Membrane Properties Underlying Patterns of GABA-Dependent Action Potentials in Developing Mouse Hypothalamic Neurons

Yu-Feng Wang, Xiao-Bing Gao, and Anthony N. van den Pol

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Wang, Yu-Feng, Xiao-Bing Gao, and Anthony N. van den Pol. Membrane Properties Underlying Patterns of GABA-Dependent Action Potentials in Developing Mouse Hypothalamic Neurons. J. Neurophysiol. 86: 1252-1265, 2001. Spikes may play an important role in modulating a number of aspects of brain development. In early hypothalamic development, GABA can either evoke action potentials, or it can shunt other excitatory activity. In both slices and cultures of the mouse hypothalamus, we observed a heterogeneity of spike patterns and frequency in response to GABA. To examine the mechanisms underlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons (n = 282) in slices and cultures of developing mouse hypothalamus. Recorded with gramicidin pipettes, GABA application evoked action potentials in hypothalamic neurons in brain slices of postnatal day 2-9 (P2-9) mice. With conventional patch pipettes (containing 29 mM Cl-), action potentials were also elicited by GABA from neurons of 2-13 days in vitro (2-13 DIV) embryonic hypothalamic cultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patterns of multiple spikes. In parallel experiments in slices, electrical stimulation of GABAergic mediobasal hypothalamic neurons in the presence of glutamate receptor antagonists [10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 µM 2-amino-5-phosphonopentanoic acid (AP5)] resulted in the occurrence of spikes that were blocked by bicuculline (20 µM). Blocking ionotropic glutamate receptors with AP5 and CNQX did not block GABA-mediated multiple spikes. Similarly, when synaptic transmission was blocked with Cd2+ (200 µM) and Ni2+ (300 µM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA excitation and were not based on local interneurons. When the pipette [Cl-] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not detected with 2 or 10 mM intracellular [Cl-]. With gramicidin pipettes, we found that the mean reversal potential of GABA-evoked current (EGABA) was positive to the resting membrane potential, suggesting a high intracellular [Cl-] in developing mouse neurons. Varying the holding potential from -80 to 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spikes, but not the GABA-evoked depolarization. Removing Ca2+ from the extracellular solution did not block spikes, indicating GABA-evoked Na+-based spikes. Although EGABA was more positive within 2-5 days in culture, the probability of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistically, this appears to be due to a greater Na+ current found in the older cells during a period when the EGABA is still positive to the resting membrane potential. GABA evoked similar spike patterns in HEPES and bicarbonate buffers, suggesting that Cl-, not bicarbonate, was primarily responsible for generatingmultiple spikes. GABA evoked either single or multiple spikes; neurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present results indicate that the patterns of multiple action potentials evoked by GABA are an inherent property of the developing hypothalamic neuron.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABA is the primary inhibitory transmitter in the hypothalamus. In mature hypothalamic neurons, GABA is generally hyperpolarizing and inhibitory (Randle et al. 1986; Strecker et al. 1997; Tasker and Dudek 1993). In contrast, in developing hypothalamic neurons GABA can exert depolarizing actions. GABA depolarization can be excitatory and lead to action potentials if the GABA-mediated depolarization exceeds the spike threshold (Chen et al. 1996). In early development, synaptic release of GABA may generate more spikes than glutamate (Gao and van den Pol 2001). On the other hand, GABA-mediated depolarization can be inhibitory if it leads to shunting of other excitatory events, a process referred to as conductance shunt type of inhibition, shunting inhibition, or depolarizing inhibition (Alger and Nicoll 1979; Andersen et al. 1980; Brickley et al. 1996, 1999; Gao et al. 1998; Staley and Mody 1992). A primary mechanism for GABA-mediated depolarization in development is a reversal potential of GABA-evoked current (EGABA) that is positive to the resting membrane potential (RMP).

Although the role of action potentials in early development has not been extensively studied in the hypothalamus, in other brain regions action potentials play an important role in refining synaptic efficacy, regulating neuronal adhesion molecule expression, modulating axon structure, branching, and segregation of connections, and fine tuning the developing synaptic interactions (Hubel and Wiesel 1998; Itoh et al. 1997; Jarecki and Keshishian 1995; Kalil 1990; Lin and Constantine-Paton 1998; Nakagami et al. 1997; Nelson et al. 1989; Seil and Drake-Baumann 1994). Thus the mechanisms underlying GABA-mediated action potentials may be of substantial importance to the developing brain.

GABA is synthesized at early stages of brain development. During embryonic development of the hypothalamus, immunoreactive GABA can be detected in neurons and their axons (Tobet et al. 1999; van den Pol 1997). GABA is found in axon growth cones (van den Pol 1997), and GABA is released from these growth cones even before synapse formation, as detected with outside-out membrane patches of plasma membrane containing GABA receptors (Gao and van den Pol 2000). Growing neurites, growth cones, and cell bodies show an increase in calcium in response to GABA (Obrietan and van den Pol 1995, 1996a, 1998). In addition to a direct excitatory action, GABA may also enhance excitation by acting synergistically with glutamate (Gao et al. 1998; van den Pol et al. 1995, 1996). GABA actions can be modulated during early development by other hypothalamic neuroactive substances including neuropeptide Y (Obrietan and van den Pol 1996b), hypocretin (van den Pol et al. 1998a), and neurotropin-3 (Gao and van den Pol 1999). In addition, GABA can act at GABAB receptors to inhibit neuronal activity; this can occur during the same period when activation of the GABAA receptor can generate an excitatory action (Obrietan and van den Pol 1998, 1999).

Although changes in intracellular chloride ([Cl-]i) may be the critical factor determining GABA-mediated depolarization, other ions, membrane conductance, and receptor expression may be critical for determining whether a GABA-mediated depolarization will propagate a spike. During neuronal development, the intracellular ionic milieu and membrane properties undergo substantial change as channels, receptors, and transporters are being synthesized. In addition to Cl-, other ions, such as Na+, Ca2+, and bicarbonate may also play a role in GABA-evoked excitation. Bicarbonate is another anion passing the GABA-gated Cl- channel, and it may underlie GABA-evoked depolarization during prolonged activation of dendritic GABAA receptor (Staley and Proctor 1999).

In nonhypothalamic cells, GABA may evoke single action potentials (Owens et al. 1999). In hypothalamic neurons, GABA often evokes a single spike, but under some circumstances may evoke complex patterns of spikes. It is not clear what mechanisms underlie the determination of spike patterns. In addition to the fact that more spikes cause more transmitter release, some patterns of spikes may elicit greater release of neuromodulators. For instance, a burst of spikes may be more effective than the same number of spikes generated at longer intervals in facilitating release of neuropeptides, as demonstrated in the case of vasopressin release from the supraoptic and paraventricular nuclei axon terminals (Bicknell and Leng 1981; Shaw et al. 1984).

In the present study, to examine the regulation and the mechanisms underlying GABA-evoked spikes in the developing hypothalamic neurons, we studied the effect of GABA on mouse hypothalamic neurons in cultures and brain slices with whole cell and gramicidin-perforated patch-clamp recording using voltage and current clamp.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of cells and slices

The methods used for culture of mouse embryonic hypothalamic neurons were the same as those for rats (van den Pol et al. 1996). Pregnant mice (CD1) were heavily anesthetized with pentobarbital sodium (Nembutal 80 mg/kg ip), and embryonic day 18-20 fetuses were quickly removed under sterile conditions. After removing the brain under hypothermia, medial hypothalami were dissected out and then incubated in a papain enzymatic solution at 37°C for 25-30 min. The digested tissue was pelleted by gentle centrifugation to remove the enzymatic solution, then resuspended in a culture medium containing 10% fetal calf serum. The cell suspension was washed three times with the culture medium and mechanically scattered by gentle aspiration with a fire-polished Pasteur pipette. The dissociated cell suspension was plated on the central area of a 35-mm Petri dish (Corning) that had been precoated with poly-D-lysine (0.3 mg/ml, 540,000 Da, Collaborative Research) in a moderate density (8,000-10,000 cells/cm2), maintained in a Napco 6100 incubator supplied with 5% CO2 at 37°C. Five hours later, the culture medium was replaced with Neurobasal medium and serum supplement (GibcoBrl, Life Technologies) favoring the growth of neurons and inhibiting the proliferation of glial cells. These cultures were used for experiments after 2-13 days in vitro (2-13 DIV). Thirty minutes before recording, the medium was changed for an artificial cerebrospinal fluid (ACSF). These experiments were approved by the Yale University Animal Use Committee.

Hypothalamic slices were prepared from the mice at postnatal days 2-13 (P2-13). The mice were anesthetized with hypothermia (neonates) or with Nembutal (100 mg/kg) and then decapitated. The brain was rapidly removed and immersed in ice-cold, oxygenated ACSF for 1-2 min before being trimmed to a hypothalamic block. The hypothalamic block was excised longitudinally from the suprachiasmatic area to the mammillary body, laterally cut at the fornix and mammillothalamic tract, and dorsally sectioned 2 mm from the ventral surface. Then the hypothalamic block was glued on the block of a vibratome, and 200-µm-thick coronal slices were cut in ice-cold, oxygenated ACSF. The slices were then transferred to a chamber filled with ACSF bubbling with oxygen and kept at room temperature (22 ± 1°C) for 1-2 h before recording. Recordings in slices were made from the mediobasal hypothalamus in the area of the arcuate and ventromedial nuclei.

Electrophysiology

Electrical measurements were carried out on an inverted microscope (Nikon, MVI) at room temperature with an EPC-9 amplifier (Heka, Lambrecht, Germany). Data were sampled at 1 kHz and saved with Pulse 8.41 software (Heka) or at 3 kHz for studies of sodium current and spike threshold. Patch pipettes were made of borosilicate glass (WPI) and polished by a vertical pipette puller. The pipettes had a tip of 1-2 µm with a DC resistance of 3-5 MOmega after filling with pipette solution. For recordings from the culture, the Petri dish was fixed on the stage of the microscope, and the culture was perfused continuously with oxygenated ACSF. Both the microinjector and patch pipette were placed close to a target neuron through micromanipulators. After forming a gigaohm seal, the membrane was ruptured by application of an additional suction, to form a whole cell recording model. The initial seal resistance was above 800 MOmega , and the series resistance was lower than 30 MOmega after compensation by the Pulse software. Current clamp and voltage clamp were used.

Gramicidin perforated patch recording was the same as that for conventional whole cell recording except that the tip of the patch pipette was filled with normal pipette solution before backfilling with gramicidin; the plasma membrane was not ruptured during gramicidin recording. Gramicidin perforation maintains the [Cl-]i at physiological levels (Ebihara et al. 1995; Quigly et al. 2000). Gramicidin recordings usually stabilized within 20-30 min with a series resistance of about 40-50 MOmega .

For recording from slices, a U-shaped steel-frame nylon net was used to hold the slice in place. Gentle cleaning of the neuronal surface was performed before establishing a gigaohm seal. Other steps were the same as those for recordings in culture. To obtain evoked responses by electrical stimulation, a bipolar electrode was positioned close to the neurons recorded in 200-µm-thick slices, and a brief rectangular pulse (50-500 µA, 0.2-0.5 ms, 0.1 Hz) was passed under computer control to trigger the release of neurotransmitters from axonal terminals. Off-line analyses were performed with Pulse Fit (Heka, Lambrecht, Germany), Axograph (Axon Instruments), and IgorPro (WaveMetrics) software.

Solutions and chemicals

The enzymatic solution used to isolate neurons for culture contained 135 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 0.5 mM EDTA, 10 mM HEPES, 10 mM glucose, 10 units/ml papain (Worthington Biochemical), and 0.2 mg/ml L-cysteine, pH 7.3 adjusted with NaOH. The culture medium was based on minimal essential medium, with 10% fetal calf serum (Hyclone), serum extender (Collaborative Research), 100 units/ml penicillin-streptomycin, and 6 g/l glucose. The standard ACSF contained (in mM) 150 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.3 NaH2PO4, 10 HEPES, and 10 glucose, pH 7.3 adjusted with NaOH. The ACSF routinely contained glutamate receptor antagonists 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 µM 2-amino-5-phosphonopentanoic acid (AP5). CNQX was dissolved in DMSO first and diluted to working solution with a final DMSO concentration <0.1%. For bicarbonate-buffered ACSF, NaCl was reduced to 135 mM, and HEPES was replaced with 26 mM NaHCO3, pH 7.3 when gassed with 5% CO2-95% O2. To remove extracellular Ca2+, Ca2+-free solution was prepared by replacing CaCl2 with 2 mM MgCl2 and 1 mM EGTA in the standard ACSF.

The standard pipette solution (29 mM Cl-) for conventional patch-clamp recording contained (in mM) 118 KMeSO4, 27 KCl, 1 MgCl2, 1.1 EGTA, 10 HEPES, 4 Mg-ATP, and 0.5 Na2-GTP, pH 7.3 adjusted with KOH. For 2, 10, 15, and 45 mM Cl--containing pipette solutions [Cl-]p, the components were the same as the standard except that the ratio of KMeSO4 to KCl was changed to vary from (in mM) 145:0, 137:8, 132:13, and 102:43, respectively. Gramicidin pipette solution was made by adding 50 µg/ml gramicidin D to 2 mM Cl--containing pipette solution. Gramicidin was dissolved in DMSO (50 mg/ml) first and diluted to working solution immediately before recordings. The diluted solution was effective for at least 3 h. Agents and chemicals used were from Sigma except where indicated.

Drug application

GABA was applied through a computer-controlled drug delivery device, Picospritzer II (General Valve) and micropipettes. GABA was applied by a brief pressure ejection (10 PSI, 100 ms) on to the neurons 10-20 µm from the tip (2 µm) of the micropipettes. Neurons in the recording chamber were perfused continuously at a rate of 1.5-2 ml/min using a gravity-fed pipe whose tip was positioned 500 µm away from the target neuron; perfusates and drugs were removed quickly by continuous aspiration through a suction needle, the tip of which was close to the neuron.

Data analysis

The excitability of the neurons recorded was expressed as the increase in spike number during a 2-s period before or during the application of GABA. EGABA was obtained by plotting GABA-evoked currents against a series of holding potentials and determining the membrane potential at which GABA evoked no current. The spike threshold was defined as the membrane potential at the acute deflection of the upstroke of a spike. The initial spike was the first spike that occurred after application of GABA; the amplitude of spikes was the difference between the peak of the initial spike and its threshold. The membrane conductance (C, in nS) was calculated by the equation C = Delta I/Delta V, where Delta I is the current (pA) injected, and Delta V is the change in the membrane potential (mV) in response to the current. Intensity of Na+ current (INa+) was the peak value of the inward INa+. Liquid junction potentials between perfusates and pipette solutions were reduced by a salt-bridge based on the auto-V0 function of the Pulse 8.41 software. All data are expressed as means ± SE. Student's t-test, ANOVA, chi 2, and regression analyses were used for statistical evaluation. Differences were considered significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The actions of GABA on 282 developing mouse hypothalamic neurons were studied in slices and cultures with whole cell or gramicidin perforated patch recordings. The primary focus of the study was mechanisms underlying mild and strong excitatory actions of GABA on hypothalamic neurons.

Diversity of excitatory effects of GABA in slices and cell cultures

HYPOTHALAMIC SLICES. Hypothalamic slices were used to observe the effect of GABA on mouse neurons. Unless otherwise noted, all experiments below were performed in the presence of AP5 (100 µM) and CNQX (10 µM) to block excitatory actions of glutamate. Excitatory responses to brief pressure injections (100 ms, 10 PSI) of GABA (100 µM) were studied in hypothalamic slices. With conventional whole cell patch-clamp recording, and using pipettes containing 29 mM Cl-, a level previously found in developing neurons of rat hypothalamus (Chen et al. 1996), an excitatory effect of GABA was observed in 20 of 20 neurons recorded from P2 to P13 slices (Fig. 1A). Because the [Cl-]i may be dictated by the pipette solution, we also used gramicidin perforated whole cell recordings to leave [Cl-]i unperturbed by the pipette solution.



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Fig. 1. Excitatory effect of GABA in slices. A-C: traces showing excitatory effects of GABA on mediobasal hypothalamic neurons in slices under different recording conditions. A: 2 neurons recorded with conventional patch pipettes containing 29 mM Cl- show an increase in spikes in response to GABA (100 µM). B: 2 neurons recorded with gramicidin perforated patch recordings at -60 mV. In response to GABA (100 µM, 100 ms, 10 PSI), a number of spikes occurred. C: single electrical stimulation (100 µA, 0.3 ms) evoked depolarization (C1) and spike (C2) in postnatal day 2 (P2) neurons recorded with gramicidin pipette. The responses were blocked by bicuculline (20 µM, middle traces) and recovered after wash out of bicuculline. All responses above were observed in 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) and 2-amino-5-phosphonopentanoic acid (AP5; 100 µM). E.S., electrical stimulation.

Excitatory responses (spikes) to GABA were observed in 11 of 13 neurons from P2 to P9 hypothalamic slices (Fig. 1B) studied with gramicidin perforated whole cell recording. In contrast, in slices older than P10 (n = 6) GABA evoked inhibitory actions or no change in potential. To determine whether synaptic GABA release would also evoke spikes during early hypothalamic development, electrically evoked responses were studied. Excitatory responses were induced by the evoked release of GABA. Electrical stimulation in the P2 to P5 mediobasal hypothalamic slice evoked depolarizing responses in 10 cells (Fig. 1C1). Some depolarizing potentials were accompanied by action potentials (Fig. 1C2). Excitatory responses were reversibly blocked by 20 µM bicuculline (Fig. 1C), suggesting that they were mediated by synaptic GABA release activating the GABAA receptor. Application of bicuculline to the bath usually evoked a hyperpolarization of the membrane potential, suggesting a tonic depolarization mediated by ongoing release of GABA. Multiple patterns of GABA-mediated excitatory responses were found in developing hypothalamic slices (Fig. 1).

CULTURE. Having established that both exogenous GABA application and synaptic GABA release can generate action potentials in hypothalamic slices, we turned to hypothalamic cultures. The responses to exogenous GABA and the recovery from these responses are faster in culture than in slices due to the immediate access of the applied transmitter to the recorded neuron. In cultured hypothalamic neurons (2-13 DIV), a dramatic depolarization of the membrane potential (28.3 ± 1.1 mV, mean ± SE, n = 93) was elicited by application of GABA (30 µM, 100 ms, 10 PSI) at a holding potential (HP) of -60 mV from all the neurons with 29 mM [Cl-]p. In those neurons, 17.2% (n = 16) showed depolarization only, with no spikes (Fig. 2A); 43.0% (n = 40) showed a single evoked spike and a more sustained depolarization (Fig. 2B); 39.8% (n = 37) showed a depolarization together with a complex pattern of multiple spikes. In the neurons with multiple spikes, the secondary spikes occurred either near the peak of depolarization following the initial spike (Fig. 2, C and D) or during the repolarizing phase (Fig. 2E). In some cells, multiple spikes occurred selectively at both the initial depolarization and during recovery during GABA wash out (Fig. 2F). In some neurons GABA evoked bursts at the depolarizing plateau (Fig. 2G). The amplitudes of the secondary spikes were usually lower than those of the initial spike and either decreased gradually before the peak of depolarization or recovered gradually with the progress of repolarization. Both the depolarization and the consequent spikes were blocked by bicuculline (20 µM), indicating a dependence on the GABAA receptor.



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Fig. 2. Effects of GABA on spike frequency in culture. Traces A-G represent different types of GABA-evoked responses in 2-13 days in vitro (2-13 DIV) neurons. Application of GABA (30 µM, 100 ms, 10 PSI, indicated by arrows) immediately evoked an obvious depolarization of membrane potential from -60 mV, which usually recovered within 2 s. The initial spike occurred during application of GABA in all neurons except in A. A: neuron showing depolarization but no spike before the return of membrane potential to baseline. B: single spike at the ascending phase of the depolarization. C-G are examples of different patterns of multiple spikes. C: double spike. D: the spikes occur before the peak of the depolarization; the spike amplitudes decreased as the peak sustained depolarization was approached. E: a 2nd group of spikes occurred after the peak of the slow depolarization, and the spike amplitudes increased as the slow depolarization decreased. F: a 2nd group of spikes occurred just after the peak of the slow depolarization; the amplitudes of the 1st group of spikes decreased before the peak of the slow depolarization and gradually recovered during repolarization. G: a set of spikes with an appearance similar to spike frequency adaptation during the sustained GABA-mediated depolarization.

The following experiments were designed to explore the mechanisms underlying this variety of GABA-mediated excitatory responses using cultured hypothalamic neurons.

Local neuronal circuits and GABA-evoked excitation

Multiple spikes induced by GABA application could be due to recurrent excitatory feedback from other neurons in the same culture that released either GABA or glutamate, or to intrinsic membrane properties. The potential facilitation of glutamate on GABA-evoked multiple spikes was evaluated first. When ionotropic glutamate receptor antagonists CNQX (10 µM) and AP5 (100 µM) were added to the bath (Fig. 3A), there was no significant change (P > 0.05, n = 12) in the excitability of the neurons, defined by GABA-evoked spike frequency. The number of spikes evoked in control buffer (2.83 ± 0.61) was nearly the same as those (2.26 ± 0.63) in the CNQX and AP5 solution. Additional experiments were undertaken to block both GABA and glutamate release. Cd2+ (200 µM) and Ni2+ (300 µM), high- and low-voltage-activated Ca2+ channel blockers, were used to block evoked and spontaneous release of neurotransmitters (Bao et al. 1998; Haage et al. 1998). These Ca2+ channel blockers did not block the occurrence of multiple spikes, although excitatory postsynaptic potentials (EPSPs) were eliminated (Fig. 3B). The number (3.80 ± 0.96) of GABA-evoked spikes and the amplitude of spikes (62.7 ± 4.4 mV) in the presence of the blockers were not significantly (n = 7, P > 0.05) different from those (4.37 ± 0.75 and 59.5 ± 4.4 mV) without the Ca2+ channel blockers. These results suggest that multiple spikes evoked by GABA can be generated by intrinsic membrane properties and can occur independent of secondary synaptic activity generated by other neurons.



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Fig. 3. Multiple spikes independent of local neuronal circuits. Traces in A and B show the influence of blockade of local neural transmission on the multiple spikes evoked by GABA (30 µM, 100 ms, arrows) at 6-9 DIV neurons. A: blocking ionotropic glutamate receptors by CNQX (10 µM) and AP5 (100 µM) did not block the depolarization and the evoked spikes compared with the responses to GABA in the absence of CNQX and AP5 (Control). B: block of general neurotransmission with Cd2+ (200 µM) and Ni2+ (300 µM) eliminated excitatory postsynaptic potentials (EPSPs) and lowered the magnitude of depolarization but did not block the generation of multiple spikes.

Role of [Cl-]i in spikes

[Cl-]i plays a critical role in determining the valence of the GABA response, with low intracellular levels leading to hyperpolarization and inhibition, and high levels leading to depolarization. We previously showed in developing rat hypothalamic neurons that the ECl was positive to the RMP and to the spike threshold (Chen et al. 1996).

By using gramicidin perforated whole cell recording, we measured the EGABA of 2-13 DIV neurons cultured from the embryonic mouse brain. During this period, EGABA showed a developmentally regulated negative trend. At periods beginning 2, 6, and 10 DIV, the EGABA was -24.0 ± 3.5 mV (n = 5), -38.0 ± 4.2 mV (n = 5), and -49.0 ± 4.8 mV (n = 4), respectively. During this period the RMP shifted negatively from -43.2 ± 1.4 mV at 2 DIV to -51.7 ± 1.1 mV at 6 DIV to -57.7 ± 1.2 mV at 10 DIV. Typical examples are shown in Fig. 4. These data confirm in mouse hypothalamic neurons that EGABA shifts in a negative direction during neuronal development. The difference between the EGABA and the RMP decreased during this period, from 19 mV at 2 DIV, 14 mV at 6 DIV, and 9 mV at 10 DIV.



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Fig. 4. Reversal potentials of GABA-evoked currents in neurons of different ages. Current-voltage (I-V) relationships of GABA-evoked currents recorded in the gramicidin-perforated patch configuration. The amplitude of the currents evoked by GABA (30 µM) at potentials varying from -70 to -10 mV by 10-mV steps was measured at the peak (A) and plotted against the membrane potential. In A, the potential to reverse GABA-evoked currents from inward to outward was shifted to a more negative level as neuronal age increased. In B, the deduced EGABA was -24, -38, and -49 mV in the groups with neuron ages beginning at 2, 6, and 10 DIV, respectively.

To examine the effects of different [Cl-]i, we varied the pipette [Cl-]. After obtaining membrane breakthrough, we waited until the ECl reached a stable level, usually after at least 10 min. The influence of different [Cl-]i on GABA-evoked excitation was observed at 6-13 DIV mouse neurons. With different [Cl-]p, GABA-evoked depolarization and excitation were highly related to [Cl-]i as presented in Table 1. At an HP of -60 mV, with low [Cl-]p (2 and 10 mM), GABA hyperpolarized the membrane potential (Fig. 5, D and E). With 15 mM or higher [Cl-]p, GABA evoked depolarization and excitation. With 29 and 45 mM [Cl-]p (Fig. 5, A and B), the neurons showed significantly (P < 0.05) higher excitability and a higher probability of spikes than the neurons with 15 mM [Cl-]p did. As the RMP and the threshold shifted in a negative direction, the magnitude of the GABA-mediated depolarization increased. There were no significant differences in the probability of spike and in the regulation of membrane potential changes of the neurons between RMP and -60 mV, except that spontaneous spikes often occurred at RMP when [Cl-]p was 45 mM, and hyperpolarization could appear at RMP when [Cl-]p was 15 mM (Fig. 5C).


                              
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Table 1. Intracellular [Cl-]: membrane characteristics



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Fig. 5. Effect of [Cl-]i on GABA actions. Traces A-E are the responses of 6-9 DIV neurons to GABA (30 µM, 100 ms, at arrows) recorded with pipettes containing different Cl--levels; those in the left column were observed at resting membrane potential (RMP), and those in the right column at -60 mV. A: 45 mM Cl- pipettes: a high magnitude of depolarization and several spikes occurred in response to GABA. B: 29 mM Cl- pipette: multiple spikes occurred with GABA-evoked depolarization. C: 15 mM Cl- pipette: a small hyperpolarization was evoked at RMP, and at -60 mV, a small depolarization was elicited by GABA. D: 10 mM Cl- pipette: hyperpolarization was induced at both RMP and -60 mV, and no spike was evoked. E: 2 mM Cl- pipette: significant hyperpolarization occurred at both RMP and at -60 mV, and no spikes appeared in response to GABA.

As a whole, these data suggest that the GABA-mediated responses were dependent on developmentally regulated intracellular Cl-. An optimal [Cl-]p for generating spikes was 29 mM, a level used in our experiments below.

Holding potential

Since membrane potential may influence the response to GABA, we studied the effect of different HPs on GABA actions.

Changing the HP had an inverted U-shaped effect on the excitability and amplitude of GABA-evoked spikes of 6-13 DIV neurons (Table 2). When HP was shifted from -80 to 0 mV in steps of 10 mV, the excitability and the amplitude of spikes at first increased and reached a peak at -60 mV, then decreased gradually at more positive HPs. Although the amplitude of spikes may have been slightly underestimated due to the sampling frequency, this would apply equally to all conditions. Spontaneous spikes gradually increased when the HP was shifted from -50 to -30 mV. On the other hand, the amplitude of the GABA-mediated depolarization (not including the spike) gradually reduced (from 46.8 ± 1.7 mV at -80 mV HP to 2.9 ± 1.4 mV at -30 mV HP, n = 18), and the threshold for spikes shifted in a positive direction as the HP was shifted in a positive direction. When the HP was positive to -27 mV, GABA evoked a hyperpolarization. Figure 6 shows representative responses of a neuron to GABA at different HPs.


                              
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Table 2. Membrane potential and GABA actions



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Fig. 6. Effect of membrane potential on GABA-evoked spikes. The 9 traces are recordings of a typical DIV 7 neuron with 29 mM Cl- in the pipette at membrane potentials of 0 to -80 mV in steps of 10 mV. GABA (30 µM, 100 ms) was applied at arrow. From 0 to -20 mV, hyperpolarization was evoked with gradually reduced magnitudes. At -30 mV, a slight depolarization was elicited. The magnitude of depolarization became bigger as the membrane potentials were held at a more negative potential. At -40 mV, spontaneous spikes with small amplitudes occurred before GABA application. These spikes disappeared during GABA-evoked depolarization. At -50 mV (the RMP of this neuron), spontaneous high-amplitude spikes were seen. At -60 mV, spontaneous spikes disappeared, but spikes were found at the beginning and after a quiet interval, at a later time point during the repolarizing phase; spontaneous EPSPs still occurred. At -70 and -80 mV, evoked spikes still occurred before the peak of depolarization but not at the repolarizing phase.

Effect of neuronal age on GABA action

Neuronal age had a strong influence on EGABA and RMP, as revealed with gramicidin perforated recordings, and possibly affected the excitability of the neurons. On the other hand, although gramicidin perforated patch recording allowed recording from cells with undisturbed [Cl-]i, a high access resistance was imposed by the perforated patch. To evaluate the relationship between development and the excitability of the neurons, we further studied the role of neuronal age on GABA action with conventional whole cell recording, where [Cl-]i could be regulated at a constant level (Table 3).


                              
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Table 3. Neuron age: membrane characteristics

Comparing three age groups, 2-5 DIV neurons, 6-9 DIV neurons, and 10-13 DIV neurons, there were significant differences between the youngest group and the two older groups in the RMP, the magnitude of depolarization, the probability of spiking, the excitability, the threshold, and the amplitude of spikes. The difference between the two older groups was less. In 2-5 DIV neurons, the number of neurons with spikes and the spike frequency, the magnitude of depolarization, as well as the amplitude of spikes were lower, whereas the RMP and the spike threshold were more positive, as shown in Fig. 7.



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Fig. 7. Neuronal maturation and GABA-evoked spike. Traces A-C show the responses of neurons of different ages to GABA (30 µM, 100 ms, at arrows) with 29 mM Cl- pipettes at -60 mV. A: 2-5 DIV neurons showed weak depolarization (neurons 1 and 2) or single spike based on the depolarization (neuron 3). B: 6-9 DIV neurons showed double spikes before the peak of depolarization (neuron 4) or many secondary spikes at the repolarizing phase with active EPSPs (neurons 5 and 6). C: 10-13 DIV neurons showed single spike (neuron 7) or multiple spikes (neurons 8 and 9).

To test the hypothesis that a substantial change in INa+ might correlate with the increase in GABA-evoked spikes, we measured INa+ in voltage clamp with depolarizing steps of 10 mV. Positive steps evoked a fast transient inward current. The transient inward current was identified as INa+ since it was abolished by replacing Na+ with choline or by adding TTX (1 µM) to the perfusion medium. The 2-5 DIV neurons had a smaller amplitude of INa+ and higher threshold for activation of the current. The intensity (399 ± 84 pA) of INa+ at 2-5 DIV neurons (n = 17) was significantly smaller than that of 6-9 DIV (1,985 ± 234 pA, n = 29, P < 0.05) and 10-13 DIV (2,859 ± 343 pA, n = 22, P < 0.01) neurons, whereas the spike threshold (-27.2 ± 2.0 mV) at 2-5 DIV was significantly more positive (P < 0.05) than threshold in 6-9 DIV (-31.5 ± 1.9 mV) and 10-13 DIV (-41.4 ± 1.4 mV) neurons. The INa+ amplitude was highly correlated with the frequency of GABA-evoked spikes in 2-13 DIV neurons (r = 0.915, n = 68).

The intensity of INa+ in cultured neurons [at embryonic day 18 (E18)] after 6-9 DIV (1,985 ± 234 pA, n = 29) was similar to that found in hypothalamic slices from cells of roughly the same age, P2-5 neurons (1,804 ± 257 pA, n = 5; add 3 days of embryonic development in situ for comparison). The intensity of INa+ and its threshold were highly correlated with the excitability of the neurons and the probability of spikes. When neurons of different ages were pooled together, high INa+ and a low spike threshold were correlated with an increase in GABA-evoked spikes regardless of the age of the recorded neuron (Fig. 8). Table 4 summarizes the relationship between INa+ and other membrane features.



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Fig. 8. Dependence of GABA-evoked spikes on sodium current. Traces in columns A-C represent the membrane electrochemical features of 3 neurons with different INa+ characteristics. The top rows show the voltage steps from -50 to -10 mV with steps of 10 mV (top) and their corresponding INa+ threshold and intensities (bottom). The inset at the left is the relationship between voltage and peak INa+ in the different groups (A-C). The traces in the middle row show the electrical responses of the neurons to GABA (30 µM, 100 ms). The bottom rows indicate the threshold of spikes revealed under current clamp with a command current that shifted membrane potential from -60 to 0 mV by injection of depolarizing current in 10 steps (2-10 pA/step), with a length of 50 ms (bottom traces). A: a neuron with low INa+ intensity: peak value of INa+ was 310 pA. A brief depolarization was evoked without subsequent spiking; the estimated threshold for spikes was -25 mV. B: a neuron with middle level INa+: peak value of INa+ was 1,850 pA. Depolarization was evoked with multiple spikes before its peak; the estimated threshold for spikes was -33 mV. C: a neuron with high INa+ intensity: peak value of INa+ was 3,210 pA. A quick depolarization was evoked with spikes before the peak of depolarization and during repolarization; the estimated spike threshold was -41 mV.


                              
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Table 4. Sodium current and membrane electrochemical characteristics

Na+, Ca2+, bicarbonate, and GABA-evoked spikes

We found that GABA-evoked spikes (n = 10) were completely blocked by TTX but the magnitude of nonspike depolarization did not change (27.6 ± 5.7 mV in control and 22.3 ± 7.6 mV in TTX) significantly (Fig. 9A), indicating that GABA-evoked spikes were dependent on INa+. However, in TTX the membrane potential hyperpolarized significantly (18.6 ± 2.7 mV, n = 13, P < 0.05), which recovered after wash out. As bicuculline also caused a membrane hyperpolarization, the TTX-mediated hyperpolarization is probably due to a block of synaptic GABA release.



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Fig. 9. Na+, Ca2+, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and GABA-evoked spikes. Traces in A-C represent the responses of neurons in TTX, Ca2+-free, and bicarbonate-buffered solution, respectively. A: GABA-evoked spikes (control) disappeared in TTX (1 µM)-containing solution, and recovered after wash out of TTX. The slow depolarization did not change markedly in TTX. B: multiple spikes still occurred in Ca2+-free plus EGTA solution, but EPSPs disappeared. The magnitude of depolarization was reduced from -32 to -44 mV, and the threshold of spikes became more negative (from -39 to -49 mV) in the absence of Ca2+. The depolarization recovered after wash out with standard solution. C: 2 neurons recorded in HEPES buffer (neuron 1) or in bicarbonate-buffered solution (neuron 2), respectively. Neurons showed the same ability to depolarize and to fire multiple spikes in both conditions.

ICa2+ may also be involved in the generation of spikes (Tennigkeit et al. 1998; Widmer et al. 1997). To reveal the relationship between extracellular Ca2+ and GABA-evoked multiple spikes, the influence of Ca2+ on GABA actions was observed in 12 neurons. Multiple spikes still occurred in the Ca2+-free solution (Fig. 9B); the RMP (-54.3 ± 1.5 mV) and the number of GABA-evoked spikes (3.23 ± 0.67) in the presence of Ca2+ were not significantly different (P > 0.05) from those (-51.7 ± 1.6 mV and 3.16 ± 0.86, respectively) in Ca2+-free solution. However, the magnitude of depolarization was reduced significantly (from 29.5 ± 1.0 mV in control to 15.1 ± 2.3 mV in Ca2+-free solution, P < 0.01), and the threshold of spikes shifted negatively (from -38.2 ± 2.0 mV to -47.8 ± 1.9 mV, P < 0.05) when the Ca2+-containing solution was changed to Ca2+-free solution.

We studied the influence of bicarbonate on GABA excitatory effect and compared the results that were observed in HEPES-buffered solution (Fig. 9C). The type of neuronal responses to GABA in bicarbonate-buffered solution was quite similar to that obtained in HEPES-buffered solution. There was no significant difference in the number of spikes evoked between neurons observed in bicarbonate-buffered solution (2.55 ± 0.67, n = 12) and in HEPES-buffered solution (2.2 ± 0.80, n = 12), although the magnitude of depolarization (31.7 ± 1.5 mV) in the former was slightly larger (0.05 < P < 0.1) than that (27.0 ± 1.1 mV) in HEPES-buffered solution.

Thus our data are consistent with the view that the action potentials observed in developing hypothalamic neurons are dependent primarily on voltage-dependent Na+ channels.

High excitability and membrane electrochemical features

GABA can depolarize the membrane potential during development; the depolarization can either be excitatory and lead to spike generation, or alternately could be inhibitory due to an increase in conductance and result in shunting other excitatory input to the same cell. We examined depolarization and membrane conductance during depolarizing responses to GABA. Of particular interest was a second set of spikes found in a number of neurons that were depolarized by GABA. These cells showed an initial spike at the onset of GABA-mediated depolarization, and then after a period of no spikes, showed a second group of spikes during the repolarizing phase of the response.

By repetitive current (30-80 pA) injection through patch pipettes at an interval of 100-300 ms, membrane conductance and potential were recorded during neuronal response to GABA. At the initial phase of GABA-induced depolarization, the membrane conductance increased from 4.7 ± 0.1 nS to 9.8 ± 0.2 nS (within 200 ms after GABA, n = 37), indicated by a sudden reduction in the magnitude of membrane potential change in response to the current injection (Fig. 10, A and B). This result was consistent with the findings of Hales et al. (1994) and Chen et al. (1996). The half recovery time (440 ± 63 ms) of the conductance was significantly (n = 20, P < 0.01) faster than that (830 ± 67 ms) of the membrane potential (Fig. 10C). The quick recovery in the conductance was accompanied by a reduction of the shunting effect. Analyzing the changes in the amplitude of the secondary spikes in sequence, we found that the amplitudes of the first, second, and third secondary spikes were 30 ± 5.8%, 68 ± 8.3%, and 81 ± 9.7% of that of the initial spikes (initial spike defined as 100%, n = 10), respectively. Moreover, the intensities of the basal and peak conductance of multiple spiking neurons were significantly (P < 0.05) less than those of single spiking neurons (Fig. 10D), suggesting that an additional mechanism acting to support multiple spikes was the greater input resistance of cells showing this increased activity.



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Fig. 10. Conductance and membrane potential. A and B: examples of the changes in the membrane potential and in the conductance of a single spiking neuron and a multiple spiking neuron, respectively. The top traces show the membrane potential change, and the bottom bar graphs show the corresponding conductance change. In A, the conductance during the control period was 5.1 nS, and increased to 24.1 nS at the peak of depolarization. The conductance recovered to the control level before the membrane potential repolarized to 50% of the depolarization; the current injected was -50 pA. B: the basal conductance was 3.2 nS, which increased to 10.2 nS when the membrane potential depolarized to its peak and then recovered to control level at about 50% repolarization of the membrane potential; the current injected was -30 pA. C: the relation between conductance and membrane potential is shown. The graph shows that the recovery of the conductance was significantly faster than that of the membrane potential: when the membrane potential recovered to about 50%, the conductance recovered more than 80% of its peak. * P < 0.05. ** P < 0.01. D shows the basal and peak conductance of neurons with different excitability. ** P < 0.01 compared with control of the same group. + P < 0.05. ++ P < 0.01 compared with single spike groups.

Neurons that showed either single or multiple spikes in response to GABA were compared. As shown in Table 5, multiple spiking neurons had a more negative spike threshold, and a greater INa+ than that found in single spiking neurons. Both differences were statistically significant (P < 0.05).


                              
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Table 5. Comparison between neurons with multiple spikes and single spike


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the developing CNS, complex patterns of action potentials alternate with periods of silence (Grossmann and Ellendorff 1986; Jones and Jones 2000; Tepper et al. 1990). As development proceeds, spike patterns and frequency also change. The present results show that GABA can evoke action potentials in developing mouse hypothalamic neurons both in brain slice and in culture, as studied with whole cell and gramicidin perforated patch recording. Complex patterns of spikes can be evoked by GABA independent of local excitatory circuits, indicating that they are an intrinsic membrane property of developing neurons. This suggests that GABA neurons may generate complex spike patterns found in the developing brain. Spike probability is dependent on ECl, amplitude of sodium current, membrane conductance, and RMP.

Development of Na+ currents

GABA-mediated depolarization can occur without an action potential, or a spike may be generated by the depolarization. At the earliest stage of development in the present experiments (2-5 DIV), although GABA was consistently depolarizing due to the relatively positive ECl, the spike probability was low. In contrast, in slightly older cells (6-9 DIV), although the ECl was less positive and closer to the RMP, thereby reducing the driving force for outward Cl- movement, a higher probability of GABA-evoked spikes was noted. A primary mechanism underlying this appears to be the development of voltage-dependent Na+ channels during this period. A substantially greater sodium current was found in 6-9 DIV neurons than in younger cells. Our finding of developmental increases in Na+ currents in hypothalamic neurons is consistent with data from the development of neurons from other brain regions where only weak sodium current and an elevated spike threshold were found early in development (Cummins et al. 1994; Walton et al. 1993; Xia and Haddad 1994). A weak sodium current may not carry enough cations into the neuron to trigger spikes even if ECl- is positive to the RMP. Although we did not study it in the present paper, potassium channels that change with development may also play an important role in the regulation of spike frequency.

Single and multiple spikes

In both slices and cultures, a single GABA application often evoked multiple spikes. That the multiple spikes were not due to secondary release of GABA, glutamate, or some other excitatory transmitter was demonstrated with experiments either blocking glutamate receptors, or by blocking synaptic release of transmitters with Cd2+ and Ni2+ (Bao et al. 1998) or by using a nominally Ca2+-free solution. These experiments support the conclusion that the GABA-evoked multiple spikes can be generated by intrinsic membrane properties of developing hypothalamic neurons. Long-lasting stimulation by GABA could result in Na+ channel inactivation or GABA receptor desensitization; both would tend to depress spike frequency.

GABA-mediated spikes; initiation by outward Cl- movement

A primary mechanism underlying GABA excitation is that EGABA is positive to the RMP. This could be due to either Cl- or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, two anions that pass the GABA-gated channel (Kaila et al. 1993). In other cells, particularly in mature hippocampal neurons or their dendrites, GABA depolarization may be at least in part dependent on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Staley and Proctor 1999; Staley et al. 1995). When those experiments were performed in a HEPES buffer, no GABA-mediated depolarization was found (Staley et al. 1995). In contrast, we find similar GABA-mediated spikes in buffers containing or lacking HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, indicating that the ionic basis for the depolarization is probably Cl- and not HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The reason that ECl is positive to the RMP in early development and negative to it in mature neurons is probably due to several factors. Outward Cl- co-transporters appear to develop late (Kakazu et al. 1999; Rivera et al. 1999). Some Cl- channels (ClC-2) that are inwardly rectifying at negative membrane potentials and may reduce Cl- accumulation in mature neurons develop late (Mladinic et al. 1999). In addition, there may be inward Cl- transport in young cells, either from specific transporters not yet identified, or from reversal of transporters that are outwardly directed in mature cells (Luhmann and Prince 1991; Misgeld et al. 1986).

Conductance

Membrane conductance plays important roles in determining spike probability. A high probability of GABA evoking multiple spikes was inversely related to the conductance of the recorded cell. Cells with large baseline conductance were less likely to spike in response to GABA. In addition, changes in conductance may also determine spike patterns. For instance, in a number of cells, a single GABA application resulted in spikes at the initial depolarization and then, after a period of silence, a second group of spikes as the membrane potential recovered. In both sets of spikes the membrane conductance was low, whereas in the silent period between the first and second set, conductance was high due to the opening of Cl- channels. As Cl- channels closed, in part due to desensitization, the conductance was decreased at a time when the membrane potential was still depolarized, resulting in the second set of spikes. That the conductance recovered faster than the membrane potential after GABA stimulation is a critical determinant of the second group of spikes occurring after an intermediate period of silence. Another line of evidence in support of this mechanism is that in the second set of spikes, the first spikes of the second set had a lower amplitude than the second spikes of the same set; this increase in spike amplitude is consistent with an ongoing reduction in the conductance as Cl- channels closed. In the silent period between the two sets of spikes, current shunting tended to reduce spiking. A low conductance was also critical in determining spike frequency and was correlated with a higher level of multiple spikes.

Another factor that changes with development is the structure of the somatodendritic complex. During development, the cell volume increases, and the somatic membrane increases in area. Substantial increases in dendritic arbor occur, in the diameter, length, and surface area of the dendrites (van den Pol et al. 1998b). The increase in membrane of both soma and dendrites may increase conductance as channels are added. GABA-mediated spikes would then be dependent on the sum of GABA actions on receptors in different locations with different local resting membrane conductances, and these would change with cellular maturity.

GABA synapses in some cells may be found preferentially near the cell body, as described in hippocampal pyramidal cells and cerebellar Purkinje cells, or may be on dendrites, as in the olfactory mitral cell (Eccles 1969). No clear subcellular localization of GABA synapses has been reported for hypothalamic neurons. Depending on the number and type of ion channel, dendritic trees may have a greater conductance than the cell body (London et al. 1999), and given the greater ratio of plasma membrane to volume in dendrites, this may also play a role in the ontogeny of conductance. Hypothalamic neurons have a lower baseline conductance than the larger cells of the hippocampus, for instance, and that may facilitate GABA exerting a greater independent effect in generating spikes in hypothalamic neurons compared with hippocampal neurons.

In the constant presence of glutamate receptor antagonists, blocking synaptic activity in synaptically coupled developing cells with TTX or with bicuculline caused a hyperpolarization of the membrane potential, in part due to the loss of depolarizing GABA activity, suggesting an ongoing GABA excitation in both cultures and slices.

Functional implications of GABA-evoked excitation

GABA may modulate different functions in the developing hypothalamus. GABA-mediated increases in cytoplasmic calcium in the cell body, neurites, and growth cone would allow GABA to modulate cell migration, neurite extension and pathfinding, and gene expression (Obrietan and van den Pol 1996a). GABA appears to influence early neuronal migration of GnRH cells; disturbances of GABA actions may alter cell movement and axon extension and may lead to disorganization of the GnRH system (Bless et al. 2000). Gender-specific responses to GABA and gender differences in GABA content in the developing hypothalamus suggest that GABA may play a role in gender-associated functions of the hypothalamus (Davis et al. 1999; Smith et al. 1996). GABA axons define the ventromedial nucleus before the nucleus can be differentiated from surrounding tissue, leading to the suggestion that GABA may play a role in hypothalamic nuclear maturation (Tobet et al. 1999). GABA may modulate gap junctions, commonly found in developing brain (Shinohara et al. 2000).

Although the present study focuses on mouse hypothalamic neurons, GABA exerts depolarizing actions in many brain regions. Obata (1974) observed that GABA induced a depolarization of chick embryonic spinal neurons, suggesting a potential excitatory role of GABA. GABA-mediated depolarization and excitation have been observed extensively in the developing CNS, particularly in rat, as found in immature CA3 hippocampal neurons (Ben-Ari et al. 1989), spinal cord (Reichling et al. 1994), cortical neurons (LoTurco et al. 1995), and olfactory bulb cells (Serafini et al. 1995). In the hippocampus, an important role for GABA during development appears to be a synergistic role in generating giant depolarizing potentials; GABA relieves the N-methyl-D-aspartate (NMDA) receptor of its voltage-dependent Mg2+ block (Leinekugel et al. 1997). In the present work NMDA receptors were blocked with AP5, indicating that the multiple spikes studied here in hypothalamic neurons can be based solely on GABA, and not on GABA as a facilitator of excitation at NMDA synapses.


    ACKNOWLEDGMENTS

We thank Y. Yang for excellent assistance with tissue culture.

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-10174, NS-34887, and NS-37788.


    FOOTNOTES

Address for reprint requests: A. N. van den Pol, Dept. of Neurosurgery, Yale University Medical School, 333 Cedar St., Box 208082, New Haven, CT 06520-8082 (E-mail: anthony.vandenpol{at}yale.edu).

Received 13 February 2001; accepted in final form 1 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society