Department of Neurosurgery, Yale University, New Haven, Connecticut 06520
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 M 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 M
, and the series resistance
was lower than 30 M
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 M
.
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 = I/
V, where
I is the current
(pA) injected, and
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,
2, and regression
analyses were used for statistical evaluation. Differences were
considered significant if P < 0.05.
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RESULTS |
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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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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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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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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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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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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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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).
|
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.
|
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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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.
|
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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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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DISCUSSION |
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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
). In other cells,
particularly in mature hippocampal neurons or their dendrites, GABA
depolarization may be at least in part dependent on
HCO
; 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
and not
HCO
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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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