Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195-7290
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
O'Brien, Jennifer A. and
Albert J. Berger.
Cotransmission of GABA and Glycine to Brain Stem Motoneurons.
J. Neurophysiol. 82: 1638-1641, 1999.
Using whole cell patch-clamp recording in a rat brain stem slice
preparation, we found that -aminobutyric acid (GABA) and glycine act
as cotransmitters to hypoglossal motoneurons (HMs). Focal application
of GABA and glycine onto a single HM revealed that GABAA
and glycine receptors are present on the same neuron. To demonstrate
that HMs receive both GABAergic and glycinergic synaptic inputs, we
simultaneously recorded GABAA- and
glycine-receptor-mediated spontaneous miniature inhibitory
postsynaptic currents (mIPSCs) in single HMs. GABAergic and glycinergic
mIPSCs were differentiated based on their kinetics and modulation by
pentobarbital. Specifically, GABAA-receptor-mediated
events decayed more slowly than glycine-receptor-mediated events.
GABAergic response decay kinetics were prolonged by pentobarbital, whereas glycinergic response decay kinetics remained unchanged. The
distinct kinetics of the glycine- and
GABAA-receptor-mediated synaptic events allowed us to
record dual component mIPSCs, mIPSCs that are mediated by both receptor
types. These data suggest that GABA and glycine are colocalized in the
same presynaptic vesicle and are coreleased from presynaptic terminals
opposed to motoneurons.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-Aminobutyric acid (GABA) and glycine are the
two main inhibitory neurotransmitters in the CNS. Each neurotransmitter
activates a different family of ionotropic receptors that are permeable to chloride ions. GABAA receptors are blocked
preferentially by bicuculline and are modulated by pentobarbital
(Macdonald and Olsen 1994
), whereas glycine receptors
are preferentially blocked by strychnine and are insensitive to
modulation by pentobarbital (Rajendra et al. 1997
).
In the spinal cord and brain stem, inhibitory synaptic transmission can
be mediated by GABA and/or glycine. Recently, Jonas et al.
(1998) demonstrated in spinal cord that GABA and glycine are
coreleased from the same presynaptic terminal, resulting in coactivation of the corresponding receptors on target spinal cord neurons. It is not known if this phenomenon is restricted to spinal cord or whether other CNS regions, such as brain-stem motoneurons, receive dual-transmitter (GABA and glycine) inhibitory synaptic inputs.
Here we demonstrate that both GABA and glycine act as cotransmitters to
visualized hypoglossal motoneurons (HMs) by using whole cell
patch-clamp techniques to record miniature inhibitory postsynaptic
currents (mIPSCs), which presumably are due to release of single
presynaptic vesicles (Katz 1969
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sprague-Dawley rats (1- to 5-days old) were anesthetized by
injection (intramuscular) of a ketamine-xylazine mixture (200 and 14 mg/kg, respectively). After decapitation, the brain stem was removed
and transverse brain stem slices (250-300 µm) were prepared. During
slicing, incubation (1 h at 37°C), and recording, the slices were
perfused by a Ringer solution containing (in mM) 119 NaCl, 26.2 NaHCO3, 1 NaH2PO4, 2.5 KCl, 11 glucose, 2.5 CaCl2, and 1.4 MgSO4. Using near-infrared DIC optics, HMs were
identified based on their characteristic location and morphology
(Umemiya and Berger 1994).
Whole cell patch-clamp recordings were performed at room temperature.
Patch electrodes (resistance 1-5 M) were filled with (in mM) 145 CsCl, 10 HEPES, 10 ethylene glycol-bis-(b-amnioethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 2 MgCl2, 2 ATP-Mg, and 0.2 GTP-tris
(pH 7.2). HMs were voltage-clamped
50 to
65 mV. Access resistance
was always <15 M
. Data were filtered at 2 kHz and digitized at 5 kHz using pCLAMP software (Axon Instruments). GABA (200 µM) and
glycine (200 µM) were applied (10-50 ms) onto the same HM using a
dual channel picospritzer (General Valve) and a double barrel glass
pipette. Five current traces were averaged and peak current amplitude
measured using pCLAMP software (Axon Instruments).
Spontaneous mIPSCs were analyzed by software developed in our
laboratory. Decay kinetics were measured as the time for the mIPSC to
decay to 37% of its peak amplitude. All results are presented as
means ± SD unless otherwise stated. The Kolmogorov-Smirnoff statistical test (KS-test) was used to assess differences in mIPSC data. An unpaired t-test was used to assess differences in
mean values from different conditions. Drugs used included: bicuculline methiodide (Sigma), strychnine hydrochloride (Sigma), tetrodotoxin (TTX, Alomone Labs), 6,7-dinitroquinoxaline (DNQX, RBI),
D()-2-amino-5 phosphopentanoic acid (AP5, RBI), and
sodium pentobarbital (Abbott labs).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine whether GABAA and glycine
receptors were present on the same HM, GABA and glycine were applied
focally via a double-barreled pipette onto a single voltage-clamped HM
(Vh = 50 mV, Fig.
1A, top).
Experiments were performed in the presence of TTX (0.5-1 µM) and
DNQX (10-20 µM) to block Na+-dependent action
potentials and non-N-methyl-D-aspartate (NMDA) glutamate receptors, respectively. Under these conditions, both GABAA and glycine receptor-mediated responses
were present in five of five HMs studied. Addition of 5 µM
bicuculline abolished almost all of the
GABAA-receptor-mediated response (Fig. 1A,
middle, n = 4). Addition of strychnine (1 µM)
abolished almost all of the remaining glycine-receptor-mediated
response (Fig. 1A, bottom, n = 4). These
data show that both types of receptors are present on the same
motoneuron.
|
Because it has been reported that bicuculline also can block
glycine-receptor-mediated responses and strychnine can block GABAA-receptor-mediated responses (Jonas
et al. 1998), we next performed dose-response experiments. Bath
application of 5 µM bicuculline blocked 96.7 ± 1.1% of the
GABAA-receptor-mediated response
(n = 4, Fig. 1B), whereas it blocked only
5.7 ± 7.5% of the glycine-receptor-mediated response
(n = 4, Fig. 1C). A greater dose of
bicuculline (10 µM) also blocked 27.5 ± 4.5% of the
glycine-receptor-mediated response (n = 4, Fig.
1C). Bath application of 500 nM strychnine blocked 97.2 ± 2.4% of the glycine-receptor-mediated response (n = 4, Fig. 1C), while inhibiting the
GABAA-receptor-mediated response by only
11.3 ± 10.5% (n = 4, Fig. 1B). A
greater dose of strychnine (10 µM) also abolished 96.6 ± 4.0%
of the GABAA-receptor-mediated responses
(n = 4, Fig. 1B).
To determine if both GABAergic and glycinergic synaptic currents are
present in the same HM, we recorded spontaneous mIPSCs in the presence
of TTX (0.5-1 µM), DNQX (20 µM) and AP5 (25 µM) with HMs
voltage-clamped at 65 mV. Under these conditions, two populations of
mIPSCs could be distinguished based on their sensitivity to antagonists
and decay kinetics (Table 1). Spontaneous
mIPSCs recorded in the absence of strychnine and bicuculline exhibited fast and slow decay kinetics (n = 6, Fig.
2, A1 and B1).
Addition of the glycine receptor antagonist strychnine (500 nM)
abolished the majority of responses with fast decay kinetics (Fig.
2A2, n = 3). The remaining mIPSCs were
blocked by 5 µM bicuculline, confirming that these events are
mediated by GABAA receptors (data not shown,
n = 3). In a separate experiment, when bicuculline was
added first, the mIPSCs with slower decaying kinetics were selectively
abolished (Fig. 2B2, n = 3). The remaining
glycinergic responses were blocked by 500 nM strychnine (data not
shown, n = 3).
|
|
We computed the cumulative probability distribution of decay times for
events recorded in control conditions (Fig. 2, A3 and B3, ) versus events recorded in the presence of either
strychnine or bicuculline. When strychnine was added, the cumulative
probability of events shifted to the right (Fig. 2A3,
,
n = 3). When bicuculline was added, the cumulative
probability of events shifted to the left (Fig. 2B3,
,
n = 3). Comparing these distributions shows that the
observed distribution in the absence of blockers lies between those of
the glycinergic and GABAergic events. This indicates that in the
control condition both types of events contribute to the overall
population of control mIPSCs.
To investigate whether GABA and glycine are contained within and released from the same presynaptic vesicle, it was necessary to distinguish GABAergic and glycinergic mIPSCs in the same cell based on differences in their decay kinetics (Fig. 3B1). To increase the difference between the GABAergic and glycinergic kinetics, we recorded mIPSCs in the presence of pentobarbital (25-50 µM). Pentobarbital prolonged the decay kinetics of GABAergic events without affecting the kinetics of glycine-receptor-mediated events (Table 1). The distributions of decay times for glycinergic and GABAergic mIPSCs recorded in the presence of pentobarbital are shown in Fig. 3B2. The prolonged GABAergic events recorded in isolation have a significantly different distribution from faster decaying glycinergic events, thus making it possible to distinguish GABAergic from glycinergic events.
|
If GABA and glycine are coreleased from the same presynaptic vesicle,
we would predict that mIPSCs should have both a fast decaying
glycinergic component and a slow decaying GABAergic component. When
recorded in the presence of pentobarbital, mIPSCs had three different
types of decay kinetics. There were fast decaying glycinergic events
(Fig. 3A1, ) and slow decaying GABAergic events (Fig. 3A1,
). There were also dual component mIPSCs having both
fast and slow decay components (Fig. 3A1,
). These dual
component mIPSCs are due to release of both GABA and glycine from
the same vesicle because presumably mIPSCs are due to release of a
single presynaptic vesicle.
We plotted the distribution of decay kinetics from events recorded in control conditions and this resulted in a skewed frequency distribution (Fig. 3A2) rather than two separate distributions indicative of isolated glycinergic and GABAergic events (Fig. 3B2). This result strongly suggests that dual component mIPSCs, involving the corelease of GABA and glycine from a single vesicle, occur under these conditions.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data suggest that GABA and glycine act as cotransmitters to
HMs. We also have demonstrated that these can be coreleased from the
same presynaptic vesicle. These data are consistent with and extend a
previous study demonstrating dual component mIPSCs in spinal cord
neurons (Jonas et al. 1998).
Biochemical and anatomic experiments suggest that GABA and glycine can
be colocalized in the same neuron and synaptic vesicle. It is known
that GABA and glycine are transported by the same vesicular transporter
(Burger et al. 1991). Colocalization of GABA and glycine
in single neurons has been shown using immunohistochemistry in many
areas including spinal cord and trigeminal nucleus. (Dumba et
al. 1998
; Taal and Holstege 1994
; Todd
and Sullivan 1990
). Although colocalization of GABA and glycine
in neurons projecting to HMs has not been demonstrated, there is
evidence that brain stem neurons, projecting to HMs, can contain either
GABA or glycine. Furthermore these neurons are located in the same
region of the brain stem (Li et al. 1997
).
Why are GABA and glycine co-released? It is possible that corelease of
GABA and glycine may be important for development because these
experiments were performed using neonatal rats. During this time in
development, the chloride gradient in neonatal HMs, causes glycine
receptor (Singer et al. 1998) and probably also
GABAA-receptor activation to depolarize HMs. A
combination of slow GABAergic responses and fast glycinergic responses
may lead to a depolarization great enough to activate voltage-gated
calcium channels. Also there is evidence that calcium influx due to
GABAA-receptor activation is important for
neuronal developmental (Cherubini et al. 1991
; Obrietan and van den Pol 1995
). There may be a
developmental change in proportions of synaptically activated
GABAA and glycine receptor as has been seen in
other systems (Gao and Ziskind-Conhaim 1995
; Kotak et al. 1998
). If the developmental switch is due
to a change in postsynaptic receptor type, corelease of both
neurotransmitters would ensure that release of vesicular contents
causes a postsynaptic effect. An important issue, and as yet unknown,
is whether GABA and glycine are coreleased throughout development or if
this phenomena is specific only to HMs of neonatal animals.
![]() |
ACKNOWLEDGMENTS |
---|
We are grateful to Dr. Jeffry Isaacson, Dr. Peter Schwindt, and E. Eggers for reading and commenting on this manuscript and to Dr. William Satterthwaite and P. Huynh for technical assistance.
J. A. O'Brien is supported by National Institute of General Medical Sciences Training Grant 5T32GM-07108. This research also was supported by a Javits Neuroscience Award (NS-14857) to A. J. Berger.
![]() |
FOOTNOTES |
---|
Address for reprint requests: J. O'Brien, Dept. of Physiology and Biophysics, School of Medicine, University of Washington, Box 357290, Seattle, WA, 98195-7290.
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 5 April 1999; accepted in final form 24 May 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|