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J Neurophysiol 77: 527, 1997;
0022-3077/97 $5.00
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The Journal of Neurophysiology Vol. 77 No. 2 February 1997, pp. 527-527
Copyright ©1997 by the American Physiological Society

Presynaptic Modulation by Metabotropic Glutamate Receptors of Excitatory and Inhibitory Synaptic Inputs to Hypothalamic Magnocellular Neurons

L. A. Schrader and J. G. Tasker

Neuroscience Training Program and Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Schrader, L. A. and J. G. Tasker. Presynaptic modulation by metabotropic glutamate receptors of excitatory and inhibitory synaptic inputs to hypothalamic magnocellular neurons. J. Neurophysiol. 77: 527-536, 1997. The effects of activation of metabotropic glutamate receptors (mGluRs) on synaptic inputs to magnocellular neurons of the hypothalamic supraoptic nucleus (SON) were studied with the use of whole cell patch-clamp and microelectrode recordings in acute hypothalamic slices. Application of the mGluR agonist trans-(±)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD, 100 µM) elicited an increase in the frequency of spontaneous excitatory postsynaptic potentials (EPSPs) and excitatory postsynaptic currents (EPSCs) in 20% of the cells, and of spontaneous inhibitory postsynaptic potentials (IPSPs) and inhibitory postsynaptic currents (IPSCs) in 50% of the cells tested in normal medium. The increased frequency of spontaneous EPSPs/EPSCs and IPSPs/IPSCs was blocked by tetrodotoxin (TTX), indicating that mGluRs act to excite the somata/dendrites of presynaptic glutamatergic and GABAergic neurons. (RS)-3,5-dihydroxyphenylglycine (50 µM), a selective group I receptor agonist, mimicked the presynaptic somatic/dendritic effects of trans-ACPD, suggesting that the presynaptic somatic/dendritic receptors responsible for increased spike-dependent glutamate and gamma -aminobutyric acid (GABA) release belong to the group I mGluRs. In the presence of TTX, trans-ACPD caused a decrease in the frequency of miniature EPSCs (up to 90%) in 13 of 16 cells, and a decrease in the frequency of miniature IPSCs (up to 80%) in 10 of 16 cells tested. Miniature EPSC and IPSC amplitudes usually did not change in trans-ACPD, suggesting that activation of metabotropic receptors located at presynaptic glutamatergic and GABAergic terminals led to a reduction in transmitter release onto SON magnocellular neurons. L(+)-2-amino-4-phosphonobutyric acid (100-250 µM), a selective group III receptor agonist, mimicked the effects of trans-ACPD at presynaptic terminals, decreasing the frequency of miniature EPSCs and IPSCs by up to 85% without affecting their amplitude. Thus the metabotropic receptors at presynaptic glutamate and GABA terminals in the SON belong to group III mGluRs. EPSCs evoked by electrical stimulation were enhanced by the group III receptor antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid, suggesting that presynaptic metabotropic receptors are activated by the release of endogenous glutamate. These data indicate that mGluRs in the hypothalamus have opposing actions at presynaptic somata/dendrites and at presynaptic terminals. Activation of group I receptors (mGluR1 and/or mGluR5) on presynaptic somata/dendrites led to an increase in spike-dependent transmitter release, whereas activation of the group III receptors (mGluR4, 7, and/or 8) on presynaptic terminals suppressed glutamate and GABA release onto SON neurons. No diffferences were seen in the effects of mGluR activation between immunohistochemically identified oxytocin and vasopressin neurons of the SON.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Magnocellular neurons in the supraoptic nucleus (SON) and paraventricular nucleus possess characteristic bursting behaviors that are responsible for the pulsatile release of vasopressin and oxytocin from axon terminals in the neurohypophysis. The patterns of bursting in these cells play a crucial role in optimizing hormone release (Cazalis et al. 1985). During the milk ejection reflex and parturition, oxytocin cells generate intermittent bursts of action potentials that are synchronized among all oxytocinergic neurons, resulting in a periodic release of hormone. Vasopressin cells exhibit phasic bursting in response to changes in blood pressure and blood osmolality (Poulain and Wakerley 1982), but the asynchrony of burst activity among individual neurons leads to a tonic increase in vasopressin secretion into the bloodstream.

Firing patterns are controlled by a combination of the intrinsic membrane conductances and synaptic inputs. Although it has been shown that the intrinsic conductances of magnocellular neurons are important for their bursting behavior (Renaud and Bourque 1991), the respective role of synaptic mechanisms in burst generation is still poorly understood. Glutamate acting at ionotropic receptors is responsible for fast excitatory synaptic transmission in hypothalamic neuroendocrine cells (Gribkoff and Dudek 1990; van den Pol et al. 1990; Wuarin and Dudek 1991, 1993). The involvement of glutamate receptors in the generation of bursting is suggested by the finding that activation of N-methyl-D-aspartate (NMDA) receptors induces rhythmic bursting in magnocellular neurons in vitro (Hu and Bourque 1992), and that phasic firing of vasopressin cells in vivo is blocked by both NMDA and non-NMDA glutamate receptor antagonists (Nissen et al. 1995).

Neuromodulation of synaptic inputs is likely to play an important role in the pattern of activation of magnocellular neurons. For example, the onset and the frequency of bursting in phasically firing magnocellular neurons are sensitive to the state of excitability of these cells, and slow changes in membrane potential are an essential part of phasic firing (Andrew and Dudek 1984). Metabotropic glutamate receptors (mGluRs), unlike the ionotropic receptors, are coupled to intracellular signal transduction mechanisms through G proteins, and mediate the slower, modulatory actions of glutamate (Schoepp and Conn 1993). Currently, there are eight identified subtypes of mGluRs (mGluR1-8), and these subtypes are divided into three groups on the basis of pharmacology and sequence homology (Pin and Duvoisin 1995). Group I includes mGluR1 and mGluR5, which have been linked to inositol triphosphate production. Group II and group III receptors have both been found to be coupled to adenosine 3',5'-cyclic monophosphate transduction pathways; group II comprises mGluR2 and mGluR3; and group III includes mGluR4, 6, 7, and 8. Membrane binding assays show that the relative density of mGluRs is comparable with the densities of NMDA and non-NMDA glutamate receptors in the hypothalamus (Meeker et al. 1994). Anatomic and biochemical data have so far provided evidence of four subtypes of the mGluRs, mGluR1 (van den Pol 1994; van den Pol et al. 1994), mGluR3 (Tanabe et al. 1993), mGluR5 (Romano et al. 1995), and mGluR7 (Kinzie et al. 1995), in the hypothalamus. Activation of mGluRs in hypothalamic neurons has been shown to cause phosphoinositide hydrolysis (Sortino et al. 1991), to inhibit adenosine 3',5'-cyclic monophosphate formation (Casabona et al. 1992), and to elicit an increase in intracellular unbound calcium (van den Pol et al. 1994).

We studied the modulatory effects of mGluR activation on the glutamate and gamma -aminobutyric acid (GABA) synaptic inputs to magnocellular neurons of the SON. We usedt r a n s - ( ± ) - 1 - a m i n o - 1 , 3 - c y c l o p e n t a n e  d i c a r b o x y l i c  a c i d(trans-ACPD), a mixture of the active and inactive ACPD isomers and a broad-spectrum mGluR agonist, to activate the three mGluR groups. (RS)-3,5-dihydroxyphenylglycine (DHPG) was used to activate group I receptors, and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) was used to activate the group III receptors selectively. (S)-2-amino-2-methyl-4-phosphonobutanoic acid (M-AP4) was applied to block group III receptors. We found metabotropic receptor activation to have opposing effects, mediated by different receptor subtypes, at presynaptic somata/dendrites and at presynaptic terminals. A preliminary account of this study has been presented in abstract form (Schrader and Tasker 1994).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slice preparation

Male Sprague-Dawley rats (40-120 g) were deeply anesthetized with pentobarbital sodium (50 mg/kg body weight) and decapitated. The brain was rapidly removed and placed in cold (0-1°C), oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM) 124 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.4 NaH2PO4, 11 glucose, and 5 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH adjusted to 7.2-7.4 with NaOH.

A tissue block containing the hypothalamus was prepared by making razor cuts rostral and lateral to the optic chiasm, and caudally at the level of the median eminence. The block was glued to the chuck of a vibrating tissue slicer (Campden Instruments) and rapidly submerged in ice-cold aCSF. Two coronal slices (400-500 µm) containing the SON were sectioned and placed on the ramp of an interface recording chamber. Heated aCSF (32-34°C) was perfused through the chamber, and a gas mixture of 95% O2-5% CO2 was humidified and directed over the surface of the slices. Slices were allowed to equilibrate in the recording chamber for 1.5-2 h before the start of experiments.

Electrophysiology

Patch electrodes (resistance 4-8 MOmega ) were pulled from borosilicate glass (1.65 mm OD, 1.2 mm ID; KG-33, Garner Glass) on a Flaming-Brown puller (Sutter Instruments). The pipette solution contained (in mM) 120 potassium gluconate, 10 HEPES, 1 NaCl, 1 CaCl2, 1 MgCl2, 2 magnesium adenosine-5'- triphosphate (ATP), 0.3 sodium guanosine-5'-triphosphate (GTP), and 10 ethyleneglycol-bis[beta -aminoethyl ether]-N,N,N'N'-tetraacetic acid (EGTA), pH adjusted to 7.2-7.4 with KOH. Potassium currents were blocked in some experiments with a patch solution containing (in mM) 110 D-gluconic acid, 110 CsOH, 10 CsCl, 10 HEPES, 11 EGTA, 1 MgCl2, 1 CaCl2, 2 Mg-ATP, and 0.3 Na-GTP; pH was adjusted to 7.2-7.4 with CsOH. Biocytin (0.1-0.3%) was included in the patch pipette as an intracellular marker.

The patch pipette was advanced through the slice in 2-µm steps with a piezoelectric microdrive (Nanostepper, Scientific Precision Instruments) set at minimum speed and acceleration settings. High-resistance seals (>1 GOmega ) were obtained before going to the whole cell configuration. Series resistance ranged from 12-40 MOmega , and was compensated 60-80%. Whole cell data were recorded with an Axoclamp 200A for current-clamp experiments and with an Axopatch 1D amplifier for voltage-clamp experiments (Axon Instruments).

Sharp microelectrodes (120-180 MOmega ) were pulled from thick-walled microfilament capillary tubes (1.0 mm OD, World Precision Instruments). The electrodes were filled with 2 M potassium acetate containing 1-2% biocytin. Microelectrodes were advanced through the tissue in 4-µm steps with the piezoelectric microdrive set at maximum speed and acceleration settings. Intracellularrecordings with sharp microelectrodes were performed with theAxoclamp 200A amplifier.

Electrical stimulation

Extracellular stimulation was applied with the use of a bipolar stimulating electrode constructed from two insulated platinum/iridium wires. The stimulating electrode was placed medial to the SON just dorsal to the optic tract. Constant-current pulses (0.5 ms, 300-800 µA) were delivered at 0.1-0.2 Hz and 8-10 evoked synaptic responses were recorded and stored on videotape for off-line averaging.

Data analysis

All data were low-pass filtered at 2 kHz, digitized at 22 kHz, and stored on video tape. Selected data were digitized and analyzed on a personal computer with the use of the Digidata 1200 interface and pCLAMP software (Axon Instruments). Current-voltage relations were acquired and analyzed on-line; synaptic events were analyzed off-line. Episodes (30-120 s) of spontaneous postsynaptic currents (PSCs) or postsynaptic potentials (PSPs) were collected at 1 kHz for frequency and amplitude analysis. PSPs and PSCs were detected by eye, selected manually, and analyzed for frequency and peak amplitude distributions with the use of pClamp software (Axon Instruments). Statistical significance of mean values was tested with the Wilcoxon signed-rank test. Cumulative fraction plots of miniature PSC amplitudes and interevent intervals were generated. Distributions of frequency and amplitude were compared with the Kolmogorov-Smirnov test. Probability values of <0.05 were considered significant. All values are expressed as means ± SE.

Pharmacology

trans-ACPD (100-200 µM), L-AP4 (10-250 µM), M-AP4 (250 µM), and DHPG (50 µM) (Tocris Cookson or RBI) were added to the perfusion bath. In some cases, trans-ACPD was applied focally in microdrops onto the surface of slices. Tetrodotoxin (TTX, 1-3 µM) (Sigma) was added to the bath in some experiments to block voltage-gated Na+ channels.

Intracellular labeling and immunohistochemistry

Cells were injected with biocytin (0.1-0.3% in patch electrodes, 1-2% in sharp microelectrodes) during each recording (Horikawa and Armstrong 1988). Slices were subsequently fixed overnight at 4°C in tris(hydroxymethyl)aminomethane-buffered saline with 4% paraformaldehyde and 10% sucrose and cryosectioned at 15-25 µm. Intracellular biocytin was labeled by incubation of free-floating sections in streptavidin conjugated to a blue fluorescent marker, 7-amino-4-methyl-coumarin-3-acetic acid (AMCA, Molecular Probes), diluted (1:500) in 0.1 M phosphate-buffered saline (PBS) containing 1% Triton X-100. Sections were screened for biocytin-labeled cells with the use of the fluorescence filter combination UV/420K under a compound microscope (Leitz) equipped with fluorescence.

Immunohistochemical double labeling of biocytin-filled cells was then performed according to methods described previously (Hoffmann et al. 1991). Briefly, sections were placed in 2% normal sheep serum in 0.1 M PBS for 15 min. Some sections were then incubated in a primary antiserum generated in rabbit against both the oxytocin- and the vasopressin-associated neurophysins of the rat for 36 h at 4°C (1:10,000 in 0.1 M PBS + 1% normal sheep serum and 0.2% sodium azide). This general neurophysin antibody, kindly provided by Dr. A. G. Robinson of the University of Pittsburgh (National Institute of Arthritis, Diabetes, and Kidney Diseases Grant AM-16166), has been fully characterized and found to label both oxytocin- and vasopressin-containing magnocellular neurons (Seif et al. 1977). Some cells were labeled for either oxytocin or vasopressin with polyclonal antibodies (VA10 or VA4, respectively; 1:1,000 in 0.1 M PBS + 1% normal sheep serum and 0.2% sodium azide), which were a generous gift from Dr. H. Gainer of the National Institutes of Health and have been thoroughly tested for specificity and cross reactivity (Altstein et al. 1988). After PBS rinses, the sections were incubated in goat anti-rabbit immunoglobulin G conjugated with fluorescein (Vector) for 1 h and rinsed in PBS again. Slices were mounted and coverslipped with Vectashield antifading mounting medium (Vector), and examined under epifluorescence for the presence of both biocytin labeling (with the UV/420K filters for AMCA) and neurophysin, oxytocin, or vasopressin immunoreactivity (with the use of a B/515W filter combination for fluorescein isothiocyanate).

The specificity of the oxytocin and vasopressin antibodies was tested in our slices with a series of preabsorption control experiments. No immunostaining was detected when the oxytocin antibody (VA10) was preincubated overnight at 4°C with three concentrations of oxytocin (10-6, 10-5, and 10-4 M; Sigma), or when the vasopressin antibody was preincubated with the same concentration series of vasopressin (10-6-10-4 M; Sigma). Additionally, the oxytocin antibody showed no cross reactivity with vasopressin (10-6-10-4 M), and the vasopressin showed no cross reactivity with oxytocin (10-6-10-4 M). The specificity of the antibodies was also confirmed empirically, because the suprachiasmatic nucleus, which contains exclusively vasopressinergic cells, did not show any immunolabeling with the oxytocin antibody, and the anterior commissural nucleus, which contains oxytocinergic but not vasopressinergic neurons, did not contain any cells labeled with the vasopressin antibody.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Supraoptic neurons were identified electrophysiologically during recordings as magnocellular neurons by the presenceof a transient, voltage-dependent potassium current (Bourque1988; Tasker and Dudek 1991). The responses of 87 magnocellular neurons to trans-ACPD were tested; 3 cells were recorded with sharp microelectrodes and 84 cells were recorded with patch electrodes. The magnocellular neurons recorded with microelectrodes had a mean membrane potential of -56 ± 2 (SE) mV and a mean input resistance of 273 ± 47 MOmega . The cells recorded with patch electrodes had a mean membrane potential, corrected for junction potential (Neher 1992), of -64 ± 1 mV and a mean input resistance of 999 ± 63 MOmega .

Evoked PSCs/PSPs

trans-ACPD (100 µM) caused a decrease in the amplitude of excitatory PSPs (EPSPs) and excitatory PSCs (EPSCs) evoked by electrical stimulation medial to the SON. Of 26 magnocellular neurons tested, 22 showed a reduction in the amplitude of the evoked excitatory synaptic response ranging from 24 to 88% in response to trans-ACPD (Fig. 1). Five of five cells recorded in voltage clamp showed a reduction in the inward current of 46 ± 10% (60.4 ± 7.1 pA to 26.2 ± 3.4 pA). Of 21 cells recorded in current clamp, 17 showed a 57 ± 8% reduction in the EPSP (10 ± 1 mV to 4 ± 0.2 mV). trans-ACPD application also caused an increase in input resistance (178 ± 46 MOmega or 18 ± 5%) in 13 of the 21 neurons recorded in current clamp. No consistent effect of trans-ACPD on evoked inhibitory PSPs (IPSPs) and inhibitory PSCs (IPSCs) was observed in six cells tested.


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  		FIG. 1. Decrease in amplitude of evoked excitatory postsynaptic potentials (EPSPs) and excitatory postsynaptic currents (EPSCs) in response to trans-(±)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD). A: amplitude of the EPSP evoked by electrical stimulation decreased from 10.8 mV in normal medium (CONTROL) to 6.8 mV in medium containing trans-ACPD (TRANS-ACPD, 100 µM); the effect was reversible (WASH). Membrane potential was -80 mV. B: amplitude of the evoked EPSC in a different cell decreased from -59.6 pA in normal medium (CONTROL) to -29.1 pA in trans-ACPD (TRANS-ACPD); this effect was reversible with washout of the trans-ACPD (WASH). Holding potential: -70 mV. Stimulus artifacts were blanked in both the current-clamp and the voltage-clamp traces. C: input resistance of the cell shown in A, calculated from the voltage responses to -50-pA current pulses, increased from 900 to 1,040 MOmega with trans-ACPD application. All traces are averages of 10 sweeps.

Spontaneous PSCs/PSPs

The effects of trans-ACPD (100 µM) were tested on spontaneous EPSCs or EPSPs in 15 cells recorded at membrane potentials of -60 to -70 mV, and on spontaneous IPSCs or IPSPs in 16 cells recorded at membrane potentials of -30 to -40 mV. trans-ACPD caused a 20-90% decrease in the frequency of spontaneous EPSCs/EPSPs in 11 of 15 cells (Fig. 2), and a 15-90% decrease in the frequency of spontaneous IPSCs/IPSPs in 8 of 16 cells. Decreased frequencies of spontaneous PSCs/PSPs were maintained throughout the 10- to 15-min application of trans-ACPD. Of the remaining four cells in which EPSCs/EPSPs were analyzed and the eight cells in which IPSCs/IPSPs were analyzed, three cells showed an increase in EPSC/EPSP frequency of 20-70% and eight cells showed an increase in IPSCs/IPSPs of 20-740% in response to trans-ACPD (Fig. 3). Thus the main effect of metabotropic receptor activation appeared to be a reduction in transmitter release from the presynaptic terminal, but a substantial number of cells showed an increase in release of transmitter.


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  		FIG. 2. Decrease in spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs) in response to trans-ACPD. A: voltage-clamp recording of EPSCs before (CONTROL) and during bath application of trans-ACPD (TRANS-ACPD, 100 µM). The cell was held at -65 mV. The reduction in EPSCs reversed partially in this cell after 20 min of washout of the trans-ACPD (WASH). B: voltage-clamp recording of IPSCs before (CONTROL) and during bath application of trans-ACPD (TRANS-ACPD, 100 µM). The cell was held at -35 mV. The reduction in IPSCs was partially reversed after 12 min of washout of the trans-ACPD (WASH).


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  		FIG. 3. trans-ACPD caused an increase in spontaneous IPSCs. A: drop application of 100 µM trans-ACPD (down-arrow ) in normal artificial cerebrospinal fluid (aCSF) caused an increase in IPSCs recorded in voltage clamp. Holding potential: -30 mV. Successive traces represent 150 s of continuous recording. B: amplitude distribution histograms of IPSCs recorded in control medium (black bars) and for 40 s after drop application of trans-ACPD (white bars). Forty s of data before and after the drop were analyzed. C: bar graph showing the mean % increase in the amplitude and frequency of spontaneous IPSCs/inhibitory postsynaptic potentials (IPSPs) of 8 cells that showed a similar enhancement of spontaneous IPSCs/IPSPs in response to trans-ACPD in normal aCSF.

Miniature PSCs

Miniature EPSCs were recorded in 16 cells and miniature IPSCs were recorded in 16 cells in the presence of TTX (1-3 µM) to block Na+ spike-mediated synaptic transmission. trans-ACPD caused a significant decrease in the frequency of miniature EPSCs (Fig. 4) in 12 of 16 cells, whereas only 3 of the 16 cells showed a significant decrease in miniature EPSC amplitude (P < 0.05, Kolmogorov-Smirnov test). The mean frequency of EPSCs calculated across all 16 cells decreased significantly (2.81 ± 0.51 Hz to 1.34 ± 0.32 Hz; 49 ± 6%) in the presence of trans-ACPD (P < 0.01, Wilcoxon signed-rank test), whereas the mean amplitude did not change (Fig. 4).


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  		FIG. 4. Decrease in the frequency of miniature EPSCs in response to trans-ACPD. A: voltage-clamp recording of EPSCs recorded in tetrodotoxin (TTX) (3 µM) before (CONTROL), during (TRANS-ACPD), and after (WASH) bath application of trans-ACPD (100 µM). Holding potential: -70 mV. trans-ACPD caused a reversible decrease in the number of inward synaptic currents recorded. B: amplitude distribution histogram of miniature EPSCs recorded in control medium (white bars) and in trans-ACPD (black bars) in the same cell as in A. Sixty seconds of data were analyzed. C: cumulative fraction plot of miniature EPSC amplitudes in control (black-square) and in trans-ACPD (black-triangle). There was no significant change in the miniature EPSC amplitude distribution caused by trans-ACPD application. D: cumulative fraction plot of the intervals between miniature EPSCs. trans-ACPD caused a significant shift in the interevent interval distribution toward longer intervals, indicating a strong reduction in the frequency of miniature EPSCs (P < 0.001 Kolmogorov-Smirnov test). E: change in mean amplitude and frequency of miniature EPSCs in trans-ACPD. Mean EPSC amplitudes and frequencies were calculated in trans-ACPD and normalized to control values for each of the 16 cells. These normalized values were averaged and expressed as percentages of mean control values. The mean population frequency was significantly different from control (**, P < 0.01), whereas the mean population amplitude was not (Wilcoxon signed-rank test).


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  		FIG. 5. trans-ACPD caused a decrease in the frequency of miniature IPSCs. A: voltage-clamp recording of IPSCs in TTX (3 µM) before (CONTROL), during (TRANS-ACPD), and after (WASH) bath application of trans-ACPD (100 µM). Holding potential: -35 mV. The number of outward synaptic currents recorded was reduced in trans-ACPD. B: amplitude distribution histogram of miniature IPSCs recorded in the same cell in control medium (white bars) and during trans-ACPD application (black bars). Sixty seconds of data were analyzed. C: cumulative fraction plot of miniature IPSC amplitudes in control (black-square) and in trans-ACPD (black-triangle). There was only a small, nonsignificant shift in the miniature IPSC amplitude distribution in trans-ACPD. D: cumulative fraction plot of the intervals between miniature IPSCs. The interevent interval plot was shifted toward longer intervals, or slower frequencies, in trans-ACPD (P < 0.05, Kolmogorov-Smirnov test). E: change in mean amplitude and frequency of miniature IPSCs in trans-ACPD. The mean frequencies and amplitudes of miniature IPSCs were calculated and normalized to mean values in normal aCSF in 16 cells. The averaged normalized values in trans-ACPD are expressed as percentages of mean control values. The mean miniature IPSC frequency was significantly different from control (**, P < 0.01), whereas the mean amplitude was not (Wilcoxon signed-rank test).

The frequencies of miniature IPSCs decreased significantly in 10 of 16 cells tested with trans-ACPD (Fig. 5), whereas only 4 of the 16 cells showed a significant decrease in the amplitude of miniature IPSCs (P < 0.05, Kolmogorov-Smirnov test). The overall mean frequency of IPSCs across all 16 cells decreased significantly (1.35 ± 0.29 Hz to 0.79 ± 0.18 Hz, 31 ± 10%) in trans-ACPD (P < 0.01, Wilcoxon signed-rank test), whereas the mean amplitude did not change. Thus the main effect of trans-ACPD in these experiments was to reduce the probability of spike-independent transmitter release by activating mGluRs on presynaptic glutamatergic and GABAergic terminals. The reduced PSC amplitude seen in some cells suggests that mGluR activation may also affect postsynaptic responsiveness to glutamate and GABA.

The increase in the frequency of spontaneous EPSCs and IPSCs recorded in 20 and 50% of cells, respectively, in normal aCSF, and the decrease in the frequency of miniature EPSCs and IPSCs in all cells recorded in TTX, suggest that trans-ACPD is acting at different sites on presynaptic neurons with opposing effects. Thus metabotropic receptor activation at presynaptic terminals inhibits the release of glutamate and GABA, whereas metabotropic receptors at the presynaptic somata/dendritic level act to excite presynaptic glutamate and GABA neurons and increase spike-dependent transmitter release. We tested the hypothesis that these effects might be mediated by actions of different mGluR subtypes.

mGluR subtypes

We studied the effects of L-AP4, the group III receptor agonist, on evoked spontaneous and miniature PSCs in SON neurons. L-AP4 (100-250 µM) reduced the amplitude of evoked EPSCs by 37 ± 6% (-45.5 ± 14.0 pA to -31.0 ± 12.1 pA; P < 0.05) in four of five cells (Fig. 6), and decreased the frequency of spontaneous EPSCs by 56 ± 16% (3.0 ± 0.5 Hz to 1.16 ± 0.23 Hz, n = 3), and the frequency of spontaneous IPSCs by 72 ± 9.2 % (2.17 ± 1.0 Hz to 0.48 ± 0.22 Hz, n = 3). No change in the spontaneous synaptic activity was seen with a concentration of L-AP4 of <50 µM (n = 2). In TTX, L-AP4 caused a significant decrease in the frequency of miniature EPSCs in three of four cells and of miniature IPSCs in five of six cells (Fig. 6), but had no effect on the amplitude of the miniature currents in any of the cells tested (Kolmogorov-Smirnov test). The overall frequency of miniature EPSCs was reduced by 52 ± 9% (1.95 ± 0.5 Hz to 0.69 ± 0.31 Hz,P < 0.05, Wilcoxon signed-rank test) and the overallfrequency of miniature IPSCs was reduced by 58 ± 13%(2.07 ± 0.54 Hz to 0.72 ± 0.17 Hz, P < 0.05).


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  		FIG. 6. Role of group III metabotropic receptor activation at presynaptic terminals. Activation of group III metabotropic glutamate receptors with L(+)-2-amino-4-phosphonobutyric acid (L-AP4) mimicked the actions of trans-ACPD at presynaptic glutamate and gamma -aminobutyric acid (GABA) terminals. A: amplitude of the EPSC evoked by electrical stimulation decreased 31% in L-AP4 (100 µM). This effect was reversible (WASH). Holding potential: -70 mV. B: group III receptor antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid (M-AP4) caused a reversible enhancement of the evoked EPSC (47%), suggesting that group III metabotropic receptors are activated by the release of endogenous glutamate. Holding potential: -70 mV. C: voltage-clamp recording of miniature EPSCs before (CONTROL), during (L-AP4), and 15 min after (WASH) bath application of L-AP4 (250 µM). Holding potential: -70 mV. The number but not the amplitiude of inward currents was reduced in L-AP4. D: miniature IPSCs recorded before (CONTROL), during (L-AP4), and 13 min after (WASH) application of L-AP4 (250 µM). Holding potential: -35 mV. The frequency but not the amplitude of outward synaptic currents was reduced in L-AP4. E: cumulative fraction plot of the intervals between miniature EPSCs in control (black-square) and in L-AP4 (black-triangle) in the same cell pictured in C. The interevent interval plot was shifted toward longer intervals, or slower frequencies, in L-AP4 (P < 0.05, Kolmogorov-Smirnov test). F: cumulative fraction plot of the intervals between miniature IPSCs for the same cell pictured in D. The interevent interval plot was shifted toward longer intervals in L-AP4 (P < 0.001, Kolmogorov-Smirnov test). G: change in mean amplitude and frequency of miniature EPSCs and IPSCs in L-AP4. Mean EPSC and IPSC amplitudes and frequencies were calculated in L-AP4 and normalized to control values for the EPSCs recorded in 4 cells and IPSCs recorded in 6 cells. These normalized values were averaged and expressed as percentages of mean control values. The mean population frequency was significantly different from control for both miniature EPSCs and miniature IPSCs (*, P < 0.05), whereas the mean population amplitude was not (Wilcoxon signed-rank test).

Application of the group III receptor antagonist M-AP4 resulted in a 45 ± 7% increase in the mean amplitude of evoked EPSCs (-25.5 ± 7.0 pA to -42.3 ± 12.9 pA) in five of five cells (Fig. 6). This implies that group III receptors are activated, leading to reduced transmitter release, by the release of endogenous glutamate, because blocking these receptors with M-AP4 enhanced evoked EPSCs. Thus group III receptors at presynaptic terminals act to suppress glutamate and GABA release.

DHPG (50 µM), a group I receptor agonist, caused an increase in the frequency of spontaneous EPSCs in one of five cells (259%) and an increase in the frequency of spontaneous IPSCs in two of seven cells (344 ± 2.7%, Fig. 7). DHPG had no effect on the frequency of miniature EPSCs in four cells or on miniature IPSCs in four cells tested. Thus the increase in the spontaneous PSCs and PSPs in response to trans-ACPD application was most likely due to the depolarization of presynaptic neurons by group I receptors. The proportion of cells that showed increases in EPSC and IPSC frequencies in response to mGluR activation may reflect the number of intact presynaptic glutamate and GABA cells in our slices.


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  		FIG. 7. Role of group I receptors in metabotropic receptor activation at presynaptic somata/dendrites. A: (RS)-3,5-dihydroxyphenylglycine (DHPG) (50 µM), a selective group I receptor agonist, caused a 342% increase in the frequency of spontaneous IPSCs (DHPG) recorded in normal aCSF. The effect was reversible (WASH). Holding potential: -35 mV. B: amplitude histogram in the same cell showing the IPSC amplitude distribution before (black bars) and during (white bars) application of DHPG. Sixty seconds of data were analyzed. C: bar graph showing the mean % increase in the amplitude and frequency of spontaneous IPSCs in the 2 cells that showed increased frequency of spontaneous IPSCs in response to DHPG.

Immunohistochemical identification of magnocellular neurons

Twenty-five cells were recovered and identified after biocytin staining and immunohistochemical double labeling with antisera to oxytocin, vasopressin, or neurophysin. Ten cells were verified as magnocellular neurons by their neurophysin immunopositivity, 10 cells were oxytocin positive (Fig. 8), 3 were vasopressin positive, and 2 were negative for oxytocin (i.e., probably vasopressinergic). No differences were found in the responses of oxytocin and vasopressin neurons to trans-ACPD.


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  		FIG. 8. Immunohistochemical identificationof an oxytocinergic magnocellular neuron. A supraoptic nucleus (SON) neuron was recorded with a sharp microelectrode and injected with biocytin. The biocytin-filled cell was labeled with a blue fluorescent marker7 - a m i n o - 4 - m e t h y l - c o u m a r i n - 3 - a c e t i c  a c i d(AMCA) and recovered in 2 serial sections. One section was treated with an oxytocin antibody and the other section was labeled with an antibody to vasopressin. A1 and B1: the 2 sections visualized after immunohistochemical treatment under ultraviolet filters to reveal the biocytin-labeled cell. A2 and B2: same sections under fluorescein filters to reveal the immunohistochemically labeled cells. A: immunohistochemical treatment of this section with theoxytocin antibody (OXY) stained the biocytin-labeled cell (), indicating that it was oxytocinergic. B: biocytin-labeled cell () stained negative for vasopressin (AVP) in the adjacent section, confirming that the recorded cell was an oxytocinergic magnocellular neuron. Calibration in A1 applies to all panels. OC, optic chiasm.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Fast excitatory and inhibitory synaptic inputs to magnocellular neurons of the SON are mediated by glutamate (Wuarin and Dudek 1993) and GABA (Randle et al. 1986). The results of this study indicate that activation of mGluRs has a modulatory effect on both the glutamatergic and GABAergic inputs to these cells. This synaptic modulation occurs at the presynaptic terminal, resulting in a reduction in spike-independent glutamate and GABA release, and at the presynaptic somatic/dendritic level, causing an increase in spike-mediated glutamate and GABA release.

mGluR activation at glutamate terminals

Several observations suggest that activation of mGluRs caused a decrease in the release of glutamate from presynaptic terminals in the SON. First, trans-ACPD application resulted in a reduction of the amplitude of evoked EPSPs and EPSCs and an increase in the input resistance of the magnocellular neurons. The increased input resistance would be expected to amplify evoked EPSPs if there were no effect of the trans-ACPD on presynaptic transmitter release or on postsynaptic ionotropic glutamate receptor sensitivity. Second, the frequency of miniature EPSCs decreased in response to trans-ACPD, without a corresponding change in the EPSC amplitude. This suggests that mGluR activation at the presynaptic terminal caused a reduction in quantal release, and a reduction in EPSC frequency, without changing significantly the sensitivity of postsynaptic ionotropic glutamate receptors. These effects were probably mediated by metabotropic receptor subtypes mGluR4 and/or mGluR7 (or mGluR8), because they were mimicked by L-AP4 (100-250 µM), the group III receptor agonist. L-AP4 concentrations <50 µM were ineffective at changing spontaneous EPSC frequency, suggesting that mGluR7, which is highly expressed in the hypothalamus and at which L-AP4 has a median effective concentration of 160 µM (Kinzie et al. 1995; Okamoto et al. 1994; Saugstad et al. 1994), probably played a more prominent role in the observed response than mGluR4, at which L-AP4 has a median effective concentration of 0.5 µM (Tanabe et al. 1993).

mGluR activation at GABA terminals

We found that metabotropic receptor activation also modulated GABAergic synaptic transmission via actions at presynaptic terminals, and that this modulation consisted of a depression of GABA release. trans-ACPD application in the presence of TTX caused a decrease in the frequency of miniature IPSCs, suggesting a reduction in transmitter release, whereas miniature IPSC amplitude, which is assumed to be susceptible primarily to postsynaptic modulation, was not changed significantly in most cells. L-AP4 also reduced the frequency of miniature IPSCs, suggesting that, like metabotropic receptors at presynaptic glutamate terminals, mGluRs responsible for inhibiting transmitter release from presynaptic GABA terminals belong to group III (i.e., mGluR4, 7, and/or 8).

mGluR activation at presynaptic somata/dendrites

trans-ACPD elicited an increase in the frequency of spontaneous IPSCs/IPSPs in normal aCSF in half the cells and an increase in the frequency of spontaneous EPSCs/EPSPs in 20% of the cells tested. TTX application blocked the trans-ACPD-evoked increase in spontaneous IPSCs/IPSPs and EPSCs/EPSPs, and resulted in a decrease in the frequency of miniature IPSCs and EPSCs in trans-ACPD in nearly all the cells. Therefore metabotropic receptor activation occurred at two levels in local presynaptic neurons, with opposing effects at each level---at presynaptic somata/dendrites it caused a depolarization and spike generation, resulting in an increase in spike-mediated GABA and glutamate release from intact GABA and glutamate neurons, and at presynaptic terminals it reduced spike-independent release of GABA and glutamate release onto magnocellular neurons. That only 50% of the SON cells showed an increase in spontaneous IPSCs/IPSPs and only 20% showed an increase in EPSCs/EPSPs in trans-ACPD probably reflected the proportion of magnocellular neurons in the slice that received inputs from intact GABA and glutamate neurons, respectively. Consistent with this interpretation is the observation that trans-ACPD elicits a 3- to 15-mV depolarization in both SON magnocellular neurons (Schrader and Tasker 1994) and in neurons recorded outside of the nucleus (unpublished observations), some of which may be GABA-containing (Theodosis et al. 1986) and glutamate-containing neurons (van den Pol and Trombley 1993). The opposing effects of trans-ACPD at the presynaptic somata/dendrites and terminals reflect activation of different mGluR subtypes, as described in GABAergic interneurons in the CA3 region of the hippocampus (Poncer et al. 1995). L-AP4 decreased the frequency of spontaneous and miniature IPSCs and EPSCs, indicating that group III receptors exist at presynaptic glutamate and GABA terminals and act to reduce the probability of quantal release. DHPG increased the frequency of EPSCs and IPSCs in normal aCSF in a fraction of the cells, but did not affect the frequency of miniature EPSCs or IPSCs, suggesting that mGluR1 and/or mGluR5 located at the presynaptic somata/dendrites were responsible for the increase in spike-dependent release of GABA and glutamate onto the magnocellular neurons. This observation correlates with anatomic data in which mGluR1proportional to was localized immunohistochemically to cells and dendrites surrounding the SON (van den Pol 1994).

Possible functional significance

Application of M-AP4, the group III receptor antagonist, in the absence of trans-ACPD or L-AP4 led to an increase in the peak amplitude of evoked EPSCs, which was caused presumably by blockade of the inhibitory actions of released glutamate at the presynaptic group III metabotropic receptors. This suggests, therefore, that the release of endogenous glutamate activates group III metabotropic receptors at the presynaptic terminal, which results in a rapid feedback inhibition of further glutamate release and a reduction in the peak amplitude of the resulting EPSC.

How the presynaptic effects of mGluR activation at glutamate and GABA terminals influence the firing patterns of oxytocin and vasopressin neurons is not yet known. The spatial organization of mGluRs with respect to glutamate and GABA synapses would be expected to have a profound effect on their activation under different physiological conditions. mGluRs on glutamate terminals appear to be close to the presynaptic release site, because their activation occurs rapidly enough to attenuate peak glutamate release. Further studies are needed to determine what role these receptors, as well as the mGluRs at GABA terminals and those on the postsynaptic membrane (Schrader and Tasker 1994), play in shaping the patterns of electrical activity characteristic of magnocellular neurons.

    ACKNOWLEDGEMENTS

  We thank K. Szabó for technical assistance, Dr. G. Schofield for critical review of the manuscript, and Dr. A. G. Robinson and Dr. H. Gainer for gifts of neurophysin, oxytocin, and vasopressin antisera.

  This work was supported by a fellowship to L. A. Schrader and a Grant-in-Aid to J. G. Tasker from the Louisiana American Heart Association, and by National Institute of Neurological Disorders and Stroke Grant NS-31187.

    FOOTNOTES

  Address for reprint requests: J. G. Tasker, Dept. of Cell and Molecular Biology, 2000 Percival Stern Hall, Tulane University, New Orleans, LA 70118.

  Received 2 February 1996; accepted in final form 20 September 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References
0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society



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