Department of Neurosurgery, Yale University Medical School, New Haven, Connecticut 06520
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ABSTRACT |
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Liu, Qing-Song,
Peter R. Patrylo,
Xiao-Bing Gao, and
Anthony N. van den Pol.
Kainate Acts at Presynaptic Receptors to Increase GABA Release
From Hypothalamic Neurons.
J. Neurophysiol. 82: 1059-1062, 1999.
Recent reports suggest that kainate
acting at presynaptic receptors reduces the release of the inhibitory
transmitter GABA from hippocampal neurons. In contrast, in the
hypothalamus in the presence of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) receptor
antagonists [1-(4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine
(GYKI 52466) and D,L-2-amino-5-phosphonopentanoic acid
(AP5)], kainate increased GABA release. In the presence of
tetrodotoxin, the frequency, but not the amplitude, of GABA-mediated
miniature inhibitory postsynaptic currents (IPSCs) was enhanced by
kainate, consistent with a presynaptic site of action. Postsynaptic
activation of kainate receptors on cell bodies/dendrites was also
found. In contrast to the hippocampus where kainate increases
excitability by reducing GABA release, in the hypothalamus where a much
higher number of GABAergic cells exist, kainate-mediated activation of
transmitter release from inhibitory neurons may reduce the level of
neuronal activity in the postsynaptic cell.
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INTRODUCTION |
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Kainate receptors are expressed widely throughout
the brain (Herb et al. 1992; Hollmann and
Heineman 1994
), including the hypothalamus (van den Pol et al.
1994
), the focus of the present experiments. Although ionotropic
glutamate receptors are generally considered as postsynaptic receptors
on the cell body or dendrites of neurons, recent evidence suggests that
kainate can activate a presynaptic receptor and that activation of this
receptor inhibits transmitter release from glutamatergic
(Chittajallu et al. 1996
) and GABAergic neurons
(Clarke et al. 1997
; Rodriguez-Moreno et al.
1997
) in the hippocampus.
In the present study, we used whole cell recordings of hypothalamic neurons in culture and in brain slices to examine kainate responses on hypothalamic neurons. In contrast to previous reports based on neurons from other regions of the brain, we find that kainate evokes an increase in the release of the inhibitory transmitter GABA by activating receptors that appear to be on the axon terminal.
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METHODS |
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Slice recording
Hypothalamic slices (400 µm thick) containing the arcuate and
ventromedial nuclei were prepared and maintained from postnatal day 10-17 rats as previously described (van den Pol et al.
1998). Whole cell patch pipettes (4-10 M
) were filled with
(in mM) 145 KCl, 1 MgCl2, 10 HEPES, 1.1 EGTA, 4 Mg-ATP, and 0.5 Na2-GTP. Membrane potentials were
maintained at approximately
70 mV.
-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) glutamate receptors were blocked with GYKI 52466 (100 µM) and
D,L-2-amino-5-phosphonopentanoic acid (AP5; 50 µM),
respectively (Donevan and Rogawski 1993
;
Paternain et al. 1996
). To examine the effect of
kainate, the frequency of spontaneous inhibitory postsynaptic
potentials (IPSPs;
5 mV, 24 s duration) was determined before,
during (30 s after initiation of kainate microapplication), and 5 min
after kainate microapplication.
Whole cell recording in cultured neurons
Primary cultures of medial hypothalamic neurons were prepared
from embryonic day 16-18 Sprague-Dawley rats as previously
described (van den Pol et al. 1998) and approved by the
Yale University Committee on Animal Use. Whole cell voltage-clamp
recordings were made with an EPC-9 patch-clamp amplifier controlled by
a Macintoish computer running Pulse v8.0 software. Data were sampled at
5-10 kHz and filtered at 1-2 kHz. Patch pipettes were filled with (in mM) 145 KMeSO4 or KCl, 2 MgCl2, 4 Na2ATP, 0.5 Na2GTP, 10 HEPES, and 1.1 EGTA, pH 7.2 with KOH.
Capacitance and series resistance (10-20 M
) were compensated
(80-85%) in all experiments except those examining miniature
postsynaptic currents (PSCs). To keep noise to a minimum during
recording of miniature PSCs, series resistance and capacitance were not
compensated in these experiments. Data were excluded if a change of
>15% in series resistance was found. Cultures were perfused (2 ml/min) with a bath solution containing (in mM) 160 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES 10, pH 7.3 with NaOH at room temperature (~22°C). Drugs were applied through large-bore flow pipes. TTX, GYKI
52466, kainate, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and AP5 were from RBI (Natick, MA), and Mg-ATP, Na2-GTP,
and bicuculline were from Sigma.
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RESULTS |
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Kainate increases GABA activity in hypothalamic slices
In the presence of 1-(4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) (100 µM) and AP5 (50 µM), kainate (1 µM) increased the mean frequency of spontaneous IPSPs from 4.6 ± 0.8 events/s (mean ± SE; range, 1.4-8.4 events/s) to 5.9 ± 1.1 events/s (range, 1.9-11 events/s; n = 9 neurons; P < 0.04; paired 1-tailed t-test; Fig. 1). When we used a minimum change criterion of 20% in IPSP frequency, we found that kainate reversibly increased the frequency of GABAA-mediated PSPs in five of nine neurons to 157 ± 11.1% of baseline activity (P < 0.03; paired 1-tailed t-test). In three of three neurons the experiment was repeated with the same response. In the remaining four neurons, a nonsignificant change in the frequency of spontaneous IPSPs was observed with kainate (97 ± 5.5% of baseline; P = 0.4). Nonresponding cells were distributed evenly in both the arcuate and ventromedial nucleus.
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Glutamate and GABA actions in cultured hypothalamic neurons
After 2-3 wk in culture, virtually all hypothalamic neurons
showed spontaneous GABA-mediated synaptic currents. At a holding potential of 70 mV, excitatory postsynaptic currents (EPSCs) were
inward currents that were blocked by the non-NMDA receptor antagonist
CNQX (10 µM, n = 7), and IPSCs were barely visible (Fig. 2, A). At a holding
potential of 0 mV, IPSCs were outward currents that were blocked by the
GABAA receptor antagonist bicuculline (10 µM,
n = 7), whereas EPSCs were barely visible (Fig.
2B). At a holding potential of
70 mV, EPSCs were
completely and reversibly blocked by the selective AMPA receptor
antagonist GYKI 52466 (100 µM, n = 7; Fig.
2A), indicating that AMPA receptors are a primary mediator
of spontaneous glutamate activity in the hypothalamus. Inhibitory PSCs
reversed at
75 mV, typical of GABA-mediated events under these
conditions (Fig. 2C). In the experiments below, GYKI 52466, instead of CNQX, was used to block the EPSCs, because it allowed us to
examine selectively kainate receptor-mediated modulation of inhibitory
synaptic transmission.
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Kainate increases spontaneous IPSCs
In the presence of GYKI 52466 (100 µM), kainate (10 µM, 3 min)
reversibly increased the spontaneous IPSC frequency to 273 ± 41%
of baseline (baseline = 100%; P < 0.01, paired
t-test) in 9 of 10 neurons (Fig. 2, D and
E). This effect had a rapid onset and often desensitized to
a stable value within 1-2 min. This stable value was still higher than
the baseline frequency. These observations suggest two possibe sites
for the action of kainate. First, specific and functional kainate
receptors may be present on the soma/dendrites of the inhibitory
hypothalamic neurons, which cause more action potentials in
response to kainate depolarization, as in the hippocampus
(Cossart et al. 1998). We found that kainate did cause a
shift in the holding current, ranging from 7 to 40 pA, providing
evidence for this possibility. Second, kainate receptors may be present
on the presynaptic axon terminals and may enhance transmitter release
by a presynaptic mechanism, as tested below.
Kainate enhances GABA release at presynaptic site
In the presence of TTX (0.5 µM) and GYKI52466 (100 µM),
miniature IPSCs (mIPSCs) were recorded at a holding potential of 70 mV. Most of the neurons (10 of 14) showed a reversible increase of
mIPSC frequency after kainate application (10 µM, 5 min, Fig. 3A), suggesting a presynaptic
mechanism. The mean frequency of mIPSCs increased to 163 ± 25%
of control values during kainate application (P < 0.05, paired t-test) and returned to baseline after wash out
of kainate (106 ± 7% of control, n = 10, Fig.
3B). In contrast, kainate had no significant effect on the
amplitude distribution of mIPSCs (Fig. 3C), suggesting that
postsynaptic sensitivity to GABA was not changed by kainate. The other
four neurons showed no significant change (103 ± 5% of control)
in the frequency of mIPSCs during kainate application.
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DISCUSSION |
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In hippocampal neurons, activation of kainate receptors caused a
decrease in transmitter release from axon terminals (Lerma 1997). In contrast, our data show that kainate presynaptically causes an increase in GABA release from cultured hypothalamic neurons.
We do not view our data as contradicting earlier reports; rather, our
data appear to indicate that the actions of kainate may be strongly
dependent on neuronal type; hippocampal neurons show a decrease,
whereas hypothalamic neurons show a substantial increase in transmitter
release in response to presynaptic actions of kainate. These appear to
be the first data showing that a kainate receptor generally exerts an
enhancing effect on transmitter release at a presynaptic site. This may
be of significant and fundamental importance related to how glutamate
may regulate general brain activity. GABA is found in a large number of
hypothalamic neurons (Tappaz et al. 1982
), is present in
at least half of all presynaptic boutons (Decavel and van den
Pol 1990
), and acts as the primary transmitter mediating
inhibition in the hypothalamus (Kim and Dudek 1992
;
Randle et al. 1986
; Tasker and Dudek
1993
) including the arcuate nucleus (Belousov and van
den Pol 1997
). That kainate can enhance GABA release in
hypothalamic neurons would have the ultimate result of increasing
inhibition by greater activation of GABA receptors on the postsynaptic
cell. The absence of a kainate-mediated decrease in GABA release in
hypothalamic neurons is interesting given that many presynaptic
neuromodulator receptors (e.g., neuropeptide Y,
GABAB, mGluRs) act to depress hypothalamic
transmitter release through different mechanisms (Chen and van
den Pol 1996
, 1998
; Obrietan and van den
Pol 1998
).
Neurons of the hypothalamic arcuate nucleus are involved in secretion
of pituitary tropins, and release of these tropins is facilitated by
bursting patterns of action potentials. On a speculative note, the
enhanced GABA inhibition evoked by kainate may hyperpolarize the
postsynaptic neuron, and a negative membrane potential has been
suggested as a mechanism to modulate the burst-responsiveness of
arcuate neurons to synaptic input (MacMillan and Bourque
1993); whether these bursting cells receive axonal input
responsive to kainate remains to be determined. A large number of
neuroactive substances are found in different neurons of the arcuate
nucleus, many colocalized with GABA (Meister and Hokfelt
1988
). Although kainate receptor mRNAs have been found in
hypothalamic neurons with in situ hybridization (van den Pol et al.
1994
), the transmitter phenotype of the neurons that express kainate
receptors and what presynaptic effect kainate has in these circuits
remains to be determined. Because kainate receptors may be on
presynaptic axon terminals or the postsynaptic somato-dendritic area of
hypothalamic neurons, or both, the cellular location of the receptors
in specific circuits would be critical for determining the action of
activated kainate receptors.
Previous reports found a decrease in transmitter release with kainate
activation of presynaptic receptors in hippocampal neurons in slice and
culture (Clarke et al. 1997; Rodriguez-Moreno et al. 1997
), whereas we find an increase in GABA release with
kainate activation of hypothalamic neurons. This raises the question as to what might be the mechanism for this difference. A recent paper (Rodriguez-Moreno and Lerma 1998
) suggested that kainate
inhibited transmitter release by a presynaptic mechanism was based on
activation of a G protein by an ionotropic receptor; kainate activated
protein kinase C (PKC), which lead to phosphorylation of a protein
involved in transmitter exocytosis and thus inhibited release. Although in that case an increase in PKC was suggested to mediate a decrease in
transmitter release, other reports have shown that a PKC increase can
lead to an increase in GABA (Capogna et al. 1995
) and
glutamate release (Malenka et al. 1986
). In fact, when
Rodriguez-Moreno and Lerma (1998)
first treated their
hippocampal neurons with phorbol esters, kainate application caused an
increase in GABA release. The increase in GABA release in hypothalamic
neurons could be due to either a different set of proteins being
phosphorylated by PKC activation, or if the idea that there are two
different pools of PKC (Rodriguez-Moreno and Lerma 1998
)
has credence, then hypothalamic neurons may have a greater pool of the
PKC that enhances neurotransmission. We recently described a parallel
mechanism whereby strong activation of protein kinase A would lead to
an inhibition of GABA activity in hypothalamic neurons, but a small increase could enhance GABA activity (Obrietan and van den Pol 1997
). Alternately, kainate may activate a different subset of kainate receptors in hypothalamic neurons that are coupled differently to G proteins in these cells.
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ACKNOWLEDGMENTS |
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Research support was provided by National Institute of Neurological Disorders and Stroke Grants NS-31573, NS-34887, NS-37788, and by the National Science Foundation.
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FOOTNOTES |
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Address for reprint requests: A. N. van den Pol, Dept. of Neurosurgery, Yale University Medical School, 333 Cedar St., New Haven, CT 06520.
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 4 December 1998; accepted in final form 30 March 1999.
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REFERENCES |
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