Kainate Acts at Presynaptic Receptors to Increase GABA Release From Hypothalamic Neurons

Qing-Song Liu, Peter R. Patrylo, Xiao-Bing Gao, and Anthony N. van den Pol

Department of Neurosurgery, Yale University Medical School, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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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 alpha -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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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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 MOmega ) 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. alpha -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 MOmega ) 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.


    RESULTS
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ABSTRACT
INTRODUCTION
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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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Fig. 1. Kainate increases the frequency of spontaneous inhibitory postsynaptic potentials (IPSPs) in hypothalamic slices. A: kainate (1 µM) reversibly increased the frequency of spontaneous IPSPs recorded from an arcuate nucleus neuron in artificial cerebrospinal fluid containing GYKI and D,L-2-amino-5-phosphonopentanoic acid (AP5). The membrane potential of this cell was held at approximately -75 mV. B: schematic diagram demonstrating the hypothalamic slice preparation and experimental configuration used. C: bar graph showing the normalized frequency of spontaneous IPSPs before, during, and 5 min after kainate application in 5 of 9 neurons that responded to kainate with a change in frequency >= 20%.

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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Fig. 2. Kainate enhances the frequency of spontaneous GABA-mediated postsynaptic currents (PSCs) in culture. A: at a holding potential (HP) of -70 mV, near the GABA reversal potential, inward currents are found. These are blocked by GYKI 52466 (100 µM) and CNQX (10 µM), and recover after antagonist wash out. Pipette solution contained KMESO4. B: when the holding potential of the same cell shown in A was shifted to 0 mV, near the reversal potential for glutamate, spontaneous outward currents were found, which were reversibly blocked by bicuculline (10 µM). C: the current reversed at around -75 mV, typical of GABA under these conditions. TTX (0.5 µM) caused a reversible block of the large spontaneous currents, indicating a dependence on sodium-mediated action potentials. D: in the presence of GYKI 52446, kainate (10 µM), caused a reversible increase in the frequency of spontaneous IPSCs. Pipette solution contained KCl. HP, -60 mV. E: analysis of the normalized frequency showed a substantial and reversible increase (to 273%) in the frequency of PSCs.

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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Fig. 3. Presynaptic site of kainate action. A: kainate (10 µM) reversibly increased the frequency of miniature GABA-mediated currents (>4 pA). Pipette solution contained KCl. B: bar graph shows the normalized increase in frequency in 10 neurons, with 1.0 being the prekainate baseline. C: cumulative probability histogram for mIPSCs before, during, and after wash out of kainate, shows no change, indicating the lack of a kainate effect on mIPSC amplitude.


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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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