Postnatal Maturation of Rat Hypothalamoneurohypophysial Neurons: Evidence for a Developmental Decrease in Calcium Entry During Action Potentials
H. Widmer,
H. Amerdeil,
P. Fontanaud, and
M. G. Desarménien
Biologie des Neurones Endocrines, Centre National de la Recherche Scientifique Unité Propre de Recherche 9055, CCIPE, 34094 Montpellier Cedex 5, France
 |
ABSTRACT |
Widmer, H., H. Amerdeil, P. Fontanaud, and M. G. Desarménien. Postnatal maturation of rat hypothalamoneurohypophysial neurons: evidence for a developmental decrease in calcium entry during action potentials. J. Neurophysiol. 77: 260-271, 1997. Action potentials and voltage-gated currents were studied in acutely dissociated neurosecretory cells from the rat supraoptic nucleus during the first three postnatal weeks (PW1-PW3), a period corresponding to the final establishment of neuroendocrine relationships. Action potential duration (at half maximum) decreased from 2.7 to 1.8 ms; this was attributable to a decrease in decay time. Application of cadmium (250 µM) reduced the decay time by 43% at PW1 and 21% at PW3, indicating that the contribution of calcium currents to action potentials decreased during postnatal development. The density of high-voltage-activated calcium currents increased from 4.4 to 10.1 pA/pF at postnatal days 1-5 and 11-14, respectively. The conductance density of sustained potassium current, measured at +20 mV, increased from 0.35 (PW1) to 0.53 (PW3) nS/pF. The time to half-maximal amplitude did not change. Conductance density and time- and voltage-dependent inactivation of the transient potassium current were stable from birth. At PW1, the density and time constant of decay (measured at 0 mV) were 0.29 nS/pF (n = 12) and 17.9 ms (n = 10), respectively. Voltage-dependent properties and density (1.1 nS/pF) of the sodium current did not change postnatally. During PW1, fitting the mean activation data with a Boltzmann function gave a half-activation potential of
25 mV. A double Boltzman equation was necessary to adequately fit the inactivation data, suggesting the presence of two populations of sodium channels. One population accounted for ~14% of the channels, with a half-inactivation potential of
86 mV; the remaining population showed a half-inactivation potential of
51 mV. A mathematical model, based on Hodgkin-Huxley equations, was used to assess the respective contributions of individual currents to the action potential. When the densities of calcium and sustained potassium currents were changed from immature to mature values, the decay time of the action potentials generated with the model decreased from 2.85 to 1.95 ms. A similar reduction was obtained when only the density of the potassium current was increased. Integration of the calcium currents generated during mature and immature action potentials demonstrated a significant decrease in calcium entry during development. We conclude that the developmental reduction of the action potential duration 1) is a consequence of the developmentally regulated increase in a sustained potassium current and 2) leads to a reduction of the participation of calcium currents in the action potential, resulting in a decreased amount of calcium entering the cell during each action potential.
 |
INTRODUCTION |
The maturation of neuronal excitability is a progressive process that relies on changes in the density and kinetics of voltage-dependent ion channels. In many vertebrate models, the main change during development is a decrease in the duration of the spike. The mechanisms underlying this decrease have been studied in detail in Xenopus spinal neurons. In these neurons, the major consequence of the action potential shortening is a diminution of the amount of calcium that enters the cells during each action potential (see review by Spitzer et al. 1994
). This process plays a key role in neuronal differentiation, because preventing or limiting calcium entry in culture perturbs axonal growth (Holliday and Spitzer 1990
), neurotransmitter synthesis (Spitzer et al. 1993
), and maturation of voltage-dependent ion channels (Desarménien and Spitzer 1991
), and may affect neurogenesis (Jones and Ribera 1994
). In addition to playing a decisive role in the development of individual amphibian neurons, electrical activity is crucial to the developmental refinement of neuronal networks of higher vertebrates (Shatz 1990
).
In the present study, we asked whether voltage-gated calcium entry is developmentally regulated in mammalian central neurons, as in lower vertebrates. To address this question, we investigated the maturation of excitability of neurosecretory magnocellular neurons, which constitute two highly distinctive populations, one synthesizing oxytocin and the other vasopressin. Both populations send their axons to the neurohypophysis, where they release their respective peptides into the blood stream. These two classes of neurosecretory neurons share a common embryonic fate until the last days of fetal life (Altman and Bayer 1978a
,b
). Yet, as maturation proceeds, they follow divergent developmental patterns that eventually lead to highly distinctive neurons, characterized by different hormonal contents and firing patterns (reviewed by Hatton 1990
).
As revealed by numerous studies of different mammalian CNS structures, an essential part of the development of neuronal properties, including synaptic refinement, occurs postnatally (reviewed by Goodman and Shatz 1993
). In particular, neuroendocrine relationships are known to be established during the perinatal period (Jost et al. 1974
). For example, developmental studies of monoaminergic neurons involved in regulation of neurosecretory nuclei revealed fiber extension and formation of synapses with several target areas during early postnatal life (Ugrumov 1992
; Ugrumov et al. 1989a
,b
). We studied the postnatal maturation of action potentials and the respective contributions of the underlying voltage-dependent currents in freshly dissociated neurons isolated from supraoptic nuclei of 0- to 21-day-old rats, with special attention to the contribution of calcium currents. The conclusions drawn from the data were checked and confirmed with the use of a computer model of individual ionic currents and resulting action potentials. A preliminary account of these data has been published elsewhere (Widmer et al. 1995
).
 |
METHODS |
Cell preparation
Experiments were performed on rats aged from 1 to 21 days. Pairs of rats from the same litter and of the same sex were killed by decapitation and the brain was quickly removed. With the use of iridectomy scissors, two blocks of tissue were dissected from the basal hypothalamus, containing part of the optic chiasm and a strip of the tissue lateral to the optic tract (Lambert et al. 1994
). Each block was cut in two to three pieces and transferred to Locke's medium (see Table 1) supplemented with proteases X and XIV (1 mg/ml, Sigma, St. Louis, MO) and DNAase I (650 U/ml, Sigma). Enzymatic dissociation was performed for 25 min at 20-24°C under gentle stirring by O2 bubbling. After thorough rinsing with Locke medium, cells were dissociated by mechanical trituration and plated in uncoated 35-mm culture dishes (Costar). The recording session began as soon as the cells attached and lasted ~2 h.
Electrophysiological recordings
Large cells (soma diameter > 14 µm) with short neurites were selected for recordings. Immunocytochemical studies reported by Oliet and Bourque (1992)
and by our laboratory (Lambert et al. 1994
) have demonstrated that most of these cells contain either oxytocin or vasopressin. Recordings were performed at room temperature, with the use of the whole cell configuration of the patch-clamp technique (Hamill et al. 1981
). Pipettes were pulled from borosilicate glass (Polylabo) and had resistances between 3 and 7 M
. Recordings were performed with a List EPC-7 amplifier; data were filtered at 5 kHz with the use of an eight-pole Bessel filter (Frequency Devices), digitized, and stored on a computer with the use of P-Clamp software (Axon Instruments). Voltage-clamp recordings were sampled at rates ranging from 3 to 20 kHz, depending on the ionic current studied. Current-clamp recordings were sampled at 16 kHz. Membrane resistance, series resistance, and cell capacity were measured with the use of a 10-mV depolarizing voltage step (from a holding potential of
80 mV). Series resistance (5.8 ± 0.09 M
, mean ± SE, n = 166) was compensated to >60% in all voltage-clamp experiments (maximal voltage-clamp error 2.3 mV/nA). Neurons with membrane resistances of <0.5 G
(measured at
80 mV) or resting potentials smaller than
45 mV were discarded from analysis. All currents were leak subtracted with the use of a P4 procedure. The results are given as means ± SE; statistical differences were assessed by Student's t-test.
Solutions
The composition of the solutions used in the present study is indicated in Table 1. Osmolarity was adjusted to 300 ± 10 mosM. Because measured liquid junction potentials were within a 4-mV range, no correction was performed.
Data analysis
Analysis of the recordings was performed with the use of Origin (MicroCal) and P-Clamp6 (Axon Instruments) softwares.
ACTION POTENTIAL.
The threshold of the action potential was defined as the inflection point, established by eye, at the foot of the regenerative upstroke. The amplitude of the spike was measured from threshold to peak. The spike duration was measured at half-maximal amplitude. The rise time was measured as the time required for the membrane potential to change from 10 to 90% of the peak of the spike. The decay time corresponded to the time required for the potential to recover from 90 to 10% of the peak of the spike. The amplitude of the hyperpolarizing afterpotential was measured from the threshold to the maximally hyperpolarized value of the membrane potential.
SODIUM CURRENT ACTIVATION.
Conductance was calculated from peak currents by the relation
|
(1)
|
where I is the sodium current amplitude, E is the membrane potential, and Erev is the reversal potential of the sodium current as measured for each conductance determination. For each neuron, maximal conductance, Gmax, was estimated from the Boltzman equation best fit to the conductance measured during current activation. The specific conductance was calculated by dividing G by the cell capacitance.
SODIUM CURRENT INACTIVATION.
For each neuron, maximal conductance was calculated from the amplitude of the peak current elicited at
10 mV from a prepulse at
90 mV.
POTASSIUM CURRENTS.
G was calculated with Eq. 1. Erev is the theoretical potential calculated from Nernst equation as a function of external and internal potassium concentration. Observation of the tail current at various holding potentials confirmed the validity of this value.
CALCIUM CURRENTS.
Because Erev could not be determined precisely, current density was evaluated by dividing the maximal intensity of the current by the cell capacitance.
Computer modeling
Individual currents and action potentials were modeled with the use of a computer software based on Hodgkin-Huxley equations. The program was a modified version of the software used by Lockery and Spitzer (1992)
, originally designed to model ionic currents and action potentials in amphibian spinal neurons. The structure and equations of the program were conserved but parameters were adjusted to fit our experimental data. The main modification concerned calcium currents, because the original study took only one class of current into account, whereas we considered the participation of both a high-voltage-activated (HVA) and a low-voltage-activated (LVA) current. Because we did not have detailed information concerning the calcium-dependent potassium current, and because this current is generally considered to participate mainly in the termination of bursts of action potentials, we did not include it in modeling single spikes.
Details concerning the basis of the calculations can be found in the original paper by Lockery and Spitzer (1992)
. Briefly, sodium and potassium currents were calculated as the result of a conductance g(V,t) multiplied by a driving force
|
(2)
|
in which Vinv is the reversal potential as calculated from the Nernst equation.
The conductance is time and voltage dependent; thus it was calculated as the product of a maximal conductance (gmax), an activation term A(V,t), and an inactivation term B(V,t)
|
(3)
|
The calcium current was calculated from the permeability and the driving force
|
(4)
|
where PCa is the membrane permeability to calcium and
Ca is the driving force, given by the constant field equation (see Lockery and Spitzer 1992
for details).
A(V,t) and B(V,t) are first-order processes, depending respectively on the steady-state activation A
(V) and inactivation B
(V) by the following relationships
|
(5)
|
|
(6)
|
They were fitted to the values of the voltage dependency of activation and inactivation rates of the current by the use of a Boltzmann equation of the form
|
(7)
|
|
(8)
|
A
(V) and B
(V) are Boltzmann functions of the membrane potential governed by equations of the following type
|
(9)
|
|
(10)
|
in which V0 represents half-maximal activation (or inactivation) and s represents the steepness of the Boltzmann relationship.
For the calculation of action potentials, we used the relationship
|
(11)
|
where
|
(12)
|
respectively, the leak, sodium, sustained potassium, transient potassium, HVA and LVA calcium currents, and C is the cell capacitance.
For each individual current, V0 and s parameters used for the calculation of
A,
B, A
, and B
were adjusted so that the currents generated by the model fit experimental data recorded from mature neurons and so that current-voltage relationships overlap. The conductances of the sustained potassium current and both calcium currents were then modified by a factor reflecting the developmental change observed to generate immature action potentials.
 |
RESULTS |
In contrast to developmental changes in the action potential, several passive properties of the neurons did not change during this period. The mean membrane capacitance of acutely dissociated neurons obtained from rats aged from 1 to 21 days was 31.7 ± 1.3 pF (n = 139) and did not change from postnatal week (PW) 1 to PW3 (PW1: 30.2 ± 1.4 pF, n = 49; PW2: 35.9 ± 3.1 pF, n = 50; PW3: 28.4 ± 1.4 pF, n = 40). The mean cellular resistance was 1.3 ± 0.1 G
(n = 139) and was also constant during this period (PW1: 1.4 ± 0.1 G
; PW2: 1.3 ± 0.1 G
; PW3: 1.1 ± 0.1 G
). Measurement of the resting potential was restricted to cells in which the action potential was studied. It was measured immediately after the patch membrane was disrupted at the electrode tip and did not change significantly from PW1 (
52.9 ± 2.7 mV, n = 10) to PW3 (
54.5 ± 1.6 mV,n = 13).
Action potentials
Action potentials recorded in neurons from rats aged 1, 10, and 21 days reveal a decrease in duration during maturation (Fig. 1). Action potentials were elicited by a 65-ms depolarizing current pulse from the resting level. The intensity of the injected current was adjusted at a slightly suprathreshold level. In most cases, 65-ms current injection elicited only one spike, although two to three spikes were occasionally observed. Action potentials could be elicited from the day of birth. Several different parameters describe the action potential as a function of age (Fig. 1). Spike width, measured at half-maximal amplitude, decreased from 2.7 ± 0.2 ms (n = 9) at PW1 to 1.8 ± 0.1 ms (n = 10) at PW3 (P < 0.05; Fig. 1D). This decrease was paralleled by a decrease in the decay time (PW1: 2.8 ± 0.2 ms, n = 9; PW3: 1.6 ± 0.1 ms, n = 10; P < 0.05) (Fig. 1E). The amplitude of the afterpotential increased from 15.7 ± 1.3 mV (n = 9) at PW1 to 23.9 ± 2.1 mV (n = 10) at PW3 (P < 0.05; Fig. 1F). Threshold, rise time, and amplitude of the spike did not change from the day of birth.

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| FIG. 1.
Developmental changes in the action potential. A-C: voltage responses elicited by current pulses at the ages indicated; bottom trace in C is the current injection monitor. D-F: action potential parameters; see text for details. Asterisks: statistically significant changes compared with postnatal week (PW) 1 (P < 0.05).
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CONTRIBUTION OF VOLTAGE-ACTIVATED CALCIUM CURRENTS TO THE ACTION POTENTIAL.
To assess the contribution of calcium inward currents to the spike, we blocked the calcium entry by adding the divalent ion blocker cadmium (250 µM). Blockade of Ca2+ currents led to a decrease in spike duration at all ages, particularly in young neurons (Fig. 2A). The spike width was reduced to 67% of its control value by Cd2+ application at PW1 (n = 7), whereas it was only reduced to 84% of control at PW3 (n = 8). Cd2+ application reduced the decay time by 43 ± 4% at PW1, and by only 22 ± 6% at PW3 (P < 0.05; Fig. 2B). The duration of the Na+/K+ spike, recorded in the presence of Cd2+, decreased only slightly and nonsignificantly during development; this indicates that calcium currents are responsible for the larger action potentials recorded in young neurons.

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| FIG. 2.
Effect of blockade of voltage-activated calcium current on spike duration and decay time during development. Number of cells tested is indicated in the bins. Asterisks: statistically significantly different compared with control.
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Voltage-gated currents
To correlate the properties of voltage-gated currents with the shape of the action potential, we studied the densities and kinetics of K+, Ca2+, and Na+ currents.
POTASSIUM CURRENTS.
Two components of the potassium current were revealed by depolarizing the neurons to
20 mV and above: a fast inactivating component, and a sustained one that showed no inactivation during a 60-ms pulse (Fig. 3A).

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| FIG. 3.
Properties of the sustained potassium current. A: currents elicited by depolarizing the membrane from a holding potential (hp) of 80 mV to 20 10, 0, +10, and +20 mV. Note that initial activation of the fast inactivating current occurred at more negative potentials than for the sustained current. B: mean density of the sustained current measured at +20 mV from a holding potential of 80 mV vs. age. C: same protocol in the same neuron, with holding potential set at 60 mV; note the absence of fast inactivating current. D: mean time to half maximum measured at +20 mV, vs. age. Numbers in columns: number of neurons. Asterisks: statistically significantly different compared with PW1.
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|
Sustained potassium current. We measured the steady-state amplitude of the sustained current at the end of a 60-ms voltage step to +20 mV, at which time the transient current was completely inactivated (see Figs. 3A and 5A). Sustained conductance density was 0.35 ± 0.04 nS/pF(n = 12) at PW1 and increased to 0.53 ± 0.07 nS/pF(n = 11) at PW3 (P < 0.05; Fig. 3B). The activation kinetics of the sustained current were characterized by measuring the time to half-maximal amplitude. Cells were maintained at a holding potential of
60 mV to fully inactivate the transient component (Fig. 3C). At PW1, the mean value was 6.2 ± 0.7 ms (n = 5), which did not change during the two subsequent postnatal weeks (PW3: 7.0 ± 1.1 ms,n = 5; Fig. 3D).

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| FIG. 5.
Voltage-dependent inactivation of the transient potassium current. A: currents elicited at 0 mV after 50-ms prepulses to 70, 50, 30, and 10 mV from a holding potential of 100 mV. The test amplitude of the current diminished as the prepulses became less negative; note the concomitant activation of the current during the prepulse. B: voltage-dependent inactivation: the peak amplitude of the subtracted current at 0 mV (see text) was plotted as a function of the conditioning prepulse. For each neuron, amplitudes were normalized to the maximal amplitude obtained after a prepulse to 80 mV. Data are the means of 8-12 neurons. Solid lines: best fit of the data with a Boltzmann equation (see details in text). Open circles: PW1. Solid circle: PW3.
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Voltage-dependent inactivation of the sustained current. Slow inactivation of the sustained current was detectable with pulse durations >100 ms. To investigate this property, neurons were held for 30 s at various depolarized potentials before stepping to +20 mV (Fig. 4A). Holding the potential at
60 mV and above led to strong inhibition of the current. In each neuron, the residual current elicited from each holding potential was normalized to the current obtained with a holding potential of
80 mV (Fig. 4B), because measurements performed at a holding potential of
120 mV indicated that the current amplitude was still maximal at
80 mV. At PW1, the amplitude of the sustained current was reduced to 68 ± 6.8% (n = 4) and 25 ± 3.7% (n = 9) of its control value by holding the potential at
60 and
30 mV, respectively. Roughly similar values were obtained at PW3 (holding potential
60 mV: 73 ± 12%, n = 4; holding potential
30 mV: 41 ± 6%, n = 5).

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| FIG. 4.
Voltage-dependent slow inactivation of the sustained potassium current. A: currents elicited at +20 mV, after holding potential was held for 30 s at 80, 60, and 30 mV; depolarizing holding potentials led to a decrease in current amplitude. B: steady-state residual current measured at +20 mV, normalized to the current elicited from a holding potential of 80 mV, plotted as a function of age.
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|
Transient potassium current. The contribution of the transient component to the total potassium current was measured by applying progressively more depolarized prepulses. This procedure resulted in a progressive decrease in amplitude and ultimately complete inactivation of the transient current elicited by the test potential (Fig. 5A). Although short prepulses were not sufficient to completely inactivate the transient current, they were not prolonged to prevent slow inactivation of sustained current. The peak amplitude of the transient current was determined as the difference between the peak amplitude of the current obtained after a given prepulse and the noninactivating current (Fig. 6, A and B). The fast transient current and the inactivated current elicited at 0 mV are superimposed in the last part of the pulse, showing that the sustained current was not affected by this protocol. This amplitude was then normalized to the maximal value obtained after a prepulse to
80 mV and plotted against the prepulse potential to construct the voltage-dependent inactivation curve (Fig. 5B). The mean data were adequately fit with a Boltzmann function, with a half-inactivation potential of
47.5 mV (slope: 7.5) at PW1. A similar value was obtained at PW3 (
49.7 mV; slope: 6.4).

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| FIG. 6.
Amplitude and kinetics of the transient potassium current. A: fast inactivating and the inactivated current elicited at 0 mV after 50-ms conditioning prepulses to 80 and 30 mV, respectively. B: subtracted trace obtained from the current traces in A, fit with a single-exponential equation ( = 15.5 ms). C: density, measured from peak amplitude of subtracted traces, reported vs. age. D: mean time constant vs. age. Numbers in columns: number of neurons.
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Amplitude and kinetics of the transient current. The conductance density, calculated from the peak amplitude of the current at 0 mV, was 0.29 ± 0.05 nS/pF (n = 12) at PW1. No developmental change was apparent (Fig. 6C). The decay of the current was adequately described by a monoexponential equation (Fig. 6B). Comparison throughout development revealed a small decrease in the time constant, from 17.9 ± 1.4 ms (n = 10) at PW1 to 12.5 ± 1.4 ms (n = 9) at PW3 (Fig. 6D).
CALCIUM CURRENTS.
Voltage dependency. Voltage-activated calcium currents constitute two functionally different classes: the LVA and HVA currents. Families of currents evoked by progressively depolarizing pulses showed that LVA current was first elicited at potentials above
50 mV; for potentials above
20 mV an HVA current was activated, superimposed on the LVA current (Fig. 7, A and B). The LVA current amplitude decreased between PW1 and PW2, whereas the HVA current amplitude increased (Fig. 7C). No shift in the voltage dependency of the activation curves was detected during the period studied, with maximal activation around
35 and +5 mV. Successive steps to
40 and 0 mV illustrate the opposite trends of LVA and HVA currents in the course of development (Fig. 7D). The duration of the first step allowed LVA to inactivate before HVA was activated.

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| FIG. 7.
Voltage-activated calcium currents. A: current traces elicited by voltage steps to 60, 30, and 0 mV from a holding potential of 100 mV at PW1. B: same protocol as A, at PW2. C: current-voltage curves measured during PW1 ( ) and PW3 ( ). Each point on the curves is the average from 12-24 neurons. Solid line was drawn by eye. D: voltage-clamped currents, illustrating the opposite development of low-voltage-activated (LVA) and high-voltage-activated (HVA) currents.
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Incidence and density. This protocol was used to assess both the incidence and the amplitude of the currents. The incidence is given as percentage of neurons expressing a current relative to the total number of neurons recorded (Table 2). Detection threshold was 10 pA. The density was averaged from the neurons expressing the currents. The incidence of HVA currents increased from 40% (50 cells recorded) to 78% (22 cells) between days 1-6 and 10-14, whereas LVA incidence increased from 66% (50 cells) to 82% (22 cells). By contrast, the density of both currents showed divergent development (Fig. 8, A2 and B2). The density of the HVA current increased from 4.4 ± 0.5 pA/pF (n = 27) to 9.7 ± 1.3 pA/pF (n = 37) between days 1-5 and 6-10, then stabilized at 10.1 ± 1.7 pA/pF (n = 23) at days 11-14. During the corresponding period, the density of LVA current decreased from 6 ± 0.7 pA/pF(n = 38) to 3.3 ± 0.3 pA/pF (n = 45), then stabilized(3.3 ± 0.5 pA/pF; n = 26). Because incidence and density of both LVA and HVA currents showed a clear tendency to stabilize between days 6-10 and 11-14, no further investigation was made during PW3.

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| FIG. 8.
Incidence and density of voltage-activated calcium currents. Results were grouped in 3 bins, corresponding to the postnatal days indicated. Left: incidence of LVA (A1) and HVA (B1) currents vs. age. Numbers in columns: number of neurons recorded. Right: density of LVA (A2) and HVA (B2) currents vs. age.
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SODIUM CURRENT.
Amplitude. Sodium current properties were studied in the presence of a reduced driving force to avoid voltage-clamp error arising from the large amplitude of the current. Measurements of peak current amplitude elicited at
10 mV yielded conductance densities ranging from 0.48 to 1.55 nS/pF [PW1: mean, 0.97 ± 0.11 nS/pF (n = 10)]. A similar variability was observed at PW3, ranging from 0.53 to 2.25 nS/pF, with a mean density of 1.22 ± 0.22 nS/pF (n = 7). No significant difference was observed between the two means.
Voltage dependence of activation and inactivation of sodium current. Sodium current was activated by depolarizing voltage steps preceded by a 100-ms conditioning prepulse to
90 mV from a holding potential of
60 mV (Fig. 9A). Plotting the peak amplitude of the current versus potential revealed that the current was activated for potentials above
40 mV and reversed polarity at 14.1 mV in this cell (Fig. 9B). Repeated steps to
10 mV from successively more depolarized prepulses revealed the voltage-dependent steady-state inactivation of the current (Fig. 9C). Inactivation and activation properties of sodium current were similar at PW1 and PW3 (Fig. 9D). Mean data of current activation were adequately fit with a Boltzmann function, with half-activation potential at
25 mV and slope of 5.5 at PW1, compared with
22 mV and 7.6 at PW3 (n = 5; data not shown). The current inactivation revealed the presence of two populations of channels, because the data could only be adequately fit with a double Boltzmann equation of the form
One population accounted for 14% of the total channel population, with a half-inactivation potential of
86.8 mV and a slope of 1. The remaining channels showed a half-inactivation potential of
51.7 mV and a slope of 5.6. Similar properties were observed at PW3, with 11% of the current showing a half-inactivation potential at
83 mV (slope: 1.2) and the remaining current showing a half-inactivation potential at
50.4 mV (slope: 5.4) (n = 5; data not shown).

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| FIG. 9.
Voltage dependence of sodium current activation and inactivation. A: current traces showing the activation of the sodium current by 8-ms voltage pulses from 44 to +28 mV (8-mV increment) from a 100-ms prepulse to 90 mV; holding potential was 60 mV. B: corresponding current-voltage curve constructed from the peak amplitude of the current. C: current traces showing the inactivation of the sodium current measured at 10 mV by progressively less negative 100-ms voltage prepulses from 90 to 34 mV (8-mV increments). D: mean activation and inactivation curve at PW1. Each point is the average of 4-5 neurons. Solid lines are the fits of the data with Boltzmann equations (see details in text).
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Computer modeling
COMPUTATION OF INDIVIDUAL CURRENTS.
The first task was to determine, for each current, the parameters providing a satisfactory fit of the voltage dependency and of the time dependency of the current; this was accomplished by comparing calculated values with experimental data obtained from individual mature cells; the experimental data were chosen from among the examples shown in previous figures. The conductance-voltage relationships and the voltage dependency of the rate of activation and inactivation of the recorded currents were used to determine the values of V0 and s used to calculate the activation and inactivation factors of each current (data not shown). An additional scaling factor f was used to adjust the calculated current to the mean amplitude of the recorded ones. Because the current densities of IKV, ICaHVA, and ICaLVA were the only ones to change during development, immature action potentials were generated by changing the scaling factor of these currents accordingly.
The equations used to produce the currents were the following.
Sodium current.INa = f*gmax*A3*B*(V
ENa) withf = 0.23; gmax = 45 nS; and ENa = +14 mV for the comparison with sodium currents and then +60 mV for the modeling of action potentials
Sustained potassium current.IKv = f*gmax*A3*(V
EK)
with f = 12.8; gmax = 26.4 nS (mature) or 14.8 nS (immature); and EK =
90 mV
Transient potassium current.IKA = f*gmax*A4*B*(V
EK) with f = 0.5; gmax = 14.3 nS; and EK =
90 mV
HVA calcium current.ICaHVA = f*PCa*A3*B*
Ca withf = 0.7 (mature) or 0.31 (immature) and PCa = 0.6.
Because the rate of inactivation of the HVA currents varied considerably from cell to cell, we chose to use an intermediate value (see Fig. 10B).
LVA calcium current.ICaLVA = f*PCa*A5*B*
Ca withf = 0.11 (mature) or 0.20 (immature) and PCa = 0.6
The currents generated with these values, superimposed on experimental data, are illustrated in Fig. 10.

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| FIG. 10.
Modeling of voltage-dependent currents with kinetics of mean experimental data recorded from mature supraoptic neurons. A: sodium current. Current-voltage relationship of the calculated current ( ) superimposed on experimental values ( ). A, right: example of recorded currents (·) superimposed on calculated currents ( ). B: calcium currents. Current-voltage relationship of the total calcium current (HVA plus LVA,  ) superimposed on that determined experimentally ( ). B, right: currents calculated with successive steps to 40 and 0 mV ( ) are superimposed on experimental data from immature (top) or mature (bottom) neurons. C and D: transient and sustained potassium currents. Calculated currents superimposed on those recorded experimentally.
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ACTION POTENTIALS.
For calculation of action potentials, we first used maximal conductances consistent with the current densities recorded from mature cells. The resting potential was fixed at
60 mV, the measured membrane capacity value of 31.7 pF was used, and the leak current was calculated assuming a membrane resistance of 1.3 G
. Action potentials were generated with the use of two different protocols. We initially examined the results of applying a 60-ms depolarization to allow a direct comparison of both active and passive properties (Fig. 11, A and B). A 2-ms pulse was used to allow an easier comparison between action potentials generated with different current densities (Fig. 11, C-E). With the use of maturelike parameters, the action potential generated had a decay time of 1.95 ms, a value close to the experimental one (1.8 ms). When current densities were changed to immature values, the decay time of the action potential increased to 2.85 ms, that is, again close to experimental values (2.8 ms). Significantly, when an immature action potential was calculated with a mature value for IKV density, its decay time decreased to the value of the mature one. This result indicates that the developmental increase of the sustained potassium current alone is sufficient to account for the observed decrease in action potential duration, despite the concomitant increase in the density of calcium currents (Fig. 11C).

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| FIG. 11.
Modeled action potentials calculated with current densities corresponding to values recorded during PW1 (A) or PW3 (B). The protocol used for stimulation is similar to that in Fig. 1. C: comparison of action potentials obtained with mature and immature current densities. Note that when the mature value is used for the current density of IKv, the duration of the immature spike decreases to that of the mature action potential. D and E: effect of suppressing the calcium currents on immature and mature action potentials. F: LVA and HVA calcium currents occurring during mature and immature action potentials. The slight increase of HVA and the large decrease of LVA result in an overall decrease of 31% of the total calcium current (area under total currents, right).
|
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We tested the contribution of calcium currents to mature and immature action potentials by suppressing them. This resulted in a decrease of action potential decay time of 35% in immature and 25% in mature conditions (Fig. 11, D and E), values to be compared with the 43 and 22% reductions induced by cadmium at PW1 and PW3, respectively. This result indicates that the contribution of calcium currents to the action potential decreases with maturation. A direct consequence should be that the amount of calcium entering the cell during an action potential decreases accordingly. We calculated this amount by calculating the integral of these currents (Fig. 11F). During an immature action potential, 986 pC entered via the LVA current and 623 pC via the HVA calcium currents, that is, a total of 1,609 pC. During mature action potentials, these values changed to 321 pC (LVA), 788 pC (HVA), and 1,109 pC (total). The total amount of calcium entering the cell during an action potential thus decreases by about one third during maturation.
 |
DISCUSSION |
In the present study, we report a developmental decrease in the duration of the action potential in supraoptic magnocellular neurons during early postnatal life, which can be attributed to a selective decrease in the decay time. In addition, we observed a concomitant decrease of the calcium component of the spike, whereas the duration of the Na+/K+ component decreased only slightly. To determine whether the developmental decrease in spike duration was due to a decrease of depolarizing currents or an increase in hyperpolarizing ones, we studied the underlying voltage-gated currents.
Voltage-activated inward currents
SODIUM CURRENTS.
The properties of the sodium current remained stable during the first three postnatal weeks, indicating that this current is already mature at birth. This is consistent with the early maturation of this current reported in other systems (reviewed by Dietzel 1995
), although postnatal maturation of sodium currents has been reported in neocortical neurons (Huguenard et al. 1988
). The presence of two distinct populations of sodium channels, as revealed here by their inactivation properties, has been reported in a variety of excitable cells and, in most cases, such a duality has been related to the presence of both tetrodotoxin-sensitive and tetrodotoxin-insensitive currents, with slightly different voltage sensitivities (see Elliott and Elliott 1993
, and references therein). Complete block of the sodium currents in our experiments required a high concentration of tetrodotoxin (10
6 M). Alternatively, it is possible that the 100-ms prepulse was not long enough to allow full recovery from the inactivation occurring at the holding potential of
60 mV.
CALCIUM CURRENTS.
We report a twofold increase in both the incidence and the density of HVA currents. Up-regulation of HVA currents density seems to be a common developmental scheme in many embryonic and postnatal excitable cells (Beam and Knudson 1988
; Dourado and Dryer 1992
; Gonoi and Hasegawa 1988
; McCobb et al. 1989
), except in rat superior cervical ganglion neurons (Nerbonne and Gurney 1989
). A large cell-to-cell variability was observed in the ratio of HVA peak to plateau currents, suggesting the presence of distinct subtypes of HVA currents. This finding is consistent with a recent study reporting the presence of at least three types of HVA currents (N-, L-, and P-type) in supraoptic magnocellular neurons of the adult rat (Fisher and Bourque 1995
). By contrast, the density of LVA current decreased, as often observed in the development of excitable cells. Its density decreases in chick embryonic motoneurons (McCobb et al. 1989
) and mouse postnatal muscle fibers (Gonoi and Hasegawa 1988
), but it increases in neurons of the rat dorsal root ganglia (Lovinger and White 1989
) and the dorsal lateral geniculate nucleus (cat: Pirchio et al. 1990
; ferret: Ramoa and McCormick 1994
). A developmental increase in the incidence of LVA, consistent with the developmental autoregulation of LVA calcium currents, is also observed in embryonic cultures of hypothalamic parvocellular neurons (Desarménien et al. 1994
). In Xenopus spinal neurons, the developmental decrease in the LVA current coincides with the end of a calcium-sensitive period of neuronal differentiation (Gu and Spitzer 1993
; Spitzer et al. 1994
). A similar finding has been reported in embryonic chick motoneurons, in which the disappearance of LVA current is concomitant with the establishment of functional synapses (McCobb et al. 1989
). It is noteworthy that we observed a developmental increase in the incidence of LVA, consistent with the developmental autoregulation of LVA calcium currents in embryonic cultures of hypothalamic parvocellular neurons (Desarménien et al. 1994
). Although an increase in incidence accompanied by a decrease in density has not been reported for calcium currents, a similar pattern was observed for potassium A current in cat retinal ganglion neurons (Skaliora et al. 1995
).
Voltage-activated outward currents
TRANSIENT POTASSIUM CURRENT.
No major changes in the properties of the transient outward current were observed during postnatal development. A fast inactivating outward potassium current has been described in supraoptic magnocellular neurons recorded in hypothalamic slices from adult rats (Nagatomo et al. 1995
), where it is considered to be responsible for delaying depolarization-induced firing (Armstrong et al. 1994
). Because of differences in experimental protocols, close comparison of the data is not relevant. This current has been recorded in a large variety of excitable cells (see review by Rogawski 1985
), and is involved in modulation of excitability (Ribera and Spitzer 1990
; Segal et al. 1984
; Tell and Bradley 1994
) and synaptic efficacy (Kaang et al. 1992
). The absence of developmental change during postnatal life suggests that most of the maturation of the current occurs before birth. A similar conclusion was drawn from postnatal development of rat sympathetic neurons (Nerbonne and Gurney 1989
) and is consistent with reports of embryonic development of A current in other nonmammalian neuronal preparations (Bader et al. 1985
; Desarménien et al. 1993
; Ribera and Spitzer 1990
).
SUSTAINED POTASSIUM CURRENT.
The density of the sustained potassium current increased between PW1 and PW3, whereas the time to half-maximal amplitude remained stable. Because only its amplitude changed, maturation may involve an increase in channel density, rather than a switch toward expression of a different subtype of potassium channels. A developmental increase in the amplitude of the sustained potassium current has been described in Xenopus spinal neurons, where it contributes to the shortening of the action potential (Barish 1986
; Lockery and Spitzer 1992
; O'Dowd et al. 1988
). The reduction of sustained current amplitude observed when the current was activated from holding potentials at
60 mV and above may facilitate firing by reducing repolarizing drive. Indeed, phasic firing lasting several seconds is frequently superimposed on small plateau depolarizations around
50 mV in magnocellular neurons (Cobbett et al. 1988
). A similar slow inactivation of sustained current has also been reported in cat retinal ganglion cells after 500-ms conditioning prepulses (Skaliora et al. 1995
).
Role of voltage-activated currents in the shaping of action potential
A reduction of the action potential duration has been reported for neurons in several other regions of the CNS during postnatal development (Hockberger et al. 1989
; Kandler and Friauf 1995
; McCormick and Prince 1987
; Ramoa and McCormick 1994
). Here we have also examined the concomitant changes of voltage-gated currents. In summary, we observed an increase in the density of both sustained potassium current (repolarizing current) and HVA calcium current (depolarizing current), and a decrease of the density of the LVA calcium current. The properties studied were evenly distributed among magnocellular neurons and consequently are most probably expressed in both oxytocin- and vasopressin-secreting neurons.
To determine the respective contribution of each individual current to maturation of action potentials, we used a mathematical model in which action potentials were generated from currents with mature or immature densities. The model was based on calculations of one sodium, two calcium (LVA and HVA), and two potassium (sustained and transient) currents. The contribution of calcium-activated potassium currents was not investigated here. Indeed, the main role of these currents in magnocellular neurons appears to lie in the termination of bursting activity (Bourque and Brown 1987
; Bourque et al. 1985
). Although its contribution to the late phases of action potentials cannot be excluded, modeling of this current requires experimental data concerning its kinetics and its dependency on intracellular calcium (as well as data concerning the regulation of intracellular calcium in the vicinity of the intracellular mouth of the channel). The role of calcium-dependent currents will await for such data to be available. However, it is noticeable that this current was not required and that action potentials with characteristics similar to the recorded ones could be generated with the five purely voltage-dependent currents. In addition, we confirmed that an increase in the sustained potassium current by an amount similar to that recorded was sufficient to decrease the action potential duration to mature values. These results strongly suggest that the developmental increase in sustained potassium density is responsible for the shortening of the spike, as has been demonstrated in Xenopus spinal neurons (Barish 1986
; Lockery and Spitzer 1992
; O'Dowd et al. 1988
).
The fact that cadmium-induced reduction of action potential decreased with development suggests that calcium influx diminishes. On the other hand, we observed an increase in the density of the HVA calcium current. The actual contribution of calcium currents to the spike is hardly predictable, because it results from a competition between depolarizing and hyperpolarizing currents, the voltage dependency of which is at least as important as their maximal conductance. The mathematical model allows an evaluation of individual currents during an action potential; we used it to calculate the amount of calcium entering the neurons via LVA and HVA channels. Results show that this amount decreased by one third, mainly as a consequence of a decreased contribution of the LVA current. Observation of prominent calcium influx in newborn magnocellular neurons supports the hypothesis that the functional role of calcium entry in neuronal differentiation, demonstrated in Xenopus spinal neurons (Gu and Spitzer 1995
), exists in mammalian neurosecretory neurons as well. Prominent calcium influx in early development, promoted either by voltage-gated or by ligand-gated calcium currents, appears to be a general scheme in neuronal differentiation (reviewed by Spitzer et al. 1994
). This influx triggers diverse regulatory mechanisms, involving gene activation as well as postranslational modifications (Bading et al. 1993
; Desarménien and Spitzer 1991
; Holliday and Spitzer 1990
; Sheng et al. 1990
; Spitzer et al. 1993
). Further experiments will determine functional role of developmentally regulated calcium entry in magnocellular neurons.
 |
ACKNOWLEDGEMENTS |
We thank Professor N. C. Spitzer for judicious suggestions and careful revisions of the manuscript, for kindly providing the program used for currents and action potential modeling, and for the help that was thus a determinant factor in the achievement of this study. We also thank Drs. P. Richard, N. Hussy, F. Moos, and G. Dayanithi for fruitful discussions in the course of this work, and A. Duvoid for skillful technical assistance.
 |
FOOTNOTES |
Present address of H. Widmer: University of Massachussets Medical Center, Dept. of Pharmacology, 55 Lake Ave. North, Worcester, MA 01655.
Address for reprint requests: M. G. Desarménien, Biologie des Neurones Endocrines, CNRS UPR 9055, CCIPE 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France.
Received 7 December 1995; accepted in final form 3 September 1996.
 |
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