons were prepared from embryonic day 11 chicks or from
postnatal day 3 mice using the following basic protocols. The region of the hypothalamus was identified on the ventral aspect of the brain with
the exterior border delineated by the optic chiasm and the posterior
border delineated by the infundibular stalk. The most lateral portions
of the demarcated area were excised, microdissected, and incubated at
37°C in divalent cation-free Earle's balanced salt solution
(EBSS; Invitrogen, San Diego, CA) containing papain (20 U/ml;
Sigma, St. Louis, MO) with L-cysteine (1 mM; Sigma). After 20-30 min of incubation, the
tissue was mechanically dispersed by repeated passage through a 10 ml
plastic pipette. Papain activity was stopped by adding 3 ml of EBSS
containing bovine serum albumin (1 mg/ml; Sigma) and DNase (0.01%;
Sigma), and a mechanical dissociation was performed with a 1 ml plastic
pipette. The homogenate was deposited on top of 4 ml of a solution with
a composition similar to that described above, except that the
concentration of bovine serum albumin was increased to 10 mg/ml. After
centrifugation (5 min at 1000 rpm), the preparation for plating was
collected by removing the supernatant and resuspending the neurons in
complete medium composed of DMEM (Invitrogen), heat-inactivated horse
serum (10% v/v; Invitrogen), chick extract (10% v/v, house-made),
penicillin and streptomycin (50 IU/ml for each; Invitrogen), and nerve
growth factor (10 nM). The equivalent "complete
medium" used for preparation and plating of LH neurons from postnatal
mice was MEM- (Invitrogen), horse serum (5% v/v), fetal calf serum
(5% v/v), putrescine (100 nM), transferrin (10 mg/ml), insulin (5 mg/ml), penicillin and streptomycin (50 IU/ml for
each; Invitrogen), and nerve growth factor (10 nM). LH neuron preparations obtained from ~12
embryonic day 11 chick brains were plated on six 35 mm
poly-L-ornithine-coated tissue-culture plastic
dishes and maintained until use in a water-saturated atmosphere (95%
air and 5% CO2) at 37°C. LH neuron
preparations obtained from ~12 postnatal mouse brains were plated on
six modified 35 mm tissue-culture plastic dishes. Each dish has a
chamber ~10 mm in diameter with a plastic ring on the bottom. As with
the chick neurons, the preferred substrate was
poly-L-ornithine.
Electrophysiological recording. Electrophysiological
recordings from either chick or mouse neurons in vitro
were conducted between 7 and 15 d after plating. Membrane currents
were recorded at room temperature (20-22°C) with an Axopatch 200B
amplifier (Axon Instruments, Foster City, CA) in the perforated
patch-clamp configuration using amphotericin B. The external solution
contained (in mM): NaCl 135, KCl 5, CaCl2 2.5, MgCl2 1, HEPES
5, and glucose 10, pH 7.3. In all experiments,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM),
DL-aminophosphonovaleric acid
(DL-APV, 50 µM) and strychnine (1 µM) (CAS) were continuously present in
the external solution. The amphotericin B (Sigma) stock solution
(30 mg/ml) was prepared in dimethylsulfoxide just before the recording
session. The pipette was first filled at the tip with internal solution containing (in mM): CsCl (or KCl) 150, HEPES 10, pH 7.3, and subsequently back-filled with the same solution containing
amphotericin B (150 µg/ml). Under these conditions, the equilibrium
potential for Cl ions
(ECl) was ~0 mV. To set
ECl to  70 mV, we replaced 141 mM CsCl or KCl with 70 mM
Cs2SO4 or 141 mM K-acetate, respectively, in the pipette
solution. Adjustment of ECl to 70 mV and
Ecations to 0 mV allowed recording of
cation-mediated components of synaptic transmission at a holding
potential (VH) of 70 mV and
recording of the Cl ion-mediated
component at a VH of 0 mV.
Eliciting monosynaptic evoked PSCs and detection of mPSCs.
To evoke PSCs, simultaneous perforated-patch-clamp recordings were obtained from synaptically connected neurons using two independent Axopatch 200B amplifiers. The presynaptic neuron was held in the current-clamp configuration at the resting membrane potential, and
depolarizing current pulses were applied at a frequency of 0.1 Hz. For
extracellular stimulations, a double-barreled electrode filled with
extracellular solution was placed in contact with the cell body of a
visually identified neuron. Stimulation was performed with short single
stimuli or pairs of stimuli [interval: 300 msec for GABAergic evoked
IPSCs (eIPSCs), 150 msec for purinergic evoked EPSCs (eEPSCs); 0.1 msec; 20 and 100 µA] delivered at 0.1 Hz. Our typical protocol
for isolation of purinergic eEPSCs and mPSCs involved setting
ECl to 70 mV (as described above) and recording
at a VH of 70 mV in the continuous
presence of CNQX, DL-APV, strychnine, and
bicuculline. Exceptions to this standard protocol are noted
throughout the text. Likewise, GABAergic eIPSCs were detected by
setting ECl to 70 mV and recording at a
VH of 0 mV in the continuous presence
of CAS. Individual spontaneously occurring mPSCs were analyzed off-line
using two commercially available softwares [Mini analysis 5.0 from
Synaptosoft Inc. (Decatur, GA) or Axograph 4.0 from Axon Instruments].
For each experiment, the threshold for detection was set to >5 pA,
except for the experiments performed at a
VH of 35. Under these circumstances,
recordings were also visually inspected to minimize data loss despite
the very small amplitude of the events. For analysis of the decay phase
of mPSCs, the events were selected on the basis of the following criteria: (1) that we were able to obtain a stable baseline recording both before and after the events were detected; (2) a minimum interval
of >100 msec between events; and (3) rise times of events that were
<3 msec (10-90% of the peak amplitude of the mPSCs). For examination
of average mPSC profiles, the events were aligned by their initial
rising phase using Mini analysis 5.0. Voltage and current traces were
stored on a videotape recorder and/or the hard drive of the analysis
computer after being filtered at 5 kHz by Axopatch 200B. Acquisition
and analysis were performed using pClamp6, Axograph 4.0 (Axon
Instruments) and Mini analysis 5.0. Student's t tests were
used to analyze the difference between parameters. The critical value
for statistical significance was set at p < 0.05. All
statistical results are given as mean ± SEM.
Immunocytochemical staining. LH neurons in vitro
from postnatal mice were fixed for 30 min at room temperature in 4%
paraformaldehyde in 0.1 M phosphate buffer, pH
7.4, and subsequently rinsed three times in PBS. Cultures were
subsequently permeabilized with 0.5% Triton X-100 for 5 min and
incubated overnight at room temperature with a mouse monoclonal
antibody against glutamic acid decarboxylase (GAD)-6 (1:200; a gift
from Dr. David I. Gottlieb, Washington University, St. Louis,
MO) and a rabbit polyclonal antibody against ATP
P2X4 receptor subunit (Alomone Labs, Jerusalem,
Israel). Cultures were rinsed three times with PBS and incubated for 1 hr at room temperature with a fluorescein isothiocyanate-conjugated
anti-mouse IgG (1:200) to reveal GAD-like immunoreactivity and a
rhodamine-conjugated anti-rabbit IgG (1:400) to reveal ATP
P2X4 receptor subunit-like immunoreactivity.
Preparation and application of drugs and other
reagents. Most reagents were prepared as 1000× concentrated stock
solutions. Bicuculline methiodide, strychnine, tetrodotoxin (TTX),
(1S,9R)- -hydrastine (all Sigma),
pyridoxalphosphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and
ARL67156 (both from Research Biochemicals, Natick, MA) were
prepared in distilled water and stored at 20°C.
DL-APV (Sigma) was prepared in 100 mM NaOH solution. CNQX (Tocris Cookson, Ballwin,
MO) was prepared in dimethylsulfoxide and stored at 4°C. Flunitrazepam (Sigma) was prepared as 10,000× concentrated
stock solution in dimethylsulfoxide. The substances to be
tested were diluted to the final concentration with extracellular
solution just before the recording session. Substances were
bath-applied at a flow rate of 2-3 ml/min. For exogenous application
of GABA and ATP, a 1.5-mm-diameter U tube controlled by a solenoid
valve was used. The threshold for responses was set to >5 pA.
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RESULTS |
Evoked ATP P2X receptor-mediated synaptic transmission
among the embryonic chick and mouse LH neurons
To examine stimulus-evoked release of ATP in isolation from other
fast transmitter responses, we first tested for synaptic transmission
using focal extracellular stimulation of potential presynaptic partners
selected on the basis of the apparent physical contact with the
recorded neuron. Such assays were conducted in the continued presence
of antagonists of ionotropic glutamate, glycine, and
GABAA receptors (in µM: CNQX 10, APV 50, strychnine 1, bicuculline 10; ECl = 70
mV; see Materials and Methods). Stimulation of presynaptic inputs
elicited postsynaptic inward currents of approximately 50 pA in
amplitude at a VH of 70 mV (Fig.
1A-C) (mean amplitude,
51.2 ± 14 pA; range, 6 to 398.4 pA; n = 31 cells). Although the magnitude of such EPSCs is small, evoked responses
were reliably detected with stimulation frequencies of 0.1 Hz for up to
30 min. The eEPSCs were reversibly but not completely inhibited by
suramin (Fig. 1A) and by PPADS (Fig.
1B). The incomplete inhibition of P2X receptors by
either suramin or PPADS is consistent with previous studies of native
receptors in the CNS (Edwards et al., 1992; Bardoni et al., 1997;
Pankratov et al., 1998; Mori et al., 2001). The incomplete blockade
could be in part a result of the expression of heteromeric P2X
channels, including P2X4 and
P2X6 receptor subunits (Le et al., 1998), that are insensitive to suramin and PPADS (Buell et al., 1996; Collo et al.,
1996).
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Figure 1.
Stimulus-evoked transmission mediated by the
activation of ATP P2X receptors at embryonic chick LH synapses
in vitro. A, B, Extracellular stimulation
of chick LH neurons in the presence of CNQX, APV, strychnine, and
bicuculline (10, 50, 1, and 10 µM, respectively;
ECl = VH = 70 mV)
elicits inward currents that are blocked by suramin (30 µM; mean inhibition, 71.2 ± 8%;
n = 10 cells) and by PPADS (50 µM;
mean inhibition, 31.4 ± 2%; n = 4 cells).
The majority of evoked synaptic currents decay in a manner that is best
fit by the sum of two exponential functions (fast = 9.4 ± 1 msec; slow = 47.2 ± 3.8 msec;
n = 16 of 26 cells); the remainder was best fit by
a single exponential function ( = 19.2 ± 2 msec;
n = 10 of 26 cells). The dotted
lines indicate the control level of the evoked EPSCs.
C, The I-V relationship of the peak
amplitude of evoked synaptic currents shows that the mean reversal
potential is 14.6 ± 2 mV (inset;
n = 3 cells; ECl = 70 mV),
consistent with the activation of a relatively nonselective cationic
conductance. The current traces shown correspond to
VHs between 80 and +40 mV in 20 mV
increments.
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The synaptic currents evoked in the continued presence of antagonists
of glutamate, glycine, and GABAA receptors have
nearly linear current-voltage (I-V) curves, with
clear outward currents recorded at the most positive potentials (Fig.
1C). These data are consistent with the previous reports of
stimulus-evoked purinergic transmission in the rat medial habenula
(Edwards et al., 1997) and spinal cord (Jo and Schlichter, 1999).
Evoked purinergic transmission is readily detected in neuronal
preparations from postnatal mouse and embryonic chick (compare Figs. 1
and 2). Under the same experimental
conditions as outlined above, extracellular stimulation of LH neurons
from postnatal mouse in vitro also elicited eEPSCs that are
reversibly but not completely blocked by suramin and PPADS (Fig.
2A,B). Mecamylamine (1 µM), a
nicotinic acetylcholine receptor antagonist, had no effect on the
amplitude of eEPSCs (data not shown). The mean amplitude, decay time
constants, and pharmacology of purinergic EPSCs at mouse LH synapses
are equivalent to those observed in in vitro preparations of
embryonic chick (Table 1). The
I-V relationship for P2X receptor-mediated currents in mice
is similar to chicks (data not shown). Together, these experiments
demonstrate robust P2X receptor-mediated purinergic transmission at
in vitro synapses from embryonic chick or neonatal mouse
LH.
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Figure 2.
Stimulus-evoked transmission mediated by the
activation of ATP P2X receptors at postnatal mouse LH synapses
in vitro. A, B, top
panels, Recordings from postnatal mouse LH neurons under the
same conditions as described in Figure 1. The eEPSCs are inhibited by
suramin (30 µM) and by PPADS (50 µM),
consistent with the eEPSCs being mediated by P2X receptor activation.
The dotted lines indicate the control level of evoked EPSCs.
A, B, bottom panels, The normalized
amplitudes of the eEPSC peaks from neurons shown in A
and B (top) are plotted as a function of
time. The solid line marks the control (baseline) level
of eEPSCs. Solid bar, Time of suramin or PPADS application,
as noted. C, Summary of inhibition of eEPSCs by
P2X receptor antagonists in chicks (14 neurons) and mice (5 neurons).
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Table 1.
Comparison of the amplitudes, decay time constants, and
antagonist profile of P2X receptor-mediated evoked synaptic currents
recorded in embryonic chick versus neonatal mouse LH neurons
in vitro
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Spontaneous ATP P2X receptor-mediated synaptic transmission within
embryonic chick LH neurons
Recordings from embryonic chick LH neurons in vitro in
the presence of TTX (1 µM) revealed that
~70% of neurons received spontaneous excitatory synaptic input that
was resistant to a mixture of glutamate receptor antagonists (in
µM: CNQX 10, APV 50, TTX 1;
VH = ECl = 70
mV; n = 16) (Fig. 3). The
amplitude of these TTX-resistant miniature EPSCs (mEPSCs) ranged
from 5.6 to 15.8 pA, with a mean of 9.3 ± 0.9 pA
(n = 11 neurons) (Fig. 3A). The decay time course of mEPSCs can be fit by a sum of two exponential functions (fast, 4.8 ± 0.9 msec;
slow, 24.3 ± 3.3 msec; n = 8 cells). Both suramin and PPADS reversibly inhibited the majority of
glutamate receptor-independent mEPSCs (Fig. 3B,C). We
propose that this population of fast mEPSCs is attributable to the
spontaneous release of ATP and consequent activation of postsynaptic
ATP P2X receptors. Unlike embryonic chick LH cultures, recordings in
postnatal mouse LH were devoid of spontaneous EPSCs, despite reliable
detection of evoked purinergic eEPSCs. The latter results are
consistent with other studies of postnatal purinergic synapses (Jo and
Schlichter, 1999).
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Figure 3.
TTX-resistant spontaneous purinergic transmission
at embryonic chick LH neuron synapses in vitro.
A, Left, Current traces
showing mEPSCs recorded in the continuous presence of TTX, CNQX, and
APV (1, 10, and 50 µM, respectively;
VH = ECl = 70 mV).
Right, Averaged trace of P2X
receptor-mediated events. Bottom, Histograms show the
distribution of mEPSC amplitudes and decay time constants from a
representative recording. B1, C1, The TTX-, CNQX-, and
APV-resistant mEPSCs were reversibly blocked by suramin and
PPADS (30 µM, n = 10; 50 µM, n = 4, respectively). B2,
C2, The frequency (number of events per 10 and 30 sec,
respectively) of mEPSCs shown in B1 and
C1 is plotted versus time. Solid bars
correspond to the time of suramin or PPADS application, as
indicated.
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Corelease of ATP and GABA from individual presynaptic neurons
Although ATP has been shown to act as a cotransmitter in the PNS
and in spinal cord (Burnstock, 1999; Jo and Schlichter, 1999), ATP
cotransmission has been difficult to study at supraspinal synapses.
Reports to date suggest that ATP is released alone, independent of
other synaptic transmitters in the medial habenula (Robertson and
Edwards, 1998).
Rigorous proof of ATP and GABA corelease is provided by concurrent
recordings from identified presynaptic and postsynaptic partners. Such
dual-perforated patch-clamp recordings from embryonic chick LH neurons
in vitro are shown in Figure
4. In the presence of antagonists of
ionotropic glutamate and glycine receptors, single presynaptic action
potentials evoke rapidly rising unitary postsynaptic currents. The
brief and constant latency (2.6 ± 0.4 msec; n = 8) as well as the absence of failures indicates that evoked PSCs are
monosynaptic in origin (Fig. 4A1). We assessed the
relative contributions of ATP P2X and GABAA
receptors to the ePSCs by dual patch-clamp recording from synaptic
pairs under control conditions and with the sequential addition of
bicuculline and suramin. In this series of experiments, we set the
ECl of the postsynaptic neuron to 0 mV and
recorded at a VH of 70 mV. Under
these conditions, the evoked currents were largely blocked by
bicuculline, and the remaining currents were significantly inhibited by
suramin (Fig. 4A2,B) (n = 5 of 5 pairs tested).
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Figure 4.
Concurrent perforated patch-clamp recordings from
connected pairs of presynaptic and postsynaptic LH neurons demonstrate
purinergic and GABAergic cotransmission. A1, Schematic
diagram of recording configuration and representative recordings.
A-C, Dual perforated-patch-clamp recordings from
synaptically coupled pairs of neurons show that single presynaptic
action potentials induced by injecting positive currents (87.5 msec;
100, 150, and 300 pA, respectively) evoke postsynaptic currents in the
presence of CAS. The resting membrane potential of the
presynaptic neuron was 50 mV. A2, A3, Postsynaptic
currents (i) evoked by single presynaptic action
potentials induced by injecting positive currents (5 msec; 300 pA) in
the presence of CAS were partially inhibited by bicuculline
(ii; Bicu; 10 µM). These
bicuculline-resistant, stimulus-evoked synaptic currents were inhibited
by the addition of the P2X receptor blocker, suramin (30 µM). Superimposition of i and
ii shows the difference in the synaptic current decay
time course of the GABAergic and/or purinergic components
(A3). B, Left,
Representative recordings of GABAergic and purinergic eEPSCs.
Right, The normalized eEPSC amplitude is plotted as a function of time.
Solid bars, Time of application of bicuculline (10 µM) with or without suramin (30 µM), as
noted. a-c (left) are sample traces from the
experiment shown in B (right). Solid
lines indicate the duration of application of the antagonists.
C, Stimulation of the presynaptic neuron of another
synaptically coupled pair of LH neurons in vitro reveals
both the inward purinergic currents and outward GABAA
receptor-mediated responses, depending on the
VH of the postsynaptic neuron
(VH = 70 mV and
VH = 0 mV, respectively).
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Additional examination of postsynaptic currents evoked by single action
potentials in individual presynaptic neurons revealed both inward and
outward currents, depending on the VH
of the postsynaptic neuron relative to ECl (Fig.
4C). Thus, with the postsynaptic neuron membrane potential
clamped at 70 mV (close to ECl), we recorded
inward P2X receptor-mediated currents. Clamping the same postsynaptic
neuron of the synaptic pair to VH of 0 mV elicited outward GABAA receptor-mediated currents
(the reversal potential, VREV, for ATP P2X
receptor-mediated currents is approximately Ecations = 0 mV; n = 3 of 3 pairs tested in this manner). These data
indicate that the recorded currents are attributable to action potential-dependent, synaptic release of both ATP and GABA from the
same presynaptic neuron. The paired-recording experiments also revealed
that a key feature of GABA and ATP cotransmitting neurons was their
strong accommodation to direct depolarization (i.e., a single action
potential rather than a burst of action potentials with a prolonged
depolarizing pulse; data not shown).
Similar assays of ATP and GABA cotransmission in mouse LH neurons
in vitro confirmed and extended the above studies (Fig. 5). When postsynaptic neurons were held
at a VH of 70 mV (close to
ECl), stimulation of the presynaptic neuron
elicited bicuculline- and (+)--hydrastine-resistant, inwardly
directed eEPSCs in the presence of ionotropic glutamate, glycine, and
GABAA receptor antagonists (Fig. 5A).
Stimulation of the presynaptic input to the same cell, held
at 0 mV, elicited no current. Subsequent removal of the
GABAA receptor antagonists led to recovery of the
outward eIPSCs mediated by GABAA receptors. The
double immunocytochemical staining with antibodies against
P2X4 receptor subunit and GAD-6 also supports the
potential overlap of a subset of GABAergic synapses with regions of P2X
receptor expression in vitro (Fig. 5B). As shown
in Figure 5, mouse LH neurons in vitro were stained with anti-P2X4 receptor subunit.
P2X4 receptor subunit-like immunoreactivity was
readily observed on a subset of neuronal somata and
occasionally on neurites. GAD-like immunoreactivity was observed in
many neurites, indicating regions of GABA-containing synapses.
GAD-positive neurites made contacts with a P2X4
receptor subunit-positive somatic area and neurites, consistent with
P2X receptor-positive neurons receiving GABAergic inputs. These
immunocytochemical data are consistent with observed monosynaptic PSCs
proposed to include both ATP P2X and GABAA
receptor-mediated components.
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Figure 5.
Corelease of ATP and GABA at postnatal mouse LH
synapses in vitro. A, Schematic diagram
of a recording configuration and representative recordings of synaptic
currents evoked by extracellular stimulation of a presynaptic input to
a mouse LH neuron in vitro. At a
VH of 70 mV, close to
ECl, in the presence of CAS and (+)--hydrastine
(10 µM) (a GABAA receptor antagonist),
extracellular stimulation elicits eEPSCs in contacted mouse LH neurons.
While the same cell was held at Ecations (close to 0 mV),
we observed no current. After washout of (+)--hydrastine from the
recording solution, the eIPSCs return, consistent with their being
mediated by GABAA receptors. B, Double
immunostaining with antibodies against GAD-6 and P2X4
receptor subunit. GAD-like immunoreactivity is observed on neurites
(green). P2X4 receptor subunit-like
immunoreactivity is observed on soma and occasionally on neurites
(red). Note GAD-positive neurites contacting
P2X4 receptor subunit-positive somata and neurites
(yellow arrow). The green arrow
indicates GAD-positive neurons that appear to contact regions that lack
P2X4-like immunoreactivity. Scale bar, 20 µm.
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A minority of synaptic pairs appeared to be solely GABAergic in the
embryonic chick preparation (30%; n = 16 of 52 pairs). In contrast, we were unable to elicit purinergic currents without concomitant GABAergic responses at any of the 52 synaptic pairs examined (i.e., we found no "ATP-only" synapses). In parallel studies assaying the effects of exogenous application of GABA and ATP
(50 and 30 µM, respectively) (Fig.
6), we found that applied GABA elicited
robust responses, whereas ATP was without effect [approximately
one-third of the neurons tested were "GABA only" (5 of 13 neurons)]. Most neurons (~60%) assayed by exogenous application responded to both GABA and ATP (Fig. 6) in the embryonic chick preparation. The average amplitudes of the responses to GABA and ATP
were 904 ± 312 pA (n = 13) and 164 ± 60 pA (n = 8) at a VH of
60 mV, respectively. The mean percentage inhibition of the response
to ATP by suramin was 70 ± 3%. Bicuculline completely blocked
the response to GABA (98.6 ± 1% inhibition; n = 5). Similar results were obtained for mice.
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Figure 6.
GABA- and ATP-induced responses in embryonic chick
LH neurons. A, Approximately 60% of neurons tested
responded to both GABA (50 µM) and ATP (30 µM). B, Approximately 30% of neurons
tested were responsive to GABA but not to ATP (n = 13).
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GABAA and P2X receptor-mediated components in
spontaneous mPSCs
We subsequently examined whether individual mPSCs are composed of
GABAA and/or P2X receptor-mediated components. We
approached this question in two series of experiments. First, we
analyzed individual mPSCs at synapses receiving both purinergic and
GABAergic mPSCs (i.e., synapses at which mPSCs were blocked only by
combined GABAA and P2X receptor antagonists.).
Under these conditions, we observed two distinct types of mPSCs: one
has a decay time course that is well fit by the single exponential
function ( = 35 ± 1 msec) and the other has a decay phase
that is best fit by the sum of two exponential functions (i.e.,
"mixed" mPSCs; fast = 7 ± 0.5 msec;
slow = 61.6 ± 3 msec) (Fig.
7A). We speculated that mixed
mPSCs may be composed of both P2X and GABAA
receptor-mediated components, because activation of two kinetically
distinct ligand-gated ion channels could yield a composite current with
a decay time course best fit by (at least) two exponential functions.
Indeed, mixed mPSCs have significantly greater peak amplitudes than
those with a monoexponential decay phase (34.5 ± 1.3 vs
14.7 ± 0.8 pA) (Fig. 7A2). Additional evidence for
the mixed nature of these mPSCs is the incomplete block by bicuculline.
The remaining bicuculline-resistant mPSCs were inhibited by suramin (30 µM), and the decay phase of the currents was
well fit by a single exponential function. The decay phase of these
suramin-sensitive mPSCs is significantly briefer than those with a
monoexponential decay phase, observed in the absence of
GABAA receptor antagonist ( = 24 ± 3 msec vs 35 ± 1 msec) (Fig. 7B). Such observations
suggest that the mixed mPSCs would be a result of the concurrent
activation of both P2X and GABAA receptors.
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Figure 7.
GABAA and P2X receptor-mediated
components in spontaneous miniature PSCs. A1, Two
distinct populations of mPSCs are detected in recordings at the
ATP-GABA cosynapse. Left, Superimposition of average of
traces (>200 events) and individual events, showing two
kinetically distinct events. Right, Average amplitude of
events classified according to the decay rate(s) of mPSCs.
A2, Mixed mPSCs have a significantly greater mean peak
amplitude than those with a monoexponential (Monoexp.)
decay phase (34.5 ± 1 vs 14.7 ± 1 pA;
p < 0.05). A3, Plot of the
percentage contribution of monoexponential decay mPSCs versus
mixed-decay mPSCs to recorded spontaneous activity (mixed component, 72%; monoexponential component, 28%).
B1, Top, Averaged traces
of bicuculline (Bicu)-sensitive
(a) and bicuculline-resistant
(b) events. Bottom,
Superimposition of the normalized traces of
a and b, showing that the decay phase of
bicuculline-resistant events is briefer than that of
bicuculline-sensitive events. B2, Comparison of the
decay time constant of ATP and GABA mPSCs (*p < 0.05). C1, C2, Normalized traces
of average amplitude of mPSCs at GABAergic and purinergic synapses
tested in the absence or presence of flunitrazepam
(Flu) (in the presence of CAS; ECl = 0 mV; VH = 70 mV). In mixed mPSCs,
flunitrazepam has no effect on the fast component,
whereas the slow component is significantly affected
(*p < 0.05; left). Flunitrazepam
(100 nM) significantly prolongs the mean decay time
constant of mPSCs with a monoexponential decay phase
(right).
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To examine the possibility that mixed mPSCs reflect the concomitant
release of GABA-ATP and the coordinate activation of both postsynaptic
receptors, we examined the effect of flunitrazepam, a selective
positive modulator of GABAA receptors, on
individual mPSCs (Fig. 7C). We reasoned that if both
GABAA and P2X receptors are present at the
postsynaptic site opposing ATP-GABA input, then flunitrazepam should
increase the duration of the time course of all GABAergic mPSCs.
Indeed, flunitrazepam (100 nM) significantly prolonged the decay time course of mPSCs with a monoexponential decay
phase (from 35 ± 1 to 53.3 ± 3 msec) (Fig.
7C1,C2). The effect of flunitrazepam on the population of
mixed mPSCs was particularly striking. Thus, the
slow component of mixed mPSCs was
significantly prolonged by a low concentration of flunitrazepam,
whereas the fast component of this mPSC
population was unaffected (Fig. 7C1,C2). Together, these
data indicate that the slow component of mixed PSCs is sensitive to bicuculline and flunitrazepam, whereas the fast component of mixed PSCs is insensitive to
GABAA receptor modulators. The
bicuculline-resistant minicomponents are blocked by P2X receptor
antagonists. Thus, the subpopulation of mPSCs identified as mixed mPSCs
arises from the coordinate activation of postsynaptic receptors that
include both GABAA and P2X receptors.
As suggested by the above experiments, spontaneous release (i.e.,
mPSCs) appears to involve concurrent activation of both ATP P2X and
GABAA receptors at a subset of synapses. Under
such circumstances, coreleased ATP and GABA may be costored within the
same synaptic vesicle, rather than in separate vesicles. We attempted
to test the hypothesis of costorage by further examination of the
mPSCs. mPSCs were recorded over a range of
VHs, including the
VREV for GABAA
(70 mV), the VREV for P2X receptor
(0 mV), and at VHs intermediate to the
reversal potentials (i.e., approximately 35 mV). If ATP and GABA are
stored within different synaptic vesicles, the spontaneous release of
each transmitter should be an independent event. Thus, under these
circumstances, we should detect mPSCs of both inward (ATP) and outward
(GABA) currents at a VH intermediate
to the reversal potentials for both receptors. In contrast, if ATP and
GABA are costored in the same vesicle, voltage clamp of the
postsynaptic neuron at a potential intermediate to
VREV for both receptors should reveal
mPSCs that are mixed (i.e., fast inward and then outward currents).
Alternatively, if the transmitters are costored, then the costored and
coreleased ATP plus GABA may elicit few detectable mPSCs, because the
concomitant activation of these two oppositely directed conductances
may sum to little net current.
Initial recordings under control conditions revealed both P2X and
GABAA receptor-mediated mPSCs at
VHs of 70 and 0 mV, corresponding to
the reversal potentials for GABAA and P2X
receptors, respectively. Subsequent recording from the same neuron
while changing the VH to 35 mV
revealed very few mPSCs in 75% of the neuron pairs tested in this
manner (Fig. 8A1).
Application of a low concentration of -hydrastine (1 µM) blocked the few outward mPSCs that were detected, as expected for this GABAA receptor
antagonist (Fig. 8A2) (n = 4). In
addition, at later times it appears that the blockade of
GABAA receptors subsequently unmasked a
population of inward mPSCs. The mean amplitude of inward mPSCs observed
at a VH of 35 mV in the presence of
-hydrastine is significantly smaller than that observed at a
VH of 70 mV (5 ± 0.1 vs
6.7 ± 0.5 pA; p < 0.01) (Fig.
8A3). The decay time constants of mPSCs observed at
VHs of both 70 and 35 mV are
similar (14 ± 0.8 vs 15 ± 0.7 msec; p > 0.05) (Fig. 8A3), whereas those observed at a
VH of 0 mV are significantly different
(28.4 ± 1 vs 14 ± 0.8 and 15 ± 0.7 msec;
p < 0.01) (Fig. 8A3). Thus, the
inward mPSCs at a VH of 35 mV plus
-hydrastine are likely to be ATP P2X receptor-mediated mPSCs.
Together, these data suggest that cotransmission of GABA and ATP may be
a result of the costorage and corelease of ATP and GABA and the
consequent coactivation of postsynaptic ATP P2X and
GABAA receptors.
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Figure 8.
Analysis of GABAA and P2X
receptor-mediated mPSCs over a range of VHs.
A1, Representative recordings of GABAergic and
purinergic mPSCs. Top, Sample traces at a
VH of 0 mV (VREV
for P2X receptors); only outward currents were seen.
Middle, Sample traces at a
VH of 70 mV
(VREV for GABAA receptors);
inward currents were observed. Bottom, Sample
traces at a VH of 35 mV
(intermediate to the reversal potentials for both receptors) with or
without -hydrastine. A2, The amplitudes of mPSCs are
plotted as a function of time. At a VH of
35 mV, few events are detected. In the continued presence of
-hydrastine (1 µM), there was an increase in the
number of inward current events detected. The solid
lines indicate noted changes in conditions (i.e., top
line,  VH from 0 to 35; bottom
line, addition of -hydrastine). A3, Histograms
of the cumulative probability of mPSC amplitudes and decay time
constants recorded at VHs of 70, 35, and
0 mV as indicated. The mean amplitude of mPSCs detected at a
VH of 35 mV in the presence of
-hydrastine is significantly smaller than that observed at a
VH of 70 mV, but there is no difference
between the decay time constants for mPSCs at 70 versus 35 mV. The
latter observation is consistent with the idea that all
mPSCs recorded at 70 mV and at 35 mV plus -hydrastine are
ATP P2X receptor-mediated. In contrast, both mean amplitude and the
decay time constants of mPSCs detected at a
VH of 0 mV are significantly different from
those observed at VHs of 70 and 35 mV,
consistent with the idea that all mPSCs detected at a
VH of 0 mV are GABAergic.
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DISCUSSION |
The major findings of the present study are that (1) ATP is
released in an action potential-dependent and in an action
potential-independent (TTX-resistant) manner at LH synapses in
vitro and (2) recordings from identified pairs of presynaptic and
postsynaptic neurons reveal that ATP and GABAA
receptor-mediated postsynaptic currents arise from the coordinate
release of both ATP and GABA from individual neurons. We also propose,
based on our findings, that ATP and GABA may be costored within the
same synaptic vesicle.
Properties of P2X receptors expressed in LH neurons
The existence of fast ATP-mediated excitatory synaptic
transmission is well documented in the peripheral nervous system (Evans et al., 1992; Galligan and Bertrand, 1994; Burnstock, 1999). Relatively little is known about purinergic transmission in the CNS, despite the
large distribution of ATP receptor subunits in many regions of the CNS.
The probability of detection of purinergic synapses in the CNS appears
to be quite low, thereby limiting detailed analysis of CNS purinergic
synapses. Bardoni et al. (1997) were able to detect an ATP P2X
receptor-mediated component of synaptic transmission at only ~5% of
neurons tested in a rat spinal cord slice preparation. Likewise, we
noted a higher incidence (i.e., more readily detectable) of ATP P2X
receptor-mediated synaptic currents in dispersed cell cultures than in
slice recordings from postnatal mice (data not shown). The relatively
low incidence of purinergic synapses in the slice and the lack of
purinergic mPSCs in recordings from dispersed LH neurons from postnatal
mice compared with the other preparations may be attributable to
several factors. One possible explanation for the differences in
purinergic mPSCs in postnatal mouse versus prenatal chick preparations
is the difference in developmental stage. Several studies show
decreases in the probability of neurotransmitter release with the
development of central synapses (Bolshakov and Siegelbaum, 1995; Choi
and Lovinger, 1997; Brenowitz and Trussell, 2001; Iwasaki and
Takahashi, 2001). Other confounders may include a higher local activity
of ectonucleotidase and soluble ectonucleotidase in slice preparations (Zimmermann, 1996), the presence of more "silent" purinergic
synapses in vitro (Khakh et al., 2001), and/or a higher
local concentration of metabolites that interfere with ATP P2X receptor
activation. Optimal detection of purinergic transmission in slice
preparations may require higher frequencies, different patterns of
presynaptic activation, or more effective perfusion of the slice.
Our present study provides evidence for robust and reliable action
potential-dependent ATP P2X receptor-mediated purinergic transmission
in two different in vitro preparations of LH, including neurons from embryonic chicks and neurons from postnatal mice. The
characteristics of the evoked purinergic transmission obtained from the
present and previous studies in culture and/or in slice preparations
(Bardoni et al., 1997; Edwards et al., 1997; Jo and Schlichter, 1999;
Mori et al., 2001) are strikingly similar (pharmacology, time course,
I vs V characteristics) despite the differences
in species, age, etc. Thus, the in vitro culture preparation
may provide a better system for the study of ATP P2X receptor-mediated transmission than the in vitro slice preparation (until the
highly selective inhibitors for ectonucleotidase and/or P2X receptors are available).
Native ATP P2X receptors are thought to be heteromeric combinations of
the various P2X receptor subunits (Lewis et al., 1995) and seem to be
assembled as trimers (Nicke et al., 1998). This complexity of P2X
receptor structure is thought to underlie the difficulties in
reconciling the well characterized pharmacological effects of
antagonists on homomeric P2X receptors with those observed in studies
with native receptors. The ATP P2X receptors found in the LH are likely
to be heteromeric ATP receptors composed of P2X2,
P2X4, and P2X6 subunits,
which are often coexpressed in the same cells (Collo et al., 1996; Le
et al., 1998; Kanjhan et al., 1999). In our experiments and in those of
others (Edwards et al., 1992; Bardoni et al., 1997; Pankratov et al.,
1998; Mori et al., 2001), the incomplete blockade by ATP P2X receptor
antagonists is likely to reflect the heteromeric nature of the
receptors involved. Of the subunits expressed in the LH, only the
P2X2 subunits show strong sensitivity to suramin
and PPADS (North and Surprenant, 2000). In addition, our
immunocytochemical data show that LH neurons from postnatal mice
express P2X4 subunits, one of several known to be
relatively insensitive to PPADS and suramin. It is likely that LH
neurons express heteromeric P2X receptors composed of suramin- and
PPADS-sensitive P2X2 as well as
antagonist-insensitive P2X4 (and/or
P2X6) subunits.
Coordinate release of ATP and GABA
GABA is copackaged with glycine within the same synaptic vesicle
by vesicular inhibitory amino acid transporter (Gasnier, 2000). Two
fast-acting inhibitory neurotransmitters, GABA and glycine, are
coreleased and subsequently activate their respective receptors,
GABAA and glycine receptors, in the spinal cord
(Jonas et al., 1998; Keller et al., 2001). Thus, GABA acts as a
cotransmitter in the developing CNS. GABA has been also shown to be
coreleased with an excitatory neurotransmitter, ATP, in the spinal cord
(Jo and Schlichter, 1999). Our present study is consistent with
previous findings of coordinate release of ATP and GABA from the
individual presynaptic neurons and provides the first demonstration of
ATP-GABA cotransmission in the brain.
Several lines of evidence support our proposal that ATP and GABA are
coreleased. Using the dual whole-cell patch-clamp technique, we
demonstrate that individual events are stimulus-locked (i.e., individual presynaptic action potentials elicit the corresponding postsynaptic events with the same time of onset and without failure). Second, the latencies for ATP and GABA components are identical. Third,
individual events are blocked by a mixture of suramin and bicuculline,
but not by bicuculline alone. Finally, intracellular stimulation of the
same presynaptic neuron while recording from the same postsynaptic
neuron at different membrane potentials elicits both cation-mediated
inward currents and Cl-mediated outward
currents that are sensitive to suramin and bicuculline, respectively.
Thus, our detection of cotransmission of ATP and GABA is attributable
to monosynaptic (rather than polysynaptic) mechanisms.
It is not known how ATP enters the synaptic vesicles or whether ATP is
copackaged with GABA within the individual synaptic vesicles. The
vesicular transporter for nucleotides has yet to be identified
(Gasnier, 2000). The current electrophysiological studies are
consistent with (but do not prove) the thesis that ATP and GABA are
costored. In experiments examining synaptic transmission at an
intermediate VH, we detected few
inward or outward current events and could not record mixed
inward/outward currents. In the same experiments, outward and inward
PSCs were readily detected at a VH of
either 0 or 70. Comparison of the time course of mPSCs indicates that
the events recorded at VH = 35 after
the inhibition of GABAA receptors are equivalent
to those detected at VH = 70 (i.e.,
both are ATP P2X receptor-mediated). The lack of mixed inward/outward
currents is also consistent with recent studies of cross-inhibition of
ATP P2X with GABAA receptors in the dorsal root
ganglions (Sokolova et al., 2001). Sokolova et al. (2001) show that P2X
and GABAA receptors can interact or
"cross-talk" at the level of the membrane, thereby resulting in
mutual inhibition of receptor activation. Inhibition by GABA is
strongly dependent on the extent of GABA-gated
Cl efflux. Previous work on NG108-15
cells also indicated that extracellular Cl may depress P2X receptor function,
probably via the inhibition of agonist binding to the receptor site
(Kaiho et al., 1997). We cannot exclude the possibility that our
inability to detect mixed mPSCs is at least in part a result of the
confounding effects of GABAA-ATP P2X receptor
interactions and/or of the small mPSC amplitude versus the relatively
high baseline noise at VH = 35.
Possible physiological significance
GABA is the primary inhibitory transmitter in the hypothalamus,
and GAD mRNA is widely expressed with the LH (Elias et al., 2001).
Recent studies demonstrate the existence of GABAergic interneuronal synapses in in vitro preparations of rat LH (Gao and van den
Pol, 2001b). These and other studies support a potentially important role of LH inhibitory circuits in the central control of feeding (for
review, see Bernardis and Bellinger, 1996). In mature hypothalamic neurons, GABA elicits hyperpolarizing responses and, as such, is
inhibitory (Randle et al., 1986; Tasker and Dudek, 1993; Strecker et
al., 1997). In contrast, GABA can exert depolarizing actions in
developing hypothalamic neurons (Chen et al., 1996). Indeed, in early
development, the synaptic release and depolarizing effect of GABA may
contribute to suprathreshold activity even more than glutamate (Gao and
van den Pol, 2001a). In view of the depolarizing effects of GABA at
early developmental stages versus GABA-mediated hyperpolarization at
mature LH synapses, the coordinate release of ATP with GABA may support
distinct mechanisms for synaptic tuning in the embryonic versus
postnatal animal. Thus, the existence of ATP-GABA cotransmission at
early stages of development may provide a synergistic mechanism for
excitatory influences on postsynaptic neurons. In mature hypothalamic
synapses, the activation of GABAA receptors may
provide an enhanced driving force for Ca2+
entry, thereby increasing the net Ca2+
influx via P2X receptor activation. The latter mechanism for GABA and
ATP interactions could provide significant synergistic enhancement of
ATP receptor-mediated events, as proposed by Robertson et al. (2001).
The net physiological effects of ATP and GABA cotransmission will
depend on the regulated changes in reversal potential of GABA-evoked
current and the localized expression of ATP P2X receptor during
development. Our present study supports novel forms of synaptic
flexibility via ATP-GABA cotransmission during the development of
avian and mammalian hypothalamic circuits.
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FOOTNOTES |
Received Dec. 4, 2001; revised Feb. 15, 2002; accepted Feb. 22, 2002.
This work was supported by National Institutes of Health Grants NS22061
and DA09366 to L.W.R. We thank Drs. Steven A. Siegelbaum, Amy B. MacDermott, and Ron Yu for helpful comments on a previous version of
this manuscript and Tom Davis for technical assistance.
Correspondence should be addressed to Dr. Lorna W. Role, Columbia
University College of Physicians and Surgeons, Center for Neurobiology,
1051 Riverside Drive, P. I. Annex, Room 807, New York, NY 10032. E-mail: lwr1{at}columbia.edu or Lrole{at}aol.com.
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TOP
ABSTRACT
INTRODUCTION
