Dopamine Inhibition: Enhancement of GABA Activity and Potassium Channel Activation in Hypothalamic and Arcuate Nucleus Neurons

Andrei B. Belousov and Anthony N. Van Den Pol

Department of Biological Sciences, Stanford University, Stanford, California 94305; and Department of Neurosurgery, Yale University, School of Medicine, New Haven, Connecticut 06520

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Belousov, Andrei B. and Anthony N. van den Pol. Dopamine inhibition: enhancement of GABA activity and potassium channel activation in hypothalamic and arcuate nucleus neurons. J. Neurophysiol. 78: 674-688, 1997. Dopamine (DA) decreases activity in many hypothalamic neurons. To determine the mechanisms of DA's inhibitory effect, whole cell voltage- and current-clamp recordings were made from primary cultures of rat hypothalamic and arcuate nucleus neurons (n = 186; 15-39 days in vitro). In normal buffer, DA (usually 10 µM; n = 23) decreased activity in 56% of current-clamped cells and enhanced activity in 22% of the neurons. In neurons tested in the presence of glutamate receptor antagonists D,L-2-amino-5-phosphonovalerate (AP5; 100 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM), DA application (10 µM) revealed heterogeneous effects on electrical activity of cells, either hyperpolarization and decrease in activity (53% of 125) or depolarization and increase in spontaneous activity (22% of 125). The DA-mediated hyperpolarization of membrane potential was associated with a decrease in the input resistance. The reversal potential for the DA-mediated hyperpolarization was -97 mV, and it shifted in a positive direction when the concentration of K+ in the incubating medium was increased, suggesting DA activation of K+ channels. Because DA did not have a significant effect on the amplitude of voltage-dependent K+ currents, activation of voltage-independent K+ currents may account for most of the hyperpolarizing actions of DA. DA-mediated hyperpolarization and depolarization of neurons were found during application of the Na+ channel blocker tetrodotoxin (1 µM). The hyperpolarization was blocked by the application of DA D2 receptor antagonist eticlopride (1-20 µM; n = 7). In the presence of AP5 and CNQX, DA (10 µM) increased (by 250%) the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in 11 of 19 neurons and evoked IPSCs in 7 of 9 cells that had not previously shown any IPSCs. DA also increased the regularity and the amplitude (by 240%) of spontaneous IPSCs in 9 and 4 of 19 cells, respectively. Spontaneous and DA-evoked IPSCs and inhibitory postsynaptic potentials were blocked by the gamma -aminobutyrate A (GABAA) antagonist bicuculline (50 µM), verifying their GABAergic origin. Pertussis toxin pretreatment (200 ng/ml; n = 15) blocked the DA-mediated hyperpolarizations, but did not prevent depolarizations (n = 3 of 15) or increases in IPSCs (n = 6 of 10) elicited by DA. Intracellular neurobiotin injections (n = 21) revealed no morphological differences between cells that showed depolarizing or hyperpolarizing responses to DA. Immunolabeling neurobiotin-filled neurons that responded to DA (n = 13) showed that GABA immunoreactive neurons (n = 4) showed depolarizing responses to DA, whereas nonimmunoreactive neurons (n = 9) showed both hyperpolarizing (n = 6) and depolarizing (n = 3) responses. DA-mediated hyperpolarization, depolarization, and increases in frequency of postsynaptic activity could be detected in embryonic hypothalamic or arcuate nucleus neurons after only 5 days in vitro, suggesting that DA could play a modulatory role in early development. These findings suggest that DA inhibition in hypothalamic and arcuate nucleus neurons is achieved in part through the direct inhibition of excitatory neurons, probably via DA D2 receptors acting through a Gi/Go protein on K+ channels, and in part through the enhancement of GABAergic neurotransmission.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

gamma -Aminobutyrate (GABA) and glutamate play major roles in hypothalamic inhibitory and excitatory neurotransmission (Tappaz et al. 1983; van den Pol et al. 1990). GABAergic neurons and terminals are distributed throughout the medial hypothalamus and in the arcuate nucleus (Decavel and van den Pol 1990; Hokfelt et al. 1987; Tappaz et al. 1983). Similarly, glutamate immunoreactivity is found in many presynaptic terminals throughout the hypothalamus, including those regions that control the neuroendocrine system (Meeker et al. 1989; van den Pol 1991; van den Pol et al. 1990). Various subtypes of glutamate and GABA receptors are widely distributed within the hypothalamus (Fritschy et al. 1994; Meeker et al. 1994; van den Pol et al. 1994).

Dopamine (DA) is also found in perikarya and axons throughout the hypothalamus (Hokfelt et al. 1987; van den Pol et al. 1984). Presynaptic dopaminergic boutons are present in many regions of the hypothalamus, some originating from dopaminergic cells groups in the medial hypothalamus and others from extrahypothalamic sources. Both D1-like (D1 and D5) and D2-like (D2-D4) DA receptors are found through the hypothalamus (Mansour et al. 1990; Weiner et al. 1991). DA plays a number of roles in hypothalamic function, including feeding, water, and plasma osmotic balance regulation and pituitary endocrine regulation (MacLead and Lamberts 1986; Reichlin 1992). DA is released by arcuate nucleus neurons from axons in the median eminence. DA inhibits hormone release and tumor formation of prolactin-secreting cells of the anterior pituitary (Einhorn et al. 1991; MacLeod and Lamberts 1986; Reichlin 1992). Disturbances in DA may lead to behavioral and psychiatric disorders (Muller et al. 1987).

Although a large number of studies has examined the functional role of DA in the hypothalamus, the cellular mechanisms of DA modulation of neurotransmission in the hypothalamus are still not certain. In preliminary experiments we found that in the absence of excitatory glutamate neurotransmission there was little effect of DA on intracellular Ca2+ levels. In contrast, in the presence of glutamate neurotransmission, DA and specific DA receptor agonists caused a large change in many neurons, in most cases causing a decrease in Ca2+ (van den Pol et al. 1996a). Electrical recordings showed that DA caused a decrease in glutamatergic neurotransmission (Belousov and van den Pol 1995). These inhibitory effects could be due to DA-mediated depression of glutamatergic neurons, or to DA modulation of other transmitter systems.

In the present set of whole cell patch-clamp experiments we tested the hypothesis that DA's action in depressing electrical activity of hypothalamic neurons can be achieved both through the direct inhibitory effect of DA on neurons and through the enhancement of inhibitory transmission, specifically by increasing the activity of inhibitory GABAergic neurons. Primary cultures of rat neurons were used. Because the arcuate nucleus has a high density of DA (van den Pol et al. 1984) and GABA (Tappaz et al. 1983) cells and terminals, and because DA plays a significant role in neuroendocrine regulation that is in large part controlled by the arcuate nucleus, some experiments focused on neurons from the arcuate nucleus and others included examination of general medial hypothalamic neurons. A preliminary report of these results has been presented (Belousov and van den Pol 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Tissue culture

The medial hypothalamus or arcuate nucleus was used in these experiments to prepare cultures. The medial hypothalamus was removed from Sprague-Dawley rats on embryonic day 18-21 and washed three times in standard minimal essential medium (Gibco), 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 6 g/l glucose). The tissue was then enzymatically digested (10 U/ml papain, 500 µM EDTA, 1.5 mM CaCl2, and 0.2 mg/ml L-cysteine in Earle's balanced salt solution) for 30 min. Once the digestion solution was removed, the tissue was resuspended in standard tissue culture medium and gently triturated to form a single cell suspension. The cells were then pelleted and resuspended three times. The suspension was plated onto 22-mm square glass coverslips coated with poly-D-lysine (540,000 Da; Collaborative Research). To maintain a high local neuronal density, the cells were plated within a 7-mm glass ring placed on top of the coverslip. The glass ring was removed 24 h after plating.

After 4 days in vitro, the proliferation of nonneuronal cells was inhibited by the application of cytosine arabinofuranoside (1 µM) to the tissue culture medium. Cytosine arabinofuranoside was usually maintained in the tissue culture medium for 10-20 days. The N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists D,L-2-amino-5-phosphonovalerate (AP5; 100 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) were added to the tissue culture medium from 4 days in vitro to enhance long-term neuronal survival. Cultures were maintained in standard (glutamate- and glutamine-free) minimal essential medium at 37°C and 5% CO2 in a Napco 5410 incubator for 15-39 days before use. Tissue culture medium was changed twice a week. In the experiments on developing hypothalamic cultures, neurons raised for 4 days in the control medium and 1 day in the AP5/CNQX-containing medium (5 days in vitro) were used.

To obtain arcuate nucleus for tissue culture, 350- to 400-µm-thick slices of the medial hypothalamus from embryonic brains were cut and the arcuate nucleus was dissected. The remaining section was saved in 4% paraformaldehyde and later stained with propidium iodide (2 µg/ml; Molecular Probes, Eugene, OR) for 3 min, and then washed in four changes of buffer. The stained slice was examined in a Biorad 500 confocal scanning microscope to verify that the tissue removed from the slice for culturing was restricted to the arcuate nucleus (for more details see Belousov and van den Pol 1997).

Whole cell recording

The patch electrodes were pulled from borosilicate glass capillaries (2 mm OD; wall thickness 0.2 mm). The electrodes had a short shank and an inside tip diameter of 1-3 µm. After being filled with an internal solution the electrodes had a resistance of 6-10 MOmega . Recordings with an Axoclamp-2B amplifier (Axon Instruments) were obtained by suitable suction during continuous single-electrode voltage clamp. The seal resistances were 4-10 GOmega . At a holding potential of -60 mV these recordings could usually be converted by further suction to satisfactory whole cell recordings. The continuous single-electrode voltage- and current-clamp (BRIDGE) modes were used in this set of experiments. To measure input resistance (Rinput) of cells, negative square-wave voltage steps 10 mV in amplitude (in the range of 10-50 mV) and 500 ms in duration were applied at voltage clamp from a -60-mV holding potential. Current-voltage (I-V) curves were made and the calculation of the linear regression for these curves was used to obtain Rinput. The crossing point of I-V curves obtained before and during DA application was used to determine the reversal potential for DA responses (see Lacey et al. 1987; Yang et al. 1991). Voltage-dependent K+ currents were isolated in cells with the use of voltage clamp and positive square-wave voltage steps 20 mV in amplitude (in the range of 20-180 mV) and 100 ms in duration from a holding potential of -90 mV [1 µM tetrodotoxin (TTX) in the external medium]. Leak currents were subtracted with the use of AxoData software. Measurements of the amplitude of K+ currents were made at the peak and in the sustained phase, between 79 and 99 ms of the voltage step application. I-V curves for K+ currents were made and the amplitude of currents recorded before and during DA application was measured.

Solutions and chamber perfusion

The external solution contained 10 µM CNQX, 100 µM AP5, 158.5 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 µM glycine, and 10 mM glucose, pH 7.3, osmolarity 325 mosmol, at room temperature (20-22°C). Increased concentration of K+ ions in the external solution (12.5 mM KCl) was used to test a change in the reversal potential of DA effects. The pipette solution contained (in mM): 145 KMeSO4, 10 HEPES, 5 MgCl2, 1.1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 4 Na-ATP, 0.5 sodium guanosine 5'-triphosphate (Na-GTP), pH adjusted to 7.2 with KOH, osmolarity 310 mosmol.

In some experiments the pipette solution also contained 0.7% neurobiotin (Vector Laboratories). Spontaneous activity and responses to application of DA were first recorded. Then positive square-wave impulses of current (50-60 pA; 2 Hz; impulse duration 250 ms) were injected through the recording electrode to fill neurons (n = 21) with neurobiotin. The application of current continued for 3-10 min. There was no difference between the electrophysiological data obtained with the use of either the control pipette solution or the solution containing neurobiotin.

A flow pipe perfusion system was used to stimulate the cells. It consisted of several inputs into a final single short output terminated by a glass pipette (0.5 mm ID). This perfusion pipette was aimed at the recorded cells (100 µm away), which were continuously perfused with the flow rate of 2 ml/min from the source containing AP5/CNQX buffer. DA- or bicuculline-containing solutions, as well as the medium that did not contain CNQX and AP5 (control buffer), were applied through switching flow pipes. Solutions could be changed in about half a second. In the present experiments we usually used 10 µM, but sometimes 5 or 30 µM, DA and bicuculline (Sigma) at 50 µM concentrations. To protect DA from degradation, all buffers included ascorbate (10 µM) and solutions containing DA were protected from bright light. TTX (1 µM) was used in some experiments to block sodium channels. Tetraethylammonium (20 mM) and 4-aminopyridine (2 mM) were applied in some experiments to block K+ channels. The DA D2 receptor antagonist eticlopride (1-20 µM; Research Biochemicals International) was used to block specific DA receptors. For pertussis toxin (PTX) experiments, cells were pretreated with 200 ng/ml PTX for 18 h immediately before experiments. As a rule, only one cell was recorded from each coverslip.


View larger version (53K):
[in this window]
[in a new window]
 
FIG. 1. Dopamine (DA) reduced glutamate-dependent electrical activity. Enhanced glutamate-dependent electrical activity was obtained in hypothalamic and arcuate nucleus cultures after removal of chronic (10-20 days) blockade of glutamate neurotransmission [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 µM and D-2-amino-5-phosphonovalerate (AP5) 100 µM]. Activity recorded in neuron in control buffer (left) was significantly reduced by application of DA (10 µM; middle). Activity was also completely blocked with reintroduction of AP5/CNQX solution (bottom right). Each trace is an immediate continuation of the previous one, located above. Middle and right, top traces are continuations of left and middle, bottom traces, respectively. Current-clamp recording.

Data acquisition and analysis

In all experiments data were monitored and stored on a Macintosh Quadra 800 computer with the use of AxoData software with the subsequent off-line analysis by AxoGraph 2.0 (Axon Instruments), Igor Pro (WaveMetrics), and InStat 2.03 (GraphPad Software). The data were acquired at 400 or 1,000 Hz. Data were compared by Student's t-test, with the use of paired data when possible. Data in the RESULTS section are presented as means ± SE. Only inhibitory postsynaptic currents (IPSCs) with an amplitude twice that of the noise level were used in calculations to determine the influence of DA on postsynaptic activity. The interval between two postsynaptic events was measured on the basis of the peak-to-peak interval. Coefficient of variation (Cv) of intervals between IPSCs was calculated as Cv = a/t, where a was the standard deviation and t was the average interval between IPSCs.

GABA immunostaining and cell labeling

Coverslips containing neurons filled with neurobiotin were fixed in 4% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M phosphate buffer overnight. After several buffer washes, the fixed neurons were treated with 0.3% t-octylphenoxypolyethoxyethanol (Triton X-100) to permeabilize the membranes, and then incubated in primary rabbit GABA antiserum at a dilution of 1:5000. The specificity of the GABA antiserum is described in detail elsewhere (Decavel and van den Pol 1990). After wash, the neurons were incubated in goat anti-rabbit antiserum conjugated to Texas Red (1:4,000; Jackson Immunoresearch Laboratories) and simultaneously with fluorescein avidin D (FITC; 1:150; Vector Labs). This double staining method stained GABA-immunoreactive cells red and neurobiotin filled cells green. A Nikon fluorescent microscope with Texas Red and FITC filters was used to detect the two colors.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Effects of DA on glutamate-dependent electrical activity

In the presence of the glutamate receptor antagonists AP5 and CNQX, little excitatory electrical activity was found in cultured neurons. Relief from AP5/CNQX resulted in an immediate increase in activity, characterized by frequent excitatory postsynaptic potentials (EPSPs), an increase in action potentials, and a sustained depolarization of the average membrane potential (Vm) from -62.26 ± 0.74 (SE) mV in the presence of AP5/CNQX to -52.61 ± 1.37 mV (P < 0.0001; n = 23) 1 min after washout of glutamate receptor antagonists.

DA (5, 10, or 30 µM) exerted primarily inhibitory effects in the presence of ongoing glutamatergic synaptic activity in 13 of 23 cells (56%; Fig. 1; Table 1). In these neurons an application of DA resulted in a hyperpolarization of Vm from -51.4 ± 1.8 mV in control buffer to -58.2 ± 1.6 mV in the presence of DA (P < 0.0001; n = 13). Action potential frequency was significantly decreased from 2.0 ± 0.3 spikes/s in control medium to 0.4 ± 0.1 spikes/s after application of DA (P < 0.0002; n = 13). Of the 13 cells, 2 showed large EPSPs (amplitude > 15 mV). DA decreased the average amplitude of these EPSPs from 20.0 ± 0.7 and 18.3 ± 0.6 mV in control to 15.2 ± 0.6 and 12.8 ± 0.6 mV, respectively (the amplitude of 20 EPSPs measured just before and 30 s after beginning of DA application was used for this calculations).

 
View this table:
[in this window] [in a new window]
 
TABLE 1. Effect of DA on glutamate-dependent activity

In 5 of 23 cells (22%) DA enhanced the glutamate-mediated electrical activity (Table 1). In three of these DA caused an increase in the frequency of action potentials from 1.3 ± 0.2 to 2.2 ± 0.2 spikes/s (n = 3). In two others the number of action potentials remained stable over time (1.7 ± 0.1 spikes/s), but the amplitude of the large EPSPs increased from 12.6 ± 2.3 mV in the control medium to 18.4 ± 1.9 mV in the presence of DA (n = 2). The remaining five neurons (22%) showed no response to DA, even at concentrations as high as 200 µM.

Effects of DA on neuronal activity in the presence of glutamate receptor antagonists

To explore the mechanisms responsible for DA-mediated responses, the experiments described below were performed in hypothalamic or arcuate nucleus cultures in the presence of glutamate receptor antagonists AP5 and CNQX. Both excitatory and inhibitory effects of DA (10 µM) on neurons were observed in either current-clamp (n = 125 cells; Table 2) or voltage-clamp (n = 28; Table 3) experiments. Neurons from the arcuate nucleus (n = 69) and hypothalamus (n = 117) showed no apparent difference in their responses to DA: 52% and 54% of neurons were hyperpolarized and 65% and 62% of neurons showed an increase in IPSCs in the arcuate nucleus and hypothalamus, respectively. Furthermore, the mean DA-mediated hyperpolarization was similar for the arcuate nucleus (9 mV) and medial hypothalamus (10 mV). Thus data from both groups of cells were pooled in the present study to generate numbers representing percentages of responding cells (n = 186).

 
View this table:
[in this window] [in a new window]
 
TABLE 2. Effect of DA on neuronal activity in the presence of glutamate receptor antagonists

 
View this table:
[in this window] [in a new window]
 
TABLE 3. Effect of DA on frequency of GABAergic IPSCs in the presence of glutamate receptor antagonists

Inhibitory effects of DA in the presence of AP5/CNQX

In 66 of 125 current-clamped cells (53%) DA (10 µM) evoked a rapid hyperpolarization that varied from 2 to 25 mV (9.6 ± 0.7 mV; n = 66). The average Vm was -59.0 ± 0.7 mV before and -68.7 ± 1.2 mV (P < 0.0001; n = 66) after application of DA (Fig. 2A, a-1). The DA-mediated hyperpolarization was always associated with a decrease in the frequency of spontaneous action potentials. In these cells the frequency of action potentials was 1.3 ± 0.4 spikes/s before and 0.1 ± 0.05 spikes/s after DA administration (n = 11; P < 0.009). All spiking activity could be blocked by the Na+ channel blocker TTX, indicating Na+-based action potentials. The amplitude of DA-mediated hyperpolarization, however, was not affected by TTX in any 16 of the neurons tested (1 µM; Fig. 2A, a-2). In these cells the average level of the hyperpolarization evoked by DA was 11.6 ± 1.5 mV before and 11.1 ± 1.4 mV in the presence of TTX (n = 16). The DA-mediated hyperpolarization was also not affected by the GABAA receptor antagonist bicuculline (50 µM). The amplitude of hyperpolarization was 13.5 ± 1.5 mV before and 12.8 ± 1.4 mV after bicuculline administration (n = 5; data not shown). The DA D2 receptor antagonist eticlopride suppressed the DA-evoked hyperpolarization in a dose-dependent manner. The amplitude of hyperpolarization was reduced by 30-65% in the presence of 1 µM eticlopride (n = 2). The hyperpolarizationwas completely blocked by 20 µM eticlopride (n = 7;Fig. 2B).


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 2. Inhibitory effects of DA on neuronal activity in presence of glutamate receptor antagonists. When cells were incubated in AP5- and CNQX-containing medium, application of DA (10 µM) evoked cell hyperpolarization (A and B). Application of tetrodotoxin (TTX; 1 µM; A, a-2) did not affect DA-evoked hyperpolarization. DA-mediated hyperpolarization was suppressed by application of 20 µM of the DA D2 receptor antagonist eticlopride (B). DA-mediated hyperpolarization was accompanied by decrease in cell's input resistance (Rinput) and increase in membrane conductance: see change in angle in current-voltage (I-V) curve obtained during DA application (bullet ) as compared with control (open circle ; C). When concentration of K+ ions (KCl) in external medium ([K+]ext) was increased from 2.5 to 12.5 mM, extrapolated reversal potential for DA-mediated hyperpolarization (EDA) measured in this neuron (-101 mV; C, top, up-down-arrow ) shifted to less negative value (-60 mV; C, bottom, up-down-arrow ), suggesting participation of K+ currents in DA-mediated hyperpolarization. Current-clamp recordings from 2 neurons are shown in A and B. I-V curves shown in C were obtained with the use of voltage clamp by application of negative 10-mV steps of voltage (in range from 10 to 50 mV, pulse duration 500 ms) from holding potential of -60 mV. All solutions contained AP5 and CNQX.

Analysis of the Rinput in neurons that responded to DA by hyperpolarization of the Vm showed a significant decrease in Rinput and, consequently, an increase in cell membrane conductance during DA application. In 22 cells tested the average Rinput was 994 ± 87 MOmega before and 791 ± 83 MOmega 30 s after application of DA (P < 0.006).


View larger version (27K):
[in this window]
[in a new window]
 
FIG. 3. DA had little effect on voltage-dependent K+ currents (A, control; B, 10 µM DA). C: voltage protocol. D: I-V curves for this neuron. Measurements of amplitude of K+ currents were made in sustained phase, between 79 and 99 ms of voltage step applications. Holding potential: -90 mV. Leak currents were subtracted with the use of AxoData software. All solutions contained AP5 100 µM, CNQX 10 µM, and TTX 1 µM.

The extrapolated reversal potential for the DA-mediated hyperpolarization, measured in 18 neurons in the external medium containing 2.5 mM K+, was -94.2 ± 4.9 mV, close to the reversal potential for K+ currents. Of these 18 cells, 3 were examined for the dependence of reversal potential on the extracellular K+ concentration. In these three neurons the reversal potential shifted from -97.4 ± 3.5 to -60.1 ± 2.3 mV (P < 0.001) when external KCl was raised from 2.5 to 12.5 mM KCl (Fig. 2C). Thus the reversal potential for DA-mediated hyperpolarization shifted in a positive direction as the concentration of K+ ions in the external medium was increased. The values of DA reversal potentials observed in our experiments are close to the equlibrium potentials of K+ currents predicted by the Nernst equation for 2.5 and 12.5 mM K+ in the external medium: -102 and -61 mV, respectively. Similar results have been previously shown for rat melanotrophs and substantia nigra neurons, suggesting participation of K+ ions in the DA-mediated hyperpolarization (Israel et al. 1987; Lacey et al. 1987; Stack and Surprenant 1991).

Voltage-dependent K+ currents were isolated in neurons with the use of voltage clamp (see METHODS section) in the presence of 1 µM TTX in the incubating medium. The amplitude of both transient and sustained components of K+ currents usually showed little change during DA application: DA evoked either little increase (n = 2) or decrease (n = 8; by 3-15%) in the amplitude of these currents. The average amplitude of K+ currents measured with the use of voltage clamp and a positive square-wave voltage step 180 mV in amplitude and 100 ms in duration from a holding potential of -90 mV showed only a slight decrease in the presence of DA. The amplitude of the fast component of K+ currents was 2.79 ± 0.41 nA before and 2.57 ± 0.37 nA (n = 13) 20 s after the beginning of DA application. The amplitude of the slow component was 2.45 ± 0.38 and 2.37 ± 0.39 nA (n = 13), respectively (Fig. 3). Both these currents were 70-80% suppressed by the joint application of tetraethylammonium (20 mM) and 4-aminopyridine (2 mM; not shown). This showed that DA-mediated hyperpolarization was primarly due to activation of voltage-independent K+ channels.

Voltage-clamp experiments also revealed inhibitory effects of DA (10 µM) on neuronal activity of some hypothalamic and arcuate nucleus cells. Because only IPSCs were detected in voltage-clamped neurons in the presence of glutamate receptor antagonists (AP5 and CNQX) (Belousov and van den Pol 1997), we focused these experiments on the effects of DA on IPSCs. The experiments were conducted at a holding potential of -25 mV because spontaneous IPSCs were partially masked at -60 mV, a holding potential closer to the reversal potential for chloride currents. The amplitude of the IPSCs was measured from the baseline to the peak of the event. The amplitude of IPSCs was dramatically increased when the holding potential was shifted to -25 mV (Fig. 4A). Spontaneous IPSCs were found in 19 of 28 neurons. Nine of 28 neurons did not show IPSCs in baseline recordings. DA depressed IPSC frequency in 4 of 28 cells (14%), showing a decrease in the frequency of IPSCs from 1.4 ± 0.3 to 0.1 ± 0.05 Hz after application of DA (P < 0.03; n = 4; Fig. 4B).


View larger version (52K):
[in this window]
[in a new window]
 
FIG. 4. DA blocked spontaneous inhibitory postsynaptic currents (IPSCs). IPSCs were usually masked at holding potential of -60 mV, close to reversal potential for chloride currents (1st 2 s of recording in A). However, IPSCs significantly increased in amplitude (A, ↙ black-down-triangle ) when holding potential was shifted to -25-30 mV. Spontaneous IPSCs (B, b-1) were depressed in some neurons during application of DA (B, b-2; 10 µM). Recording shown in B, b-2 was made 1 min after beginning of DA application. Recovery of IPSC activity shown in B, b-3 was recorded in this cell in 40 s after washout of DA from external medium. All solutions contained AP5 and CNQX. Voltage-clamp recordings were conducted at holding potential of -25 mV.

Depolarizing effects of DA in the presence of AP5/CNQX

In a second group of current-clamped cells (27 of 125; 22%) tested in the presence of glutamate receptor antagonists, the application of DA (10 µM) caused a modest depolarization of 6.2 ± 0.8 mV (from 2 to 17 mV), from a Vm baseline of -59.8 ± 1.1 mV to -52.6 ± 1.3 mV (P < 0.0001; n = 27) in the presence of DA (Fig. 5, A and B). Only 7 of 27 cells in this group had spontaneous action potentials. DA increased the activity of all these neurons (Fig. 5A) and evoked action potentials in the other 11 cells. Thus, of the 27 cells that responded to DA by depolarization of the Vm, 18 neurons revealed an increase in spike frequency. The average frequency of action potentials in these neurons was 0.3 ± 0.1 spikes/s before and 1.3 ± 0.2 spikes/s after application of DA (P < 0.002; n = 18). DA-mediated depolarization was not affected by TTX (1 µM; Fig. 5B, b-2; n = 3). No significant change in Rinput (Fig. 5C) was found in cells during DA-mediated depolarization. The average Rinput (810 ± 124 MOmega ) showed little change in the presence of DA (831 ± 119 MOmega ; n = 10). In this regard, because our neurons had extensive dendritic trees, it is possible that with inadequate space clamp, conductance changes in distal dendrites might not be detected with recordings from the cell body.


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 5. Excitatory effects of DA on neuronal activity in presence of glutamate receptor antagonists. Current-clamp recordings of 2 neurons responding to DA (10 µM) by increase in action potential frequency (A) and cell depolarization (A and B) are shown. DA-mediated depolarization was sustained during application of the sodium channel blocker TTX (1 µM; B, b-2). I-V curves presented in C were obtained with the use of voltage clamp from neuron shown in B. DA application did not affect Rinput in this cell (C). All solutions contained AP5 and CNQX.

As mentioned above, little excitatory electrical activity was found in current-clamped neurons in the presence of glutamate receptor antagonists. EPSPs were not usually detected in these conditions and only 9 of 125 neurons revealed spontaneous inhibitory postsynaptic potentials (IPSPs). In seven of these nine cells DA (10 µM) increased the frequency of spontaneous IPSPs from 1.2 ± 0.3 to 2.7 ± 0.4 Hz (P < 0.001; n = 7; Fig. 6A), with either no change in the Vm (5 cells), or a small depolarization by 3 mV (1 cell), or hyperpolarization by 6 mV (1 neuron) of the Vm.


View larger version (70K):
[in this window]
[in a new window]
 
FIG. 6. DA increased frequency of spontaneous IPSCs and inhibitory postsynaptic potentials (IPSPs). Frequency of spontaneous IPSPs recorded in current-clamped neuron was increased during application of DA [membrane potential (Vm) of this neuron was -59 mV; A]. DA (10 µM) also increased frequency of spontaneous IPSCs, which could be reversibly blocked by the GABAA antagonist bicuculline (BIC; 50 µM; B, voltage-clamp recording at holding potential of -25 mV). Level of activity recovered to background level after washout of DA. Each trace in Figs. 6-8 is immediate continuation of the previous one, located above. Histograms of integrated postsynaptic activity for these neurons are presented under corresponding neuronal recordings in A and B. One bin in histogram equals 8.3 s. Frequency of potentials was measured in Hz. DA administration is shown as bold line above histogram bars. All solutions contained AP5 and CNQX.

In voltage-clamp experiments performed at a holding potential of -25 mV, DA (10 µM) enhanced IPSCs in 18 of 28 cells (64%), increasing the frequency of spontaneous IPSCs in 11 of 19 neurons (Fig. 6B) and eliciting IPSCs in 7 of 9 cells that had not previously shown spontaneous postsynaptic activity (Fig. 7). The average frequency of IPSCs was 0.5 ± 0.1 Hz before and 1.7 ± 0.2 Hz after DA application (P < 0.0001; n = 18). In 9 of 11 neurons responding to DA with an increase in the frequency of spontaneous IPSCs, the enhancement in activity of IPSCs was also accompanied by an increase in their regularity. The Cv of intervals between IPSCs in these neurons was 0.8 ± 0.1 in the control and 0.2 ± 0.02 in the presence of DA (P < 0.01; n = 9).


View larger version (49K):
[in this window]
[in a new window]
 
FIG. 7. DA activates GABA-dependent IPSCs. Application of DA (10 µM) caused appearance of IPSCs in cultured neuron that had not previously shown spontaneous postsynaptic currents. These DA-evoked IPSCs were reversibly blocked by bicuculline (50 µM), verifying their GABAergic origin. Voltage-clamp recordings were conducted at holding potential of -25 mV. All solutions contained AP5 and CNQX.

The amplitude of 20 inhibitory postsynaptic events measured just before and 30 s after the beginning of DA application was compared. In 15 of 19 cells showing spontaneous IPSCs during baseline recordings, the average amplitude of IPSCs was not affected by DA (81.1 ± 11.2 pA; n = 15). In the other four cells the amplitude of IPSCs increased significantly from 68.0 ± 10.3 pA before to 165.5 ± 36.4 pA (P < 0.04; n = 4) after DA application (Fig. 8). Two of these four neurons did not show change in the level of IPSC activity and two others demonstrated an increase in the frequency of IPSCs.


View larger version (46K):
[in this window]
[in a new window]
 
FIG. 8. DA increased amplitude of spontaneous IPSCs. Amplitude of spontaneous IPSCs was significantly increased after application of DA (10 µM). Voltage-clamp recordings were conducted at holding potential of -25 mV. All solutions contained AP5 and CNQX. Histogram of amplitude of postsynaptic activity for this neuron is presented under corresponding neuronal recordings. One bin in histogram presents average amplitude (mean ± SE, error bars) of 20 IPSCs. Each following bin includes data of amplitude measurements of 20 IPSPs. DA administration is shown as bold line atop histogram bars. Data obtained for 1st and last bars are located out of shown neuronal recordings. All solutions contained AP5 and CNQX.

Coapplication of DA with bicuculline (50 µM) completely and reversibly blocked both spontaneous and DA-evoked IPSCs and IPSPs (Figs. 6 and 7) in all 21 neurons tested, demonstrating that the inhibitory postsynaptic activity was due to GABA release.

Both DA-mediated depolarization (n = 6) and hyperpolarization (n = 9) were detected with DA concentrations as low as 300 nM (data not shown), suggesting specific activation of DA receptors rather than other catecholamine receptors. The amplitude of these responses was usually 50-80% lower than that evoked by 10 µM DA.

IPSP frequency and hyperpolarization can be independently modulated by DA

As indicated above, 9 of 125 neurons had spontaneous IPSPs in current-clamp recordings. DA application increased the IPSP frequency in seven of these nine neurons and this effect was recorded both in the neurons with no change in the Vm (Fig. 6A) and in cells showing a shift in the Vm. In two cells DA did not change the frequency of spontaneous IPSPs, but significantly hyperpolarized the neurons by 9 and 11 mV (Fig. 9). These data show that the changes in the level of spontaneous IPSPs and in Vm can be independent events, reflecting a direct DA effect on the recorded cell (change in the Vm) and the excitatory effect of DA on presynaptic GABAergic neurons (increase in the frequency of spontaneous IPSPs). The only interaction of these two events was a decrease in the amplitude of IPSPs during DA-mediated hyperpolarization when the Vm was shifted from -60-65 mV to -75-80 mV, closer to the reversal potential for chloride-dependent inhibitory postsynaptic activity. Because of limitations of space clamp, IPSPs on more distal dendrites of the recorded cells may not have been clearly detected in these studies. Thus the effects of DA on the total cell in increasing IPSPs may have been even greater than we detected.


View larger version (27K):
[in this window]
[in a new window]
 
FIG. 9. Little change in activity of spontaneous GABAergic IPSPs during DA-mediated cell hyperpolarization. When cell was hyperpolarized by DA, frequency of IPSPs did not significantly change, showing that changes in level of spontaneous IPSPs and in level of membrane potential (Vm) are probably independent events. Current-clamp recording. All solutions contained AP5 and CNQX.

In 32 of 125 current-clamped neurons (25%) neither Vm (-59.1 ± 0.8 mV) nor Rinput (831 ± 98 MOmega ) changed during application of DA (10 µM) in the presence of glutamate receptor antagonists. Similarly, 6 of 28 cells (22%) tested in voltage clamp also were insensitive to DA.

Effects of PTX pretreatment on DA-mediated responses

PTX, which inactivates the GTP binding proteins Gi and Go by ADP ribosylation (Katada et al. 1984), was used to investigate the role of G proteins in the DA-mediated responses in cultured arcuate nucleus and hypothalamic neurons. Preincubation of cells with PTX (200 ng/ml; 18 h; n = 15 neurons) did not virtually change either average Vm (-58.5 ± 1.0 mV) or Rinput (898 ± 116 MOmega ) as compared with the control neurons (see above), but abolished the ability of DA to elicit a hyperpolarization of Vm. Whereas 53% of control neurons responded to DA with hyperpolarization of Vm, none of the 15 PTX-treated cells we examined showed this DA effect (Fig. 10A). In contrast, PTX treatment did not affect the excitatory DA effects: DA increased action potentials and depolarized 3 of 15 PTX-treated neurons (20%; Fig. 10B) and increased IPSCs in 6 of 10 neurons pretreated with PTX (60%; Fig. 10C). This percentage of neurons was similar to that obtained in controls not treated with PTX: 22% of 125 cells in control cultures showed DA-evoked depolarization and 64% of 28 neurons showed an increase in frequency of IPSCs.


View larger version (50K):
[in this window]
[in a new window]
 
FIG. 10. Effects of pertussis toxin (PTX) treatment on DA-mediated responses. Application of DA (10 µM) evoked hyperpolarization of Vm in control but not PTX-treated neuron (A). DA elicited Vm depolarization and increase in action potentials and IPSCs in 2 PTX-treated neurons presented in B and C. Recordings shown in middle traces in B and C were made 40-60 s after beginning of DA application. Current-clamp (A and B) and voltage-clamp (C) recordings are presented. Voltage-clamp recordings were conducted at holding potential of -25 mV. All solutions contained AP5 and CNQX.

Responses of immunocytochemically identified GABAergic neurons

To determine whether there was a morphological difference between neurons that showed hyperpolarizing responses versus those cells that showed depolarizing responses, we compared the dendritic arbors of cells characterized physiologically and then filled with the tracer molecule neurobiotin. Most neurons had two to four dendrites that branched once or twice. The mean diameter of the perikarya varied from 10 to 22 µm, with a mean of 15 µm (n = 21), determined by measuring the long and short axis of the cell body and taking the mean of the two. We found no obvious differences between the two response types (depolarizing and hyperpolarizing) relative to their general dendritic or axonal arbors. We also filled cells with neurobiotin, and then used GABA antiserum to characterize the transmitter identity of neurons that responded to DA. GABA-immunoreactive cells that responded to DA had a mean of 15.1 ± 0.4 µm (n = 4), and non-GABAergic immunoreactive cells that responded to DA had a diameter of 15.9 ± 0.5 µm (n = 9). Interestingly, 4 of 13 DA-responsive neurons were immunoreactive for GABA (Fig. 11, A and B), and all 4 showed depolarizing responses to DA (10 µM). Of nine that were not immunoreactive for GABA (Fig. 11, C and D), six showed a hyperpolarizing response to DA, and three showed a depolarizing response. These data support the hypothesis that DA can be excitatory to GABAergic neurons.


View larger version (125K):
[in this window]
[in a new window]
 
FIG. 11. Combined neurobiotin labeling and GABA immunocytochemistry. Neurons were labeled with neurobiotin during recording, and were revealed with fluorescein avidin D (A). GABA immunostaining showed that neurobiotin-labeled cell was immunoreactive for GABA (B). This cell is representative of GABAergic neurons that showed depolarizing responses to DA (10 µM). Axon of GABA-immunoreactive neuron is indicated in both A and B. C: neurobiotin-labeled neuron (horizontal arrow). D: same field as in C after GABA immunostaining. Neurobiotin cell was not labeled with GABA antiserum, but other cells in same field were. This cell is representative of neurons that showed hyperpolarizing response to DA (10 µM). Calibration bar: 20 µm.

Effects of DA on developing neurons

Neuronal cultures, raised for 4 days in the control medium and 1 day in the medium containing AP5/CNQX, were tested in our experiments to investigate effects of DA on developing neurons. Both excitatory and inhibitory responses of neurons to DA were detected with the use of current and voltage clamp. Of 13 neurons tested in current clamp, 4 cells showed a small (2-4 mV) hyperpolarization and decrease in action potentials during DA application (Fig. 12A), 3 neurons showed a depolarization (2-3 mV), and 6 cells were insensitive to DA. Two of four neurons studied with voltage clamp showed an increase in frequency of IPSCs (Fig. 12B) and two others were DA insensitive.


View larger version (35K):
[in this window]
[in a new window]
 
FIG. 12. Effects of DA on neuronal activity during early development. DA depressed action potentials (A) or increased IPSC frequency typical of GABA-mediated activity (B) in 2 neurons on day 5 in vitro. Current-clamp (A) and voltage-clamp (B) recordings are presented from different neurons. Recordings shown in A and B, bottom traces, were made in the 30 s after beginning of DA application. Voltage-clamp recordings were conducted at holding potential of -25 mV. All solutions contained AP5 and CNQX.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

DA inhibition of glutamate-dependent electrical activity

A modulatory influence of DA on glutamatergic neurotransmission has been previously found in many brain regions of humans and animals. DA depressed both NMDA and non-NMDA EPSPs in rat neocortex and nucleus accumbens (Law-Tho et al. 1994; Pennartz et al. 1992; Pralong and Jones 1993), inhibited NMDA-evoked rhythmic oscillations in rat ventral tegmentum (Seutin et al. 1994), and increased in some neurons and decreased in others the excitatory effects of glutamate, NMDA, and quisqualate in human neocortex (Cepeda et al. 1992) and in rat neostriatum (Cepeda et al. 1993). Dose-dependent DA modulation of glutamate-evoked currents and glutamate-mediated intracellular calcium rises was found in our previous experiments with hypothalamic cultures, suggesting modulation of DA on glutamate activity (van den Pol et al. 1996a).

In the present study we found that glutamate-dependent activity obtained in hypothalamic and arcuate nucleus neurons after removal of a chronic (15-39 days) blockade of glutamatergic neurotransmission was significantly decreased during application of DA in the majority of cells. In these neurons DA evoked hyperpolarization and suppressed the frequency of action potentials and EPSPs.

The DA-mediated depression of glutamate-mediated activity could be 1) a result of a direct inhibitory DA influence on a recorded cell or on glutamatergic cells presynaptic to the recorded neuron, 2) a result of an excitatory effect of DA on GABAergic neurons terminating on either the recorded neuron or surrounding glutamatergic cells, or 3) a result of both these events.

Inhibitory effects of DA on hypothalamic and arcuate nucleus neurons, G proteins, and K+ channels

Inhibitory effects of DA or its agonists on the activity of neurons both in brain slices and in cell cultures were previously shown for substantia nigra (Lacey et al. 1987, 1988), entorhinal cortex (Pralong and Jones 1993), and pituitary of rats (Einhorn and Oxford 1993; Einhorn et al. 1991; Israel et al. 1987; Lledo et al. 1990b; Stack and Surprenant 1991; Williams et al. 1989), as well as for guinea pig nucleus accumbens (Uchimura et al. 1986), frog melanotrophs (Valentijn et al. 1991a,b), and cells from human pituitary prolactinomas (Israel et al. 1985). Application of DA to cells usually evoked a hyperpolarization of the Vm, a blockade or reduction in action potentials, and a decrease in the cell's Rinput (Einhorn and Oxford 1993; Einhorn et al. 1991; Israel et al. 1985, 1987; Lacey et al. 1987, 1988; Lledo et al. 1990b; Pralong and Jones 1993; Stack and Surprenant 1991; Uchimura et al. 1986; Valentijn et al. 1991a,b; Williams et al. 1989). DA-evoked hyperpolarization was probably a result of a postsynaptic effect of DA, because it was sustained during the blockade of Na+ or Ca2+ channels with TTX and Cd2+ or Co2+ ions (Lacey et al. 1987; Pralong and Jones 1993; Uchimura et al. 1986; Valentijn et al. 1991a; Williams et al. 1989) that would block transmitter release.

In our experiments on hypothalamic and arcuate nucleus neurons maintained in the presence of glutamate receptor antagonists (AP5 100 µM and CNQX 10 µM), the DA-mediated hyperpolarization was accompanied by a significant decrease in Rinput and, consequently, an increase in cell membrane conductance. Our experiments suggest that the DA-mediated hyperpolarization was dependent on an activation of voltage-independent rather than voltage-dependent K+ currents. Voltage-dependent K+ currents have been previously described in hypothalamic neurons (Muller et al. 1992). Our data suggest that voltage-independent K+ currents are also found in hypothalamic neurons.

The DA-mediated hyperpolarization was suppressed by the application of the DA receptor antagonist eticlopride, suggesting that the D2 receptor was responsible for the hyperpolarization. PTX treatment, that blocks G proteins of the Gi/Go classes, also blocked DA-mediated hyperpolarization. Our data suggest that the DA-evoked hyperpolarization in hypothalamic and arcuate nucleus was probably the result of activation of voltage-independent K+ channels coupled to DA D2 receptors.

These data obtained on hypothalamic or arcuate nucleus neuronal cultures are in agreement with those reported previously for other neuronal and endocrine tissues (Einhorn and Oxford 1993; Einhorn et al. 1991; Israel et al. 1985, 1987; Lacey et al. 1987, 1988; Lledo et al. 1990b; Stack and Surprenant 1991; Valentijn et al. 1991a,b; Williams et al. 1989), showing that the D2 receptor-G protein-K+ channel coupling mechanism is responsible for DA-mediated hyperpolarization. D2-mRNA has been detected in hypothalamus (Mansour et al. 1990; Weiner et al. 1991), consistent with our results suggesting D2 mediation of hyperpolarization.

In the present study, the percentage of cells responding to DA by hyperpolarization was relatively similar when DA was applied either in the control (AP5/CNQX-free) solution (56% of cells) or in the solution containing glutamate receptor antagonists (53%). Thus the DA modulation of glutamate-independent electrical activity via the direct inhibition of the recorded neurons during the activation of K+ currents appears likely. However, the inhibition of glutamatergic neurons innervating the recorded cell, and the resultant decrease in excitatory glutamatergic input to this neuron, can also contribute substantially to the general DA-mediated depression of electrical activity recorded in current-clamp experiments (van den Pol et al. 1996a,b).

We did not examine the effects of DA on Na+ or Ca2+ currents, depression of which could also potentially contribute to DA-mediated inhibition of glutamate-dependent neuronal activity (Lledo et al. 1990a; Stack and Surprenant 1991). However, we did find a significant decrease in Na+-dependent action potentials in the majority of neurons (56%) during application of DA in control (AP5/CNQX-free) buffer. This DA effect could be explained by a DA-mediated increase in the threshold for generation of Na+-dependent action potentials, or a decrease in conductance of Na+ and some Ca2+ currents, as shown previously in neurons from other regions of the brain (Lledo et al. 1990a; Rutherford et al. 1988; Stack and Surprenant 1991; Surmeier et al. 1992; Valentijn et al. 1991a,b), or by a reduction in glutamatergic activity (van den Pol et al. 1996a,b).

In our experiments a rapid 3- to 22-mV DA-evoked hyperpolarization was observed in almost half of the hypothalamic and arcuate nucleus neurons cultured in the conditions of blockade of glutamatergic activity. Hyperpolarization was found even in the presence of the Na+ channel blocker TTX and the GABAA receptor antagonist bicuculline. This suggests that the hyperpolarization can be the result of a direct DA influence on the recorded cell.

DA enhancement of activity in hypothalamic and arcuate nucleus neurons

Excitatory effects of DA on neuronal activity were found previously in brain slices of rat supraoptic nucleus (Yang et al. 1991) and prefrontal (Law-Tho et al. 1994; Penit-Soria et al. 1987) and entorhinal cortex (Pralong and Jones 1993), and guinea pig nucleus accumbens (Uchimura et al. 1986). DA was shown to depolarize cells, sometimes increasing the frequency of spontaneous action potentials. DA-mediated depolarization was sustained during the blockade of both sodium and calcium currents (Pralong and Jones 1993; Uchimura et al. 1986; Yang et al. 1991).

The mechanisms responsible for excitatory effects of DA are probably different in different brain structures. In the nucleus accumbens the DA-mediated depolarization of the Vm was due to a decrease in K+ conductance (Uchimura et al. 1986), and in the entorhinal cortex there was an increase in the membrane conductance and a decrease in cell's Rinput (Pralong and Jones 1993). In the prefrontal cortex no change in the membrane conductance or Rinput was reported (Law-Tho et al. 1994), although both increases and decreases in Rinput in different cells of rat prefrontal cortex were found in another study during DA-dependent depolarization of neurons (Penit-Soria et al. 1987).

Despite the blockade of DA-mediated hyperpolarizing responses in PTX-treated cultures, PTX treatment did not affect the DA depolarizing effects on cells. Interestingly, the percentage of cells responding by an increase in action potentials and Vm depolarization as well as by an increase in IPSC frequency was very similar in cells not treated and treated with PTX: 20% versus 22 and 60% versus 64%, respectively. The DA-mediated excitation of the neuronal activity was therefore not achieved through the Gi/Go classes of G proteins, which were reported to be coupled in many cells to DA D2 receptors. The Gs class of G proteins that increases activity of adenosine 3',5'-cyclic monophosphate and that is coupled to DA D1 and beta 1 and beta 2 noradrenaline receptors is not affected by PTX treatment. It is possible that excitatory effects of DA found in this study could be the result of activation of some single or multiple DA receptors other than D2 receptors. The identity of the DA receptor(s) responsible for the depolarizing action was not studied here, but bears further experimental analysis. The crossreactivity of DA and beta -adrenergic receptors reported previously (Malenka and Nicoll 1986) appears unlikely in our experiments because a low concentration (300 nM) of DA could evoke depolarization, and this concentration was probably not sufficient to generate a substantial activation of beta -adrenergic receptors.

DA-mediated modulation of GABAergic neurotransmission

Both excitatory and inhibitory effects of DA on GABAergic activity were shown previously in a number of studies. DA was found to reduce GABAA-dependent IPSPs in rat prefrontal cortex (Law-Tho et al. 1994) and nucleus accumbens (Pennartz et al. 1992), and to depress GABAA- and GABAB-mediated IPSPs in entorhinal cortex of rats (Pralong and Jones 1993). On the other hand, DA was shown to increase activity of GABAA- or GABAB-mediated IPSPs as well as GABAA-dependent neuronal activity in rat prefrontal cortex (Penit-Soria et al. 1987; Pirot et al. 1992) and guinea pig ventral tegmental area (Cameron and Williams 1993).

Modulatory effects of DA on the inhibitory postsynaptic activity were found in our experiments with the use of both current- and voltage-clamp approaches. Spontaneous IPSPs and IPSCs recorded in arcuate nucleus and hypothalamic cell cultures in the presence of glutamatergic antagonists were GABAergic in nature and could be completely and reversibly blocked by the GABAA antagonist bicuculline. In current-clamp experiments DA evoked cell depolarization and enhanced action potential activity in 22% of the cells and increased the activity of spontaneous GABA-mediated IPSPs in 64% of voltage-clamped neurons. Experiments with GABA immunostaining showed that the neurons that revealed depolarizing responses to DA were usually GABAergic, whereas non-GABAergic neurons responded more frequently by hyperpolarization of the Vm. These experiments with electrophysiology on cytochemically identified GABAergic neurons support the hypothesis that DA may be more likely to exert excitatory actions on hypothalamic GABAergic neurons than on non-GABAergic neurons.

In experiments (Gellman and Aghajanian 1993) on brain slices from rat piriform cortex, DA increased both the spontaneous IPSPs in 24% of pyramidal neurons and the action potential frequency in the majority of interneurons. Also, as in our study on hypothalamic and arcuate nucleus neurons, IPSPs recorded in the experiments of Gellman and Aghajanian were blocked by bicuculline and were GABAA dependent. However, although in the study by Gellman and Aghajanian the excitatory effect of DA on IPSPs was reduced by antagonists of glutamatergic neurotransmission, we observed a DA-mediated depolarization in some neurons during glutamatergic blockade. This difference could be due either to the use of different brain structures in these two studies or to a chronic culturing of our neurons in the presence of glutamate receptor antagonists, or to a combination of these factors. The chronic blockade of glutamate neurotransmission in cell cultures increases the sensitivity of neurons to endogenously released glutamate (Furshpan and Potter 1989; van den Pol et al. 1996b). The activity of GABAergic neurons was also increased after relief from a chronic glutamatergic blockade (Belousov and van den Pol 1997; Grantyn et al. 1995; van den Pol et al. 1996b).

The DA-mediated changes in Vm (mainly hyperpolarization) and in frequency of spontaneous IPSPs (mainly increase) found in our experiments were probably independent events, reflecting, respectively, a direct DA effect on the recorded neuron and an excitatory effect of DA on presynaptic GABAergic neurons. Both DA-mediated excitatory (increase in action potentials and IPSCs; Vm depolarization) and inhibitory (decrease in action potentials and hyperpolarization) responses could be recorded in cultures already by day 5 in vitro, showing that the mechanisms responsible for DA modulatory effects were present during early development.

    CONCLUSIONS

Taken together, the results obtained in this study support the hypothesis that the inhibitory effects of DA on activity in hypothalamic and arcuate nucleus neurons in culture are achieved in part through a hyperpolarization of excitatory neurons, likely via DA D2 receptors acting on K+ channels, and in part through the activation of inhibitory GABAergic neurons.

    ACKNOWLEDGEMENTS

  We thank V. Cao for excellent technical assistance; Drs. K. Obrietan, J. M. Weimann, and N. A. Otmakhov for suggestions and help; and Dr. H. C. Heller for providing facilities and encouragement.

  This work was supported by the Air Force Office of Scientific Research; National Institute of Neurological Disorders and Stroke Grants NS-10174, NS-31573, and NS-34887; the National Science Foundation, and a National Alliance for Research on Schizophrenia and Depression Young Investigator Award to A. B. Belousov. A. B. Belousov is a Scientific Researcher in the Institute of Cell Biophysics of the Russian Academy of Sciences, Puschino-on-Oka, Moscow Region 142292, Russia.

    FOOTNOTES

  Address for reprint requests: A. N. van den Pol, Section of Neurosurgery, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520.

  Received 3 July 1996; accepted in final form 22 April 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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