Physiological Evidence for Local Excitatory Synaptic Circuits in the Rat Hypothalamus

Cherif Boudaba1, Laura A. Schrader2, and Jeffrey G. Tasker1, 2

1 Department of Cell and Molecular Biology and 2 Neuroscience Graduate Program, Tulane University, New Orleans, Louisiana 70118

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Boudaba, Cherif, Laura A. Schrader, and Jeffrey G. Tasker. Physiological evidence for local excitatory synaptic circuits in the rat hypothalamus. J. Neurophysiol. 77: 3396-3400, 1997. We conducted whole cell voltage-clamp and current-clamp recordings in slices of rat hypothalamus to test for local excitatory synaptic circuits. Local excitatory inputs to neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were studied with the use of electrical and chemical stimulation. Extracellular electrical stimulation provided indirect evidence of local excitatory circuits. Single stimuli evoked multiple excitatory postsynaptic potentials (EPSPs) or excitatory postsynaptic currents (EPSCs) in some PVN and SON cells, invoking polysynaptic excitatory inputs. Repetitive stimulation (10-20 Hz, 2-10 s) elicited long afterdischarges of EPSPs/EPSCs, suggesting a potentiation of upstream synapses in a polysynaptic circuit. Bath application of metabotropic glutamate receptor agonists provided more conclusive evidence for local excitatory circuits. Metabotropic receptor activation caused an increase in the frequency of EPSPs/EPSCs that was blocked by tetrodotoxin, suggesting that it was mediated by activation of local presynaptic excitatory neurons. The local excitatory inputs to SON and PVN neurons were mediated by glutamate release, because the EPSPs/EPSCs elicited with electrical stimulation and metabotropic receptor activation were blocked by ionotropic glutamate receptor antagonists. Finally, glutamate microstimulation furnished the most direct demonstration of local excitatory synaptic circuits. Glutamate microstimulation of perinuclear sites elicited an increase in the frequency of EPSPs/EPSCs in 13% of the PVN and SON neurons tested. Two sites provided most of the local excitatory synaptic inputs to PVN neurons, the dorsomedial hypothalamus and the perifornical region. These experiments provide converging physiological evidence for local excitatory synaptic inputs to hypothalamic neurons, inputs that may play a role in pulsatile hormone release.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Pulsatile hormone release, which is a common characteristic of neuroendocrine systems of the hypothalamus, is often a result of synchronized bursting among neurosecretory cells (Belin and Moos 1986; Poulain and Wakerley 1982). How the electrical activity of individual hypothalamic neurons is synchronized is not known. Mechanisms such as electrotonic coupling, electric field effects, and increased extracellular potassium may contribute to the synchronization of hypothalamic neurons (Hatton 1990; Theodosis and Poulain 1993), but local synaptic circuits are likely to be critical in synchronizing neuronal activity, especially among neurons located in separate hypothalamic nuclei (Moos and Richard 1989). We have reported in previous studies that both magnocellular and parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) receive local inhibitory synaptic inputs from perinuclear gamma -aminobutyric acid (GABA) neurons (Boudaba et al. 1996a; Tasker and Dudek 1993). In the current study we tested for the presence of local glutamatergic inputs, defined here as intrahypothalamic projections, to magnocellular and parvocellular neurons of the PVN and to magnocellular neurons of the supraoptic nucleus (SON) with converging electrical and chemical stimulation techniques. Magnocellular and parvocellular neurons were distinguished on the basis of their specific electrical properties (Hoffman et al. 1991; Tasker and Dudek 1991). A preliminary description of these data has been presented in abstract form (Boudaba et al. 1996b).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slices were prepared according to methods described previously (Boudaba et al. 1996a). Briefly, male Sprague-Dawley rats (Charles River) were deeply anesthetized with pentobarbital sodium (50 mg/kg ip) and decapitated, and the brains were removed and immersed in cooled (1-2°C), oxygenated artificial cerebrospinal fluid consisting of (in mM) 140 NaCl, 3 KCl, 1.3 MgSO4, 1.4 NaH2PO4, 11 glucose, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.4 CaCl2, and 3.25 NaOH, pH 7.2-7.4. Hypothalamic slices (400 µm) containing the PVN in the coronal, horizontal, or parasagittal plane, or containing the SON in the coronal plane, were cut in cooled artificial cerebrospinal fluid with a vibrating microtome (WPI) and transferred to a ramp-style interface recording chamber (32-34°C) or to a holding chamber (room temperature).

Sharp microelectrodes were made from microfilament glass capillaries (0.6 mm ID, 1.0 mm OD; WPI) and patch pipettes were pulled from borosilicate glass (1.65 mm OD, 1.2 mm ID; Garner Glass) with a Flaming/Brown micropipette puller (Sutter Instruments). Sharp microelectrodes were filled with 2 M KCl or 2 M potassium acetate; patch pipettes had an inner diameter of ~2 µm and were filled with (in mM) 120 potassium gluconate, 10 HEPES, 1 NaCl, 1 CaCl2, 1 MgCl2, 2 Mg-adenosine-5'-triphosphate, 0.3 Na-guanosine-5'-triphosphate, and 10 ethyleneglycol-bis[b-aminoethyl ether]-N,N,N',N'-tetraacetic acid, pH adjusted to 7.2-7.4 with KOH.

Electrical stimulation of the areas dorsolateral or dorsomedial to the SON and lateral to the PVN was administered with an insulated bipolar platinum-iridium electrode to evoke excitatory postsynaptic potentials (EPSPs)/excitatory postsynaptic currents (EPSCs) in SON and PVN neurons, respectively. Constant-current pulses (0.5 ms, 0.3-0.8 mA) were delivered as single (<= 0.1 Hz) or repetitive stimuli (10-20 Hz for 2-10 s) with an isolated stimulator (Winston Electronics).

The following drugs were applied in the perfusion bath: the metabotropic glutamate receptor agonists trans(±)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD, 75-100 µM) and (RS)-3,5-dihydroxyphenylglycine (DHPG, 50 µM; Tocris Cookson) to activate local presynaptic neurons (Schrader and Tasker 1997); the N-methyl-D-aspartate (NMDA) receptor antagonistD,L-2-amino-5-phosphonovalerate (AP5; 100 µM) and the non-NMDA receptor antagonist 5,6-dinitroquinoxaline-2,3-dione (DNQX; 50 µM; Tocris Cookson) to block ionotropic glutamate receptors; bicuculline methiodide (30-50 µM; Sigma) to block GABAA-receptor-mediated inhibitory postsynaptic potentials; and tetrodotoxin (TTX) (1.5-3 µM; Sigma) to block spike-mediated synaptic activity.

Glutamate microdrops (10-20 mM) were applied via a pipette (5-10 µm ID) on the surface of slices with a picospritzer (General Valve). The microdrops were applied under visual control at different sites around the PVN and SON. Janus green (0.1%) was included in the pipette to monitor the position and spread of the microdrops, which measured 200-250 µm diam after radiating on the surface of the slice.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

A total of 221 neurons was recorded in the PVN and 57 in the SON; 152 cells were classified as putative magnocellular and 126 cells were classified as putative parvocellular neurons on the basis of electrophysiological characterization of each cell (Hoffman et al. 1991; Tasker and Dudek 1991). The mean membrane potential was -58 ± 0.7 (SE) mV(n = 96) in the cells recorded with sharp electrodes (this and the following means expressed as the mean ± SE) and -59 ± 0.7 mV (n = 92) with patch electrodes (values uncorrected for junction potentials). The mean input resistance was 276 ± 13 MOmega (n = 120) for cells recorded with sharp electrodes and 741 ± 23 MOmega (n = 101) for cells recorded with patch electrodes. Mean spike amplitude (threshold to peak) was 67 ± 0.6 mV (n = 125) in sharp-electrode recordings and 64 ± 0.9 mV (n = 96) in patch recordings.

Activation of local excitatory circuits with electrical stimulation

Extracellular electrical stimulation was performed in coronal slices. Single-shock stimulation at resting potential generally elicited an initial EPSP/EPSC with a latency of 2-3 ms, considered a monosynaptic response. The first evidence suggestive of local excitatory circuits derived from the finding that 6 of a total of 13 cells (4 of 7 in the PVN and 2 of 6 in the SON) generated multiple, long-latency EPSPs/EPSCs, in addition to the monosynaptic EPSP/EPSC, in response to single stimuli (Fig. 1A). The long-latency EPSPs/EPSCs were occasionally seen even when there was failure of the monosynaptic EPSP/EPSC. Further indirect evidence came from repetitive stimulation at 10-20 Hz for 2-10 s, which resulted in an afterdischarge of EPSPs/EPSCs following the stimulus train (Fig. 1B). This windup of EPSPs/EPSCs during repetitive stimulation was seen in all the cells tested (n = 11; 7 putative magnocellular neurons in the SON and 4 in the PVN), regardless of whether they showed multiple EPSPs/EPSCs in response to single stimuli, and occurred in the presence of bicuculline methiodide(10-30 µM, n = 2; Fig. 1B). The multiple EPSCs were blocked by the ionotropic glutamate antagonists DNQX (50 µM) and AP5 (100 µM; n = 3). These observations provided the first indication, albeit indirect, that SON and PVN neurons receive synaptic inputs from local glutamatergic neurons.


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 1. Evidence for polysynaptic excitatory circuits with electrical stimulation. A: multiple excitatory postsynaptic currents (EPSCs) with variable latencies were evoked by single stimuli (down-arrow ) in a putative magnocellular neuron in supraoptic nucleus (SON). Nine successive responses are superimposed. B: repetitive stimulation (20 Hz, 3 s) caused an afterdischarge of EPSCs in a putative magnocellular neuron. Frequency of spontaneous EPSCs was low before repetitive stimulation (1), but increased dramatically for several hundred ms following stimulus train (2). This cell was voltage clamped at -50 mV in presence of bicuculline (30 µM) to block inhibitory postsynaptic currents.

Stimulation of local excitatory circuits by activation of presynaptic metabotropic receptors

In a recent study we found that the metabotropic glutamate receptor agonists trans-ACPD and DHPG have an excitatory effect on the somata/dendrites of cells presynaptic to magnocellular neurons (Schrader and Tasker 1997). We therefore used bath application of trans-ACPD (75-100 µM) and DHPG (50 µM) to activate local presynaptic neurons in the present study. Of 19 SON and 47 PVN neurons (46 putative magnocellular and 20 putative parvocellular neurons) tested for synaptic activation with trans-ACPD (n = 61) and DHPG (n = 5), 19 cells (29%; 13 putative magnocellular and 6 putative parvocellular neurons) showed an increase in the frequency of EPSPs/EPSCs with a latency-to-onset of 1-3 min (Fig. 2). Of 21 SON and PVN cells tested directly in TTX without prior trans-ACPD or DHPG application, none showed an increase in EPSPs/EPSCs. Of three PVN neurons in which EPSPs/EPSCs were elicited by trans-ACPD in normal medium, TTX completely blocked the response in all three cells, indicating that this excitatory effect of presynaptic metabotropic receptor activation on transmitter release occurred at the somata/dendrites of local presynaptic neurons. The EPSPs/EPSCs elicited by trans-ACPD were maintained in the presence of bicuculline methiodide (30 µM, n = 14), but were abolished when the ionotropic glutamate receptors were blocked with AP5 (100 µM) and DNQX (50 µM) (n = 3), indicating that they were mediated by glutamate release (Fig. 2).


View larger version (14K):
[in this window]
[in a new window]
 
FIG. 2. Stimulation of local excitatory circuits by metabotropic glutamate receptor activation. Frequency of excitatory postsynaptic potentials (EPSPs) recorded in a putative magnocellular neuron in paraventricular nucleus (PVN) increased after 2 min in trans(±)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD, 100 µM). This response reversed after 10 min in normal artificial cerebrospinal fluid (Wash). EPSPs elicited by trans-ACPD were blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphonovalerate (AP5) (100 µM) and the non-NMDA receptor antagonist 5,6-dinitroquinoxaline-2,3-dione (DNQX) (50 µM), suggesting that they were mediated by activation of presynaptic glutamatergic neurons. This neuron was current clamped at -70 mV in the presence of bicuculline methiodide (30 µM).

Activation of local excitatory circuits with glutamate microstimulation

Glutamate microstimulation was used to selectively activate local neurons (Christian and Dudek 1988; Goodchild et al. 1982; Tasker and Dudek 1993). Eighty-two putative magnocellular neurons in the PVN and SON and 110 putative parvocellular neurons in the PVN were tested for synaptic responses with glutamate microstimulation of sites throughout the hypothalamus in three slice planes: coronal, horizontal, and parasagittal. A total of 25 cells (13%)---12 putative magnocellular neurons (15%) and 13 putative parvocellular neurons (12%)---showed an increase in the frequency of EPSPs/EPSCs in response to glutamate microstimulation (Fig. 3A). This response was observed in 6 of 49 neurons (12%) in coronal slices, in 9 of 80 neurons (11%) in horizontal slices, and in 10 of 63 neurons (16%) in parasagittal slices. The response was abolished when spike-dependent transmitter release was blocked with TTX(1.5-3 µM; n = 3), confirming that the glutamate-evoked increase in EPSPs/EPSCs was caused by activation of local presynaptic neuronal somata/dendrites. The response to glutamate microstimulation was not blocked by bicuculline methiodide (30-50 µM) in four of four cells recorded with KCl-filled microelectrodes, indicating that it was not due to GABA release.


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 3. Activation of local excitatory synaptic circuits with glutamate microstimulation. A: glutamate microstimulation of perifornical region (down-arrow ) in a horizontal slice caused an increase in frequency of EPSPs at resting potential in a putative magnocellular neuron in PVN. This response was blocked after 10 min in tetrodotoxin (TTX) (2.5 µM). B: topographic distribution of presynaptic excitatory sites (gray circles) in coronal (1), horizontal (2), and parasagittal planes (3). Each dark and light gray circle represents a site at which glutamate microstimulation elicited EPSPs in a PVN magnocellular (dark gray) or parvocellular neuron (light gray). Sites were concentrated primarily in perifornical and dorsomedial/posterior hypothalamic areas. Rostral is up in 2 and to the right in 3. Areas tested for excitatory circuits stretched from posterior hypothalamic area (PH) caudally to ventral limb of diagonal band of Broca (VDB) rostrally, from retrochiasmatic area (Rch) ventrally to fornix (Fx) dorsally, and laterally around lateral horn of fornix. 3V, third ventricle; AC, anterior commissure; AHA, anterior hypothalamic area; BNST, bed nucleus of stria terminalis; DA, dorsomedial hypothalamic area; DMD, dorsomedial hypothalamic nucleus; LA, lateral hypothalamic area; LPO, lateral preoptic area; MnPO, median preoptic nucleus; MPA, medial preoptic area; MS, medial septum; Mt, mammillothalamic tract; OT, optic tract; OX, optic chiasm; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta.

A detailed topographical analysis of the local excitatory synaptic projections to PVN neurons was conducted in the three slice planes. Glutamate microstimulation elicited EPSPs in PVN magnocellular and parvocellular neurons primarily at two sites, a perifornical site and a site caudal to the PVN (Fig. 3B). The perifornical site corresponded to the posterior division of the bed nucleus of the stria terminalis and the anterior hypothalamic nucleus, and the caudal site was located in the dorsomedial hypothalamus (Paxinos and Watson 1986; Swanson 1992). Only a limited topographical analysis, in the coronal plane, was performed in the SON. This revealed a site directly dorsal to the SON that evoked EPSCs in two of eight (25%) magnocellular neurons tested (data not shown).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We present here for the first time evidence of local excitatory synaptic circuits in the hypothalamus. Three converging and increasingly conclusive series of experiments revealed excitatory synaptic inputs to magnocellular and parvocellular neurons originating from hypothalamic regions surrounding the SON and PVN. Electrical stimulation often elicited multiple EPSPs of variable latency in cells of the SON and PVN. This is suggestive of polysynaptic excitatory circuits in our slices, because a single stimulus to a cut axon (i.e., from a neuron located outside the slice), which makes a monosynaptic connection with an SON or PVN neuron, would not be expected to generate multiple action potentials and cause multiple EPSPs. Furthermore, high-frequency stimulation resulted in an afterdischarge of EPSPs/EPSCs. It is unlikely that the multiple EPSPs/EPSCs were caused by repetitive firing in monosynaptic inputs, because axons do not express conductances capable of generating bursts of spikes (e.g., T-type Ca2+ channels), and because endogenous glutamate binding to presynaptic terminals in the hypothalamus leads to a reduced probability of glutamate release (Schrader and Tasker 1997). Thus the probable cause of this windup of EPSPs/EPSCs was a potentiation of upstream synapses in a polysynaptic circuit, leading to increased spike generation in the presynaptic cells and multiple EPSPs in the target cells. A more direct indication of local excitatory synaptic inputs to SON and PVN neurons was found in the increased frequency of EPSPs/EPSCs with metabotropic glutamate receptor activation. The increase in EPSPs/EPSCs was detected in ~29% of the cells tested and was blocked by TTX, suggesting that it was caused by the stimulation of local presynaptic excitatory neurons and the spike-dependent release of excitatory neurotransmitter. The low percentage of responsive SON and PVN cells may reflect the limited number of excitatory synaptic projections that are maintained in our slices, or may be a function of the proportion of presynaptic excitatory neurons that express metabotropic glutamate receptors. Glutamate microstimulation provided the most conclusive evidence for local excitatory synaptic inputs to SON and PVN neurons, although these experiments were also the most prone to false negative results because only a limited number of sites could be tested for each recorded cell. Glutamate microstimulation revealed perinuclear sites containing presynaptic neurons that provided TTX-sensitive, excitatory synaptic inputs to 13% of PVN and SON neurons.

The local excitatory synaptic inputs to SON and PVN neurons were likely to be glutamatergic for several reasons. First, as in other regions of the brain, most fast excitatory synaptic events in the hypothalamus are mediated by glutamate release and the activation of ionotropic glutamate receptors (van den Pol et al. 1990; Wuarin and Dudek 1991). Furthermore, multiple EPSPs/EPSCs evoked by electrical stimulation (i.e., polysynaptic EPSPs/EPSCs) were blocked by ionotropic glutamate receptor antagonists. Finally, EPSPs/EPSCs elicited by presynaptic metabotropic receptor activation were completely abolished by blocking ionotropic glutamate receptors. Thus we suggest that some locally projecting hypothalamic neurons, which are generally thought of as expressing peptide or other modulatory neurotransmitters, sythesize and release glutamate. Consistent with this is the finding that hypothalamic cell cultures contain intrinsic glutamate-producing neurons (van den Pol and Trombley 1993).

The strongest local excitatory projections to the PVN came from the dorsomedial hypothalamus and the perifornical area. The dorsomedial hypothalamus neurons may serve as an excitatory relay in the milk ejection reflex, because dorsomedial hypothalamic cells project to SON magnocellular neurons and display bursts of spikes in synchrony with bursts generated by oxytocin neurons during milk ejection, and dorsomedial hypothalamic lesions block the reflex (Takano et al. 1992). Our glutamate microstimulation experiments are consistent with this hypothesis, and demonstrate that the excitatory projections from the dorsomedial hypothalamus to the PVN are not due to passing axons, but originate in neurons located in the region. Similarly, perifornical neurons in the bed nucleus of the stria terminalis have been implicated in a feedback control of the milk ejection reflex, because they are excited by exogenous oxytocin, and unit activity in the bed nucleus of the stria terminalis has been found to correlate with bursts in oxytocin neurons (Ingram et al. 1995). The role that local excitatory inputs from dorsomedial hypothalamic and perifornical neurons may play in the synchronization and gating of SON and PVN neurons, and the respective role of local inhibitory circuits in pattern generation (Boudaba et al. 1996a), need to be explored further with the study of local synaptic circuits in vivo.

    ACKNOWLEDGEMENTS

  We thank K. Szabó for technical assistance.

  This project was supported by grants from the National Institute of Neurological Disorders and Stroke (NS-31187), The National Science Foundation (IBN-9315308), the Louisiana Board of Regents, and the American Heart Association, Louisiana Affiliate. L. Schrader was supported by a fellowship from the American Heart Association, Louisiana Affiliate.

  Permanent address of C. Boudaba: Institut des Sciences de la Nature, USTHB, BP 39 Dar El Beida, Algiers, Algeria.

    FOOTNOTES

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

  Received 7 October 1996; accepted in final form 10 March 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society